Extraction and culture of rat adipose-derived mesenchymal stem cells
Adipose-derived mesenchymal stem cells (ADSCs) were isolated from 6-week-old male SD rats. The rats were intraperitoneally anesthetized with 3% sodium pentobarbital solution and sterilized by immersion in 70% ethanol. Subcutaneous adipose tissue was collected from the inguinal region, placed in PBS buffer and sheared to a celiac shape. The adipose tissue was then digested with 0.1% type I collagenase (C8140; Solarbio, China) in a rotary shaker for 60 min at 37°C. DMEM containing 10% FBS was added to stop the enzymatic reaction. After being filtered through a 70 µm filter and centrifuged at 1200 rpm for 5 min, the cells were resuspended in DMEM containing 10% FBS, inoculated in Petri dishes and incubated at 37°C and 5% CO2.
Extraction and culture of primary rat chondrocytes
Rat knee cartilage tissue was clipped to a size of 1 mm³ and placed in PBS buffer. The tissues were then digested with 0.1% type II collagenase (C8150, Solarbio, China) at 37°C for 6 h. The digestion was terminated by adding DMEM supplemented with 10% FBS. After being filtered through a 70 µm filter and centrifuged at 1200 rpm for 5 min, the cells were resuspended in DMEM containing 10% FBS, inoculated in Petri dishes and incubated at 37°C with 5% CO2.
Flow cytometry-mediated identification of ADSCs
ADSCs cultured to the third to fifth generation were collected and washed three times with PBS, and the percentage of positive cells was analyzed by flow cytometry using antibodies against CD45 (P0096, Beyotime, China), CD90 (P0096, Beyotime, China), and CD105 (P0096, Beyotime, China), which were incubated with ADSCs for 25 min in the dark at 4°C. The cells were subsequently washed three times with PBS.
Cellular immunofluorescence analysis
Chondrocytes were seeded on the cell slides, fixed with 4% paraformaldehyde for 15 min, washed with PBS, incubated with immunostaining permeabilizer (P0096, Beyotime, China) for 10 min, and washed again with PBS. PBS solution containing 5% BSA was added to block the slides for 1 hour, the slides were washed with PBS, and the primary antibodies were added and incubated overnight at 4 ℃. On the next day, the primary antibody was added, and the second antibody, which was fluorescently labeled, was added and incubated with the slides for 1 hour. After being washed with PBS, the slides were sealed with anti-fluorescence quenching solution containing DAPI (P0131; Beyotime, China) and observed under a fluorescence microscope. The following antibodies were used: anti-collagen II (AF0135; Affinity, China) and anti-IL-1β (WLH3903; Wanleibio, China).
Transwell assay
ADSCs and chondrocytes were cultured in the upper and lower transwell chambers, respectively, for 24 hours. After the cells were attached, the transwell chamber containing ADSCs was placed in the bottom chamber in which the chondrocytes were cultured, and culture medium supplemented with 1 µg/mL LPS was added to the bottom chamber to simulate the environmental state in KOA.
Cytopathology
After the intervention, the chondrocytes were seeded on slides and fixed with 4% paraformaldehyde for 15 min. After fixation, the slides were washed with PBS, stained with dye, and then washed with running water. After drying, the cells were sealed with neutral glue and observed under a light microscope.
Western blotting
Proteins were extracted from chondrocytes and articular cartilage of the knee using RIPA buffer (89901, Thermo Scientific, USA). After the sample concentration was determined using a NanoDrop spectrophotometer, 20 µg of protein was loaded onto the gel, and the protein bands were transferred from the gel to the PVDF membrane using electrophoresis. The membranes were then blocked using 5% BSA. The primary antibody was added and incubated with the membrane, and the secondary antibody was added to the membrane. Finally, the luminescence intensity of the protein bands was observed with a chemiluminescence imager.
