Abstract Temporomandibular joint osteoarthritis (TMJOA) urgently needs regenerative therapies due to the limited effects of traditional treatments. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are considered a potent alternative for MSC therapy for the treatment of TMJOA. However, the specific mechanisms remain inadequately investigated. In this study, we explored how EVs from adipose-derived stromal/stem cells (ASCs) influence the TMJOA model triggered by Complete Freund’s Adjuvant in rats and their impact on the state of dendritic cells (DCs) under pathological conditions. Subsequently, we conducted transcriptomic and metabolomic analyses to elucidate the specific mechanisms by which EVs affect DCs. Mechanistically, we demonstrate that EVs transferred functional mitochondria to DCs, which reverses their metabolic states. The internalized functional mitochondria from EVs activate the MAPK/ERK1/2/FoxO1/autophagy pathway, which causes the metabolic reprogramming of DCs and facilitates the achievement of therapeutic effects. These findings provide a mechanistic rationale for utilizing ASCs-EVs as cell-free alternatives to MSC transplantation in TMJOA therapy. Graphical Abstract [42]graphic file with name 12951_2025_3478_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03478-9. Keywords: Extracellular vesicles, Mitochondria, Dendritic cells, Autophagy, Reprogramming metabolism Introduction Temporomandibular joint osteoarthritis (TMJOA) is a notable category of temporomandibular joint conditions [[43]1], marked by the gradual breakdown of cartilage, bone erosion, and ongoing discomfort, severely affecting the well-being of those who suffer from it [[44]2]. At present, the treatment approach for TMJOA mainly aims to alleviate symptoms, with only modest effectiveness in repairing the compromised temporomandibular joint [[45]1, [46]3]. Therapies centered on mesenchymal stem cells (MSCs) are showing great potential in treating osteoarthritis [[47]4, [48]5]. Adipose-derived stromal/stem cells (ASCs), in particular, are notable for their strong abilities to multiply and differentiate, and they can be sourced at a comparatively low expense [[49]6]. Despite promising clinical outcomes of ASCs injection in osteoarthritis trials [[50]7–[51]9], safety concerns associated with whole-cell therapies necessitate the identification of key paracrine mediators. Extracellular vesicles (EVs) have emerged as cell-free alternatives that recapitulate MSC therapeutic effects [[52]10], yet their mechanism of action requires systematic elucidation. A multitude of studies have explored the function of EVs across a range of biological activities and pathological conditions. Within the realm of immune regulation, it has been observed that EVs engage with a variety of immune cells [[53]11, [54]12]. For example, research has indicated that EVs have the capacity to influence the behavior of macrophages [[55]13]. They can direct macrophages towards an anti-inflammatory state, which results in a decrease in the generation of pro-inflammatory cytokines like TNF-α and IL-6, and a boost in the release of anti-inflammatory elements such as IL-10. The influence of EVs on macrophage activity is believed to contribute to the mitigation of inflammatory responses across multiple disease scenarios [[56]14, [57]15]. Dendritic cells (DCs), pivotal regulators of immune tolerance and inflammation, have emerged as key contributors to osteoarthritis pathogenesis through their immunomodulatory functions [[58]16, [59]17]. While research hints at the potential of MSCs-EVs to shape immune reactions through interactions with immune cells, the detailed mechanisms on DCs activation, maturation, and antigen-presenting capacity are still unclear. Notably, metabolic reprogramming is crucial for the transition of different states in DCs [[60]18–[61]20], yet how MSC-EVs modulate DC metabolism to ameliorate TMJOA remains unexplored. Emerging evidence suggests mitochondrial transfer between cells can exert anti-inflammatory effects through metabolic reprogramming, a process particularly relevant to DC functional states [[62]21, [63]22]. However, the therapeutic effects of MSCs-EVs in modulating DC metabolism for TMJOA treatment remain unaddressed. In this study, EVs were effectively isolated from ASCs and demonstrated potent anti-inflammatory properties in an experimental TMJOA