Rat ASCs and DCs
Adipose tissue was obtained from the groin of 4-week-old male Sprague-Dawley (SD) rats. ASCs were obtained from adipose tissue using the explant culture method with alpha-minimal essential medium (α-MEM, M4526-500ML, Sigma-Aldrich) + 10% fetal bovine serum (FBS, FSP500, Excell) + 1% penicillin-streptomycin (PS, SV30010, HyClone). The method was performed as described [23]. When the cells reached 80–90% confluence, they were passaged using a 0.05% trypsin-EDTA solution (SH30042.01, HyClone). ASCs were used at passages 3–4. DCs were isolated from the femoral bone marrow of male SD rats. Afterward, the thigh bones were carefully separated in a sterile environment and immersed in RPMI 1640 medium (R8758-500ML, Sigma-Aldrich) + 1% PS. The epiphyses were removed, and the bone marrow was rinsed with RPMI 1640 medium. After centrifugation at 450× g for 5 min, red blood cell lysate (C3702-120ML, Beyotime) was added for 2 min, followed by 8 mL of normal saline to terminate the lysis. The cells were washed 2–3 times with phosphate buffer saline (PBS, P1003-2 L, Solarbio) and then cultured in RPMI 1640 medium supplemented with 10% FBS, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF, RP01207, ABclonal), and interleukin 4 (IL-4, RP01874, ABclonal). Treatment commenced on day 6, with the medium being replaced every other day.
Preparation and characterization of EVs
EVs were obtained by sequential centrifugation: 300× g for 10 min, 2000× g for 15 min, and 12,000× g for 40 min to remove debris, followed by 100,000× g for 2 h, and 10 min at 4 °C to pellet the EVs. EVs were then suspended in sterile PBS to form ASCs-EVs for the following experiments. To obtain EVs generated by ASCs treated with Rhodamine 6G (R-EVs), ASCs were exposed to 1 µg/mL Rhodamine 6G (56226-25MG, Sigma-Aldrich) for 48 h. The medium was then changed to exosome-free serum configured medium for another 48 h before supernatant collection and extraction as described. The ASCs-EVs were analyzed using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot to determine size distribution, morphological characteristics, surface markers (CD90, CD63, TSG101, and Calnexin), and the mitochondrial marker (TOMM20).
Rat TMJOA models
4-week-old male SD rats were used to create the TMJOA models. In the short-term inflammation group, male rats were given 50 µL [24] of complete Freund’s adjuvant (CFA, F5881-10 M, Sigma-Aldrich) to pretreat the temporomandibular joint for 10 days, followed by injections of 50 µL PBS, 106/mL ASCs, 50 µg/mL EVs, or R-EVs. One week later, the rats were euthanized, and their condyles were immersed in PBS. In the chronic inflammation group, the rats were injected with CFA every two weeks and given weekly injections of the test substances after four weeks. Samples were collected two weeks later.
Adipogenic, osteogenic and chondrogenic differentiation
1 × 105/mL of ASCs were inoculated in 6-well plates. For adipogenic differentiation, after the cells reached 80–90% confluence, 10% FBS was supplemented with α-MEM containing 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), 10 µM insulin (Sigma-Aldrich), 200 µM indomethacin (Sigma-Aldrich) and 1 µM dexamethasone (Sigma-Aldrich). The culture medium was changed every 2 days. After one week of culturing, the cells were treated with 10% neutral buffered formalin fixative (G2161, Solarbio) for 15 min, then stained with Oil Red O (08010-5G, Solarbio). For osteogenic differentiation, the cells were cultured in α-MEM containing 10% FBS, 100 µM dexamethasone (Sigma-Aldrich), 10 mM L-glycerophosphate (Sigma-Aldrich) and 50 µg/mL ascorbic acid (Sigma-Aldrich). The culture medium was changed every other day. After three weeks of culturing, the cells were treated with 10% neutral buffered formalin fixative for 15 min, then stained with a 1% solution of Alizarin Red (A5533, Sigma-Aldrich) for 30 min. To induce chondrogenic differentiation, follow the protocol outlined in the instruction manual for the stem cell chondrogenic induction differentiation kit (RAXMX-90041, Ori Cell). Proteoglycan secretion was detected in the samples via paraffin embedding, sectioning, and staining with Alcian Blue 8GX (Sigma-Aldrich). The phase-contrast inverted microscope (Olympus) was utilized for conducting observations and capturing photographs.