The following antibodies were used: beta-actin (1:1000, 4967, CST, USA), Collagen II (1:1000, AF5139, Affinity, China), ACAN (1:1000, DF7561, Affinity, China), MMP3 (1:1000, 14351, CST, USA), MMP13 (1:1000, 18165-1-AP, Proteintech, China), Sox9 (1:1000, 67439-1-Ig, Proteintech, China), Cox2 (1:1000, 12282, CST, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), Adamts5 (1:1000, DF13268, Affinity, China), PINK1 (1:1000, DF7742, Affinity, China), Parkin (1:1000, 32833, CST, USA), SQSTM1/p62 (1:1000, 5114, CST, USA), LC3 (1:1000, 14600-1-AP, Proteintech, China), and Beclin1 (1:1000, AF5128, Affinity, China). Western blotting was performed with antibodies against CD9 (1:1000, AF5139, Affinity, China), CD63 (1:1000, AF5117, Affinity, China) and CD81 (1:1000, DF2306, Affinity, China) to evaluate the protein markers of ADSCs-Exos.
Isolation and extraction of exosomes from ADSCs
Exos were collected from the supernatants of ADSCs using ultracentrifugation. ADSC supernatants were collected and sequentially centrifuged at 300 ×g, 2000 ×g, 10,000 ×g and 110,000 ×g. The residual cells, debris and impurities in the supernatants were removed by the different centrifugation steps. ADSC-Exos were ultimately isolated and resuspended in PBS and stored at -80°C for further experiments. The protein concentration of ADSC-Exos was determined by a BCA kit (P0010, Beyotime, China). The OD values of the standard proteins and ADSC-Exos were measured by a microplate reader, and standard protein curves were drawn according to the obtained values.
Nanoparticle tracer analysis (NTA)
A particle size analyzer (NanoSight NS300, Malvern, China) was used to analyze the size distribution of ADSC-Exos. The instrument was first cleaned with deionized water and then with polystyrene microspheres 100 nm in size. After calibration, the instrument was cleaned with PBS, and ADSC-Exos were diluted with PBS and injected for analysis.
Exosomes observation by transmission electron microscopy
The ADSC-Exo suspension (20 µL) was placed on a carbon membrane copper mesh and incubated for 3 min. The excess liquid was removed with filter paper, and 2% phosphotungstic acid was added to the carbon support membrane copper mesh and incubated for 1 min and dried at room temperature. Transmission electron microscopy and image analysis were performed.
Exosome tracing
ADSCs were labeled using the cell membrane red fluorescent probe DiL (C1036, Beyotime, China) according to the manufacturer's instructions, and labeled ADSCs were cocultured with rat primary chondrocytes for 24 h. Chondrocyte mitochondria were labeled using the mitochondrial far-infrared fluorescence probe MitoTracker Deep Red FM (C1032, Beyotime, China), and the cytoskeleton was labeled using the microfilament green fluorescence probe Actin-Tracker Green-488 (C2201s, Beyotime, China) after cell fixation. After being labeled, the cells were observed and imaged with an inverted fluorescence microscope.
CCK-8 assay
Chondrocytes were seeded in 96-well plates at a density of 4000 cells per well in 100 µL of medium. Cell culture media containing of 0, 1, 5, 10, 15, and 20 µmol/L GW4869 were prepared. After 24 h of culture, the medium was replaced with a solution containing 10 µL of CCK-8 for 2 h, after which the OD at 450 nm was measured by a microplate reader.
Mitochondrial membrane potential
After the treatment, chondrocytes were subjected to in situ probe loading using a JC-1 mitochondrial membrane potential detection kit (C2003, Beyotime, China) according to the manufacturer’s instructions. The cells were incubated at 37°C for 20 min, washed with PBS and observed under an inverted fluorescence microscope.
Reactive oxygen species
Reactive oxygen species (ROS) analysis (R252, DOJINDO, Japan) was performed according to the manufacturer’s instructions, and after the ROS working solution was added at a 1:1000 ratio, the cells were incubated with the DCFH-DA probe for 20 min at 37°C. Then, the cells were washed with PBS, and the images were observed under an inverted fluorescence microscope.