model by transferring functional mitochondria. The results showed that DCs activated the MAPK/ERK1/2/FoxO1/autophagy pathway after internalizing the functional mitochondria from EVs, which then altered cellular metabolism. This reprogramming promoted the transition of DCs from an activation state to a tolerance state, thereby reducing inflammation. The primary objective is to elucidate the molecular mechanisms of ASCs-EVs modulates the immune response of DCs in TMJOA, aiming to develop novel targeted therapeutic approaches. Materials and methods 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 [[64]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 [[65]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, 10^6/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 × 10^5/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 [66]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 [[67]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 × 10^7 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 [[68]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 [[69]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. Results EVs from ASCs improve TMJOA in rat Initially, ASCs and their EVs were effectively isolated and characterized. After adipogenic induction of ASCs, Oil Red O staining revealed a large number of lipid droplets within the cells. After osteogenic induction of ASCs, Alizarin Red staining showed the formation of calcified nodules within the cells. After chondrogenic induction of ASCs, Alcian Blue staining demonstrated significant glycosaminoglycan synthesis and deposition in the extracellular matrix (Fig. [70]S1A). Subsequently, Real-time PCR experiments indicated that the expression of Runx2 increased after osteogenic induction, while the expression of aP2, PPARγ2, and Adiponectin increased after adipogenic induction (Fig. [71]S1B). Flow cytometry analysis showed that more than 99% of the cells expressed CD29 and CD90, while less than 5% of the cells expressed CD34 and CD45 (Fig. [72]S1C). We used differential centrifugation to isolate EVs, and the specific steps of the procedure are shown in the schematic diagram (Fig. [73]S1D). TEM revealed the double-layered membrane structure of EVs (Fig. [74]S1E). The NTA results showed that the average diameter of EVs is 143 nm (Fig. [75]S1F). Western blot analysis confirmed the presence of CD90, CD63, and TSG101 in EVs, but did not detect the endoplasmic reticulum marker Calnexin (Fig. [76]S1G). In this research, we evaluated the therapeutic potential of ASCs and ASC-EVs in a short-term (10 days) rat model of TMJOA induced by CFA (Fig. [77]1A and Fig. [78]S2). After treatment with EVs and ASCs, the degree of swelling and hyperplasia in the perichondral tissue of TMJ was considerable (Fig. [79]1B). The presence of the FC and the CC was observed through the application of H&E and Safranin O/Fast Green Staining to mandibular condylar cartilage. Compared with the NC group, the FC showed considerable reduction and the CC was notably thinner in the PBS group. However, the damage was significantly reduced in both the EVs and ASCs groups. (Fig. [80]1C-D). Real-time PCR analysis of temporomandibular joint disc showed that MMPs, which promote cartilage matrix degradation, including Mmp-9, Mmp-3 and Mmp-13 were up-regulated in the PBS group and were reversed in both the EVs and ASCs groups. The effect of the EVs group was as that of the ASCs group. Similar patterns were observed in factors related to inflammation, including Il-6 and Tnf-α (Fig. [81]1E). Immunofluorescence staining showed that the expression trend of intra-condylar related factors was the same as that of Real-time PCR (Fig. [82]1F). Fluorescence density and intensity were quantitatively analyzed (Fig. [83]1G). These findings underscore that ASCs can treat TMJOA in rats through EVs. Fig. 1. [84]Fig. 1 [85]Open in a new tab EVs from ASCs improve TMJOA in rat. (A) The rat TMJOA model was divided into four groups: NC, PBS, ASCs and EVs (n = 6 in all groups). (B) Representative images of condyles from the NC, PBS, ASCs and EVs groups. (C) Representative H&E and safranin O-fast green staining of condyles from the NC, PBS, ASCs and EVs groups. FC (F: fibrous zone; P: proliferative zone; M: mature chondroblast zone), CC (H: hypertrophic layer), SB: subchondral bone. (D) Calculation of FC/CC ratio based on H&E