Real-time PCR
Total RNA from cells and tissues was extracted by the RNAiso Plus instrument. Reverse transcription was performed using the PrimeScript™ RT reagent Kit (RR036A, Takara Biomedical Technology). Expression levels of the target genes (Mmp13, Mmp3, Il-6, Mmp9, Runx2, Tnf-α, aP2, Pparγ2, Adiponectin, Gapdh, Glut1, Hk2, Ldha, Adamts5, Col II and Actin) were determined using SYBR Premix (1129280, QIAGEN). The relative expression of normalized mRNA was measured using 2-ΔΔCT, with Gapdh or Actin serving as the internal reference genes. Real-time PCR primer sequences are shown in Supplementary Table S3. Each experiment included a minimum of three technical replicates.
TEM
10 µL drops of the EVs sample were absorbed and deposited on the copper grid for 1 min. The filter paper wicked away the excess liquid. 10 µL of uranyl acetate was added to the copper grid to precipitate for 1 min, and the filter paper wicked away the excess liquid. The sample was dried at room temperature for a few minutes. The imaging results were obtained using transmission electron microscopy (HT-7700, Hitachi) at 100 kV.
NTA
The size and distribution of EV samples in liquid were tracked by NTA technique. EVs were diluted with 1×PBS and the NTA device (ZetaVIEW, PARTICLE METRIX) was used for NTA detection. After the sample was thoroughly mixed, 1 mL of the sample was slowly loaded into the chamber of the NTA device. The NTA system was then started for tracking, and the NTA software was used for data acquisition and analysis.
Histological analyses
Condyle processes were extracted at specified time points (day 7 and day 14 after injection). Specimens were examined under a microscope (Zeiss), then immersed in 10% neutral buffered formalin fixative for 2 days, then subjected to 17% ethylenediaminetetraacetic acid (EDTA, E9884, Sigma-Aldrich) for decalcification, and embedded. Sections were treated with xylene to remove paraffin, hydrated with ethanol, and finally stained with H&E (G1120, Solarbio) and safranin O-fast green (G1371, Solarbio). Cartilage degeneration was evaluated by comparing the ratio of the fibrocartilage layer (FC) to the calcified cartilage layer (CC).
Immunofluorescence analyses
Following deparaffinization and rehydration, the sections were immersed in trypsin (ZLI-9010, ZSGB-BIO) at 37 °C for 15–20 min for antigen retrieval, treated with 3% hydrogen peroxide (PV-6001, ZSGB-BIO) for 15 min, and then blocked with 1% sheep serum for 30 min (SAP-9100, ZSGB-BIO) at 37 °C. Sections were then incubated with primary antibodies against IL-6 (ab9324, Abcam), TNF-α (ab307164, Abcam), and MMP13 (ab219620, Abcam) overnight at 4 °C. Alexa 488 dye-labeled secondary antibodies were used to stain the sections, along with DAPI (C1002, Beyotime) for nuclear counterstaining. The fluorescence microscope (Zeiss) was used to capture images.
DCs phagocytosis in vitro
EVs were marked with PKH26 (D0030, Solarbio) and added to the DCs, and staining was performed at multiple time points (15 min, 2 h, and 24 h). Initially, cells were fixed with 10% neutral buffered formalin fixative for 10 min, followed by Ghost Cyclic Peptide (40736ES75/300T, Yeasen) staining of the cytoskeleton for 60 min, and DAPI staining of the nucleus for 10 min. In the mitochondria phagocytosis experiment, the mitochondria of ASCs were first labeled with red mitochondria tracker (C1032-50µg, Beyotime) for 20 min. The dye was then washed off, the cells were cultured for 2 days, and collected supernatant to extract EVs, which were then added to the DCs. The mitochondria in the DCs were then marked with green (C1048, Beyotime). The fluorescence microscope (Zeiss) was used to capture images. In addition, after adding PHK26-labeled EVs to DCs, we also used confocal microscopy (Leica) to capture the live-cell uptake experiment.
Flow cytometry
DCs from different groups were digested with pancreatic enzymes and washed twice with PBS containing 2% FBS. The cells were then incubated with 1% Goat Serum (C01-03001, Bioss) at room temperature for 30 min. Subsequently, antibodies against CD11b/c (201809, Biolegend), CD80 (200205, Biolegend), CD86 (200307, Biolegend) and MHC-II (205405, Biolegend) were added to each group, incubated for 30 min in darkness, and subsequently rinsed 2–3 times. The cells were then filtered through a filter screen and tested using a flow cytometer (BD Biosciences).
Mitochondrial membrane potential (JC-1)
In accordance with the guidelines provided by the JC-1 staining kit (C2006, Beyotime). DCs were cleansed and then exposed to 5 µM JC-1 for 30 min at 37 °C. The cells were then rinsed three times with PBS. Images were captured using a fluorescence microscope (Zeiss). The fluorescence intensity ratio between the red and green channels was analyzed using ImageJ.