ATP content
Chondrocytes and cartilage tissues were collected after the indicated treatments, and changes in ATP levels were determined using an ATP detection kit (S0027, Beyotime, China). A standard curve was first established according to the standard protein protocol provided by the manufacturer. Then, the samples were lysed with ATP solution. The chemiluminescence intensity was measured by an enzyme-labeled instrument, and the ATP concentration of the sample was calculated based ona standard curve.
Chondrocytes were observed via transmission electron microscopy
Chondrocyte autophagosomes and mitochondria were observed under a transmission electron microscope. The chondrocytes were fixed with 2% glutaraldehyde for 2 h and then with 1% osmic acid in PBS for 2 h at room temperature. After dehydration in graded ethanol, the chondrocytes were embedded in 1,2-epoxypropane, sectioned and placed under a transmission electron microscope for observation and imaging.
Establishment of the KOA rat model and intervention
All animal experiments were reviewed by the Nanjing University of Chinese Medicine Animal Ethics Committee (202306A054). Thirty SD male rats (6 weeks old, 150 ± 10 g) were purchased from China Jiangsu Biotechnology Co., Ltd., and all animals were kept in a constant temperature and humidity environment (25 ± 5°C, 50%) with a 12-hour dark and 12-hour light cycle and free access to food and water. After three days of adaptive feeding, the rats were randomly divided into the sham group, KOA group and ADSC-Exos group (n = 10) using the statistical software SPSS20.0. In our previous study, after the rats were intraperitoneally anesthetized with 3% pentobarbital (2 mL/kg), rats in the KOA and ADSC-Exo groups were subjected to ACLT in both knees for 2 weeks to simulate knee cartilage wear in KOA. Subsequently, 100 µg/mL ADSC-Exos in 50 µL of PBS were injected twice per week into both knees, and equal amounts of PBS were injected into rats in the sham and KOA groups. Eventually, the rats were anesthetized and euthanized with pentobarbital to reduce pain, as shown in Fig. 6A.
Pathological and immunofluorescence staining of tissue sections
Rat knee cartilage samples were fixed with 4% paraformaldehyde overnight, embedded and sectioned at a thickness of 4 µm. The sections were stained with dyes, and after being washed, the sections were sealed with a neutral adhesive and observed under a light microscope to assess staining and for image analysis. When immunofluorescence analysis was performed, the sections were dewaxed and antigen repaired, and after being blocked, the sections were incubated overnight with SQSTM1/P62 (1:400, 23214; CST, USA) and LC3B (1:200; SC-271625; Santa Cruz, USA) antibodies. Then, fluorescently labeled secondary antibodies were added, the slices were incubated at room temperature for 1 hour, and a fluorescence microscope was used for observation and image collection.
Micro-CT
Micro-CT scanning and three-dimensional reconstruction of the rat knee were performed. Knee tissue was fixed in 4% formaldehyde for 24 h, placed in PBS for preservation, and analyzed using high-resolution micro-CT (SkyScan 1276, SkyScan, Belgium). Knee tissue was placed in the sample chamber and remained relatively still during the scan, and 3D image reconstruction was performed using CTvox software provided by the manufacturer.
ELISA
Serum levels of IL-1β (AF2923-A, Afantibody, China), IL-6 (AF3066-A, Afantibody, China), IL-15 (AF2962-A, Afantibody, China) and TNF-α (AF3056-A, Afantibody, China) were measured with ELISA kits. According to the manufacturer’s instructions, standard wells, sample wells and blank wells were prepared in the plate. After the corresponding samples were added, 100 µl of enzyme-labeled reagent was added to each well except for the blank wells, and the plate was incubated at 37°C for 60 min. Then, 250 µL of washing liquid was added to each well, after which the plate was washed 5 times. Color developing solution was added and incubated at 37 ℃ in the dark for 15 min, 50 µL of termination solution was added to each well, the OD value was measured at 450 nm, and the standard curve was used to calculate the concentrations of the samples.
Statistical analysis
Statistical comparisons between groups were performed using one-way analysis of variance (ANOVA) or paired t tests. A P value less than 0.05 (two-tailed) was considered to indicate statistical significance. All the statistical analyses and data visualizations were performed using GraphPad Prism 8.0.