and safranin O-fast green staining in the NC, PBS, ASCs and EVs groups. (E) Expression levels of Mmp9, Il-6, Mmp3, Tnf-α and Mmp13 mRNA in relation to each other from articular disc treated with PBS, ASCs and EVs. (F-G) Representative immunofluorescence pictures (F) and quantification fluorescence intensity (G) for IL-6, TNF-α and MMP13 in condyles from the NC, PBS, ASCs and EVs groups The significant role of DCs in the EVs-mediated treatment process To further explore the specific mechanisms of EV treatment, we found that in the rat TMJOA model, the number of activated DCs significantly increased, while after EV treatment, their number markedly decreased (Fig. [86]2A-B). Additionally, we isolated synovial fluid from different experimental groups and analyzed it using flow cytometry. The results showed that EVs could significantly reduce the number of activated DCs (Fig. [87]2C-D). Subsequently, we investigated the effects of EVs on DCs in vitro. We effectively isolated and characterized DCs (Fig. [88]S3A). Using PKH26 fluorescence labeling, we observed the uptake of EVs by DCs, and the results showed that DCs could easily capture ASC-derived EVs within 15 min, 2 h and 24 h (Fig. [89]2E and Video S1). To verify the effects of EVs on DCs in vitro, EVs were administered to DCs at concentrations of 10 µg/mL, 50 µg/mL, and 100 µg/mL respectively, demonstrating significant anti-inflammatory effects across all doses (Fig. [90]S3B). Subsequent experiments were conducted using 50 µg/mL of EVs. The results showed that EVs could restore the decreased membrane potential of DCs (Fig. [91]2F-G). Additionally, we found that EV treatment down-regulated the expression of inflammatory markers Cd80, Cd86, and Mhc-II as confirmed by Real-time PCR analysis (Fig. [92]2H). The ELISA results showed that EVs could reduce the inflammatory cytokines IL-6 and TNF-α in the supernatant of DCs (Fig. [93]2I). Real-time PCR analysis of TMJ chondrocytes showed that treatment with the supernatant of DCs from the EVs group led to decreased expression of the inflammatory factors Il-6 and Tnf-α. Additionally, the expression of the catabolism-related molecules Adamts5 and Mmp13 was reduced. While the expression of Col II was increased (Fig. [94]2J). In summary, the results of this part of the study indicate that DCs play a significant role in the treatment of rat TMJOA mediated by EVs. Fig. 2. [95]Fig. 2 [96]Open in a new tab The significant role of DCs in the EVs-mediated treatment process. (A-B) Representative confocal laser scanning microscope images (A) and quantification of fluorescence intensity (C) showing colocalization between CD11c and MHC-II in condyles with different treatments. Arrows indicate colocalization of CD11c and MHC-II. (C-D) Flow cytometry analysis (C) and quantification (D) of the surface antibody MHC-II on DCs derived from synovial fluid. (E) Schematic diagram and fluorescence images that show the in vitro tracing of EVs. (F) JC-1 fluorescent images showing mitochondrial membrane potential in DCs treated with LPS alone and LPS combined with EVs. (G) Flow cytometry analysis of mitochondrial membrane potential in DCs treated with LPS alone and LPS combined with EVs. (H) Relative mRNA expression levels for Cd80, Cd86, and Mhc-II in DCs treatment with LPS alone and LPS combined with EVs. (I) ELISA quantification of IL-6 and TNF-α in supernatants of DCs treatment with LPS alone and LPS combined with EVs. (J) Relative mRNA expression levels for Il-6, Tnf-α, Mmp13, Adamst5 and Col II in chondrocytes treated with conditioned media from DCs RNA-seq and metabolomics analysis in DCs To obtain further insights into the mechanisms underlying EV function, we conducted comprehensive omics analyses on DCs subjected to various treatments (Fig. [97]3A). RNA-seq revealed a total of 3514 genes altered in the LPS group compared to the Control group, with 1840 genes being up-regulated and 1736 genes down-regulated. Additionally, there were 95 genes changed between the EVs group and the LPS group, with 74 genes up-regulation and 83 genes down-regulation. Moreover, there were 62 overlapping genes that displayed consistent changes in both comparisons (Fig. [98]3B and Fig. [99]S4A). The volcano map displays the top 10 genes with increased and decreased (Fig. [100]3C-D). As for metabolomics, OPLS-DA technique was employed to analyze the data for each individual