ELISA
In accordance with the guidelines provided by the ELISA kit. The supernatant from various cellular components was gathered, subjected to centrifugation at 4 °C and 1000× g for 15 min. The liquid above was kept for analysis using the IL-6 (RK00020, ABclonal) and the TNF-α ELISA kit (RK00029, ABclonal).
Chondrocyte culture
Cartilage tissue was obtained from the TMJ of the rat and cut into small pieces with a scalpel. The tissue was then incubated in 0.5 mg/mL collagenase type II (40508ES60, Yeasen) at 37 °C in a shaking incubator for 2 h. After digestion was complete, the mixture was centrifuged at 1000–1200 rpm for 5–10 min to pellet the cells, and the supernatant was removed. The cell pellet was resuspended in DMEM/F12 (C11330500BT, Gibco) containing 20% FBS (A5669402, Gibco) and seeded into culture dishes.
Chondrocyte inflammation induction
The supernatants of DCs treated with different conditions for 24 h (untreated DCs, LPS, LPS + EVs, LPS + 2-DG, LPS + C75, LPS + FCCP) were collected. The concentration of 2-DG used was 100 mM. The concentration of C75 used was 20 µM. The concentration of FCCP used was 1 µM. The amounts of reagents used were all referenced from the literature [20]. The supernatants from each group were then added to the chondrocytes, and after 24 h, the chondrocytes were collected for subsequent experiments.
Oxygen consumption rate (OCR)
Seahorse XF HS Mini (Agilent) was utilized for measuring OCR. The OCR rate was assessed following the introduction of various inhibitors: oligomycin 2 µM, FCCP 1 µM, antimycin A and rotenone 1 µM (Agilent). The Wave software (Agilent) was utilized to compute basal respiration, ATP generation, respiratory capacity, and respiratory reserve.
RNA sequencing
After quickly draining the medium, pre-cooled PBS buffer at 4 °C was added to rinse the cells repeatedly for 2 to 3 times. The cells were then treated with 1 mL RNAiso Plus (9109, Takara Biomedical Technology) for 1 min, homogenized thoroughly and evenly transferred to 1.5 mL EP tubes for storage at -80 °C. The NanoDrop 2000 spectrophotometer (Thermo Scientific) was utilized for assessing the purity as well as quantification of RNA samples. The Agilent 2100 Bioanalyzer (Agilent Technologies) was employed for evaluating the integrity of RNA. Subsequently, libraries generated from the VAHTS Universal V6 RNA-seq Library Prep Kit were prepared in accordance with the guidelines provided by the manufacturer Oebiotech (China) and used for RNA sequencing.
Metabolite sample preparation
Drain the solution rapidly, then rinse with pre-cooled PBS buffer at 4 °C or normal saline 2 to 3 times (if using a pipette, direct the solution against the petri dish wall to prevent cell flushing). Absorb the excess PBS buffer, then bring the liquid nitrogen to the outer surface of the petri dish in order to induce cell freezing (1 × 107 cells are recommended), with a freezing time of approximately 1 min. Next, pour 500 µL of chilled methanol-water (4:1, v/v) into the petri dish, then collect the cells. Subsequently, change them to 1.5 mL frozen storage tube or a thick-walled centrifuge tube using a pipette. Then, add another 500 µL of chilled methanol-water (4:1, v/v) into the petri dish, transfer any remaining cells to the storage tube, and seal it with a sealing film before storing it at -80 °C. The analysis was performed at Oebiotech (China). Metabolomics normalization was achieved through the method of Internal Standard Normalization.
WGCNA and module-traits relationships
As a research tool, the WGCNA package from R is employed to analyze metabolomics data across distinct groups [25]. By establishing a network of genes that are co-expressed, our objective is to uncover significant metabolites linked to traits and gain deeper insights into the underlying mechanisms. The determination of topological overlap measures involves the use of an adjacency matrix, and the application of the DynamicTree Cut algorithm from the WGCNA package is utilized to partition metabolites into distinct modules. The modules were considered significant if|r| ≥ 0.5 and P < 0.05. To identify core metabolites, we applied stricter criteria of module > 0.8 and gene significance > 0.2. Finally, core metabolites were subjected to functional annotation and pathway enrichment analysis using the KEGG database.
Proteomics
EVs and R-EVs were extracted separately according to the aforementioned methods. An appropriate amount of each sample was transferred to an ultrafiltration tube, followed by centrifugation at 14,000 × g for 15 min, and the filtrate was discarded. The ultrafiltrated samples were then transferred to a 1.5 mL centrifuge tube, and DB solution (6 M urea, 100 mM TEAB, pH 8.5) was added to equilibrate the protein solution. The supernatant was collected, and an appropriate volume of 1 M DTT was added to react at 56 °C for 1 h. After that, the samples were placed in an ice bath for 2 min, followed by the addition of an excess of IAM for a 1-hour reaction in the dark at room temperature. Detection was performed using the Thermo Orbitrap Astra mass spectrometer.
Lipophilic membrane dye labeling
Following the guidelines provided by the manufacturer, PKH26 was used for labeling the EVs. The EVs were incubated for 5 min. To halt the staining reaction, the termination solution of PKH26 (1% BSA) was added and incubated for 1 min. Subsequent steps, including PBS washes and EV extraction, were performed in a dark environment as previously described.
mIHC
After dewaxing and rehydrating, the tissue sections were subjected to trypsin treatment at 37 °C for 15–20 min to restore antigenicity. Subsequently, they were exposed to a solution containing 3% hydrogen peroxide for 15 min and then blocked with 1% sheep serum at 37 °C for 30 min. Following the blocking step, primary antibodies were applied and incubated at room temperature for 1 h. The sections were submerged in 1×TBST Buffer for 2 min and underwent three consecutive repetitions. Excessive liquid was removed from the sections, followed by the addition of secondary antibodies (10079100020, PANOVUE) labeled with HRP. The sections were subsequently placed in a room-temperature environment with adequate moisture for a period of 30 min. Subsequently, the sections were immersed in 1×TBST Buffer for a period of 2 min and this process was repeated three times. Monochromatic TSA fluorescent dye (10079100020, PANOVUE) was gradually introduced. The sections were incubated at ambient temperature and kept moist for a duration of 15 min, while the sections were submerged in 1×TBST Buffer for a period of 2 min, which was repeated three times. Subsequently, the antibody eluate was applied for 5 min (10108001010, PANOVUE), followed by immersion in 1×TBST Buffer for another 2 min, also repeated three times. Next, another primary antibody was applied after the blocking step. Following the completion of the last 1×TBST washes, 10 min DAPI staining was performed and then rinsed with sterilized water for 2 min. Subsequently, the sections were gently dried and treated with an enhanced anti-quench sealant. Confocal laser scanning microscopy (Leica) was used to capture images.
Western blot
In different treatment groups of DCs, the treatment time and concentration for LPS and EVs are the same as mentioned above. The treatment time for the FoxO1 inhibitor (AS1842856) is 24 h, and the concentration is 100 nM [26]. The RIPA lysis buffer (P0013B/100 ml, Beyotime), supplemented with protease inhibitor, was used to extract the total protein. The BCA protein assay kit (P0012, Beyotime) was utilized to ascertain the concentration of the protein extracted. The protein extracts were transferred to a polyvinylidene fluoride (1620177, Bio-Rad) after SDS-PAGE. The membrane was incubated at a temperature of 4 °C for the duration of the night with primary antibody: anti-FoxO1 (2880, Cell Signaling Technology), anti-P-FoxO1 (9461, Cell Signaling Technology), anti-LC-3 (83506, Cell Signaling Technology), anti-p62 (39749, Cell Signaling Technology), anti-ERK1/2 (sc-514302, Santa Cruz Biotechnology), anti-P-ERK1/2 (sc-81492, Santa Cruz Biotechnology), anti-JNK (9252, Cell Signaling Technology), anti-P-JNK (9251, Cell Signaling Technology), anti-P38 (8690, Cell Signaling Technology), anti-P-P38 (28796-1-AP, Proteintech), or Actin (4970, Cell Signaling Technology). The primary antibody was changed to secondary antibodies labeled with HRP (1706515, 1706516, Bio-Rad). The ChemiDoc MP system (Bio-Rad) was employed for the detection of the images. The ImageJ software was utilized to quantify the grayscale values of each band, which were subsequently normalized to obtain the corresponding Actin expression. Original images of representative Western blots are provided in the supplementary information (Data. S1).
Measurements of free fatty acid and lactic acid
The supernatants from DCs in different treatment groups were collected, centrifuged at 4 °C and 1000× g for 15 min. Free fatty acids were detected in accordance with the guidelines provided by the Amplex Red Free Fatty Acid Assay Kit (S0215S, Beyotime). Lactic acid was identified based on the provided guidelines of the L-Lactic Acid (LA) Colorimetric Assay Kit (E-BC-K044-M, Elabscience).
Quantification and statistical analysis
The data reported as mean ± SEM. Statistical significance was indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001 in all conducted tests. A significance level of P < 0.05 was employed to establish the statistical significance. GraphPad Prism was employed for statistical analysis. Statistical significance was assessed using one-way ANOVA and Tukey’s post hoc test for comparing multiple groups.