sample. This analysis revealed minimal variability within groups but significant differences between groups (Fig. [101]S4B). Subsequently, further analysis revealed that 258 metabolites showed altered levels in the LPS group compared to the control group, with 93 metabolites upregulated and 165 down-regulated. Moreover, EV treatment induced changes in 209 metabolites compared to the LPS group, including 147 upregulated and 62 down-regulated metabolites. Interestingly, 107 metabolites were found to overlap between these two comparisons (Fig. [102]3E and Fig. [103]S4C). The heat map visualized the top 50 metabolites across different comparison groups (Fig. [104]3F-G). All differential genes and metabolites can be obtained in the supplemental material (Table [105]S1, 2). WGCNA was conducted to identify key metabolites associated with specific traits. Three modules were identified between the Control and the LPS group, whereas 17 modules were identified between the EVs and the LPS group (Fig. [106]3H-I). Ultimately, two crucial metabolites were discovered: one in the blue module for Control vs. LPS and another in the yellow-green module for EVs vs. LPS, both showing a correlation coefficient (|r|) ≥ 0.5 and statistical significance (P < 0.05) (Fig. [107]S4D). Finally, pathway mapping of differentially expressed genes and metabolites revealed a total of 120 common pathways between the LPS group and the Control group. Additionally, there were 50 shared pathways observed in the EVs group and the LPS group (Fig. [108]3J-K). A bubble diagram illustrating the top 30 pathways was constructed (Fig. [109]3L-M). Overall, these findings indicate that EV treatment induces changes in the genes and metabolism of DCs. Fig. 3. [110]Fig. 3 [111]Open in a new tab RNA-seq and Metabolomics analysis in DCs. (A) Schematic diagram illustrating the approach for RNA-seq and Metabolomics analysis. (B) Pie chart showing cumulative gene alterations identified from RNA-seq data in LPS vs. Control and EVs vs. LPS groups. (C-D) Volcano plots displaying the top 10 gene names both up-regulated and down-regulated from RNA-seq in LPS vs. Control and EVs vs. LPS groups. (E) Pie chart illustrating cumulative metabolite alterations identified from metabolomics in LPS vs. Control and EVs vs. LPS groups. (F-G) Heat maps showing the top 50 changed metabolite names from metabolomics in LPS vs. Control and EVs vs. LPS groups. (H-I) The correlation between metabolites characteristic of different modules and various traits is represented by columns, while rows represent the metabolites of each module. The upper value in each cell indicates the correlation coefficient, while the lower value represents the corresponding P-value. (J-K) Pie charts illustrating common pathway alterations identified from RNA-seq and Metabolomics analyses in LPS vs. Control and EVs vs. LPS groups. (L-M) Bubble plots displaying the top 20 common pathway names identified from RNA-seq and Metabolomics analyses in LPS vs. Control and EVs vs. LPS groups EVs change the state of DCs to reduce injury via functional mitochondria transfer By analyzing the RNA-seq data, we found that one of the main changes following EV treatment is the alteration in energy metabolism (Fig. [112]4A). Therefore, we speculated that this may be due to the action of mitochondria within the EVs. To investigate whether EVs exert their effects through transferring mitochondria to DCs. 5-10% of the particles within the EVs that are larger than 500 nm in diameter fall within the size range typical of mitochondria. Meanwhile, electron microscopy and Western blot results showed that EVs contained mitochondria (Fig. [113]4B-C). However, R-EVs, which inhibit the mitochondrial function, have less than 1% of particles larger than 400 nm, corresponding to the size range of mitochondria and showed no detection of the TOMM20 protein (Fig. [114]4C-D). This result is consistent with previous studies [[115]27]. Subsequently, we conducted proteomic analysis on EVs and R-EVs. The results showed that the changes in mitochondrial proteins were highly significant, and they were downregulated in R-EVs (Fig. [116]4E and Fig. [117]S5). Subsequently, the mitochondria of ASCs and DCs were fluorescently stained to prove that EVs contained mitochondria of ASCs and could be engulfed by DCs (Fig. [118]4F). In order to ascertain the mechanism of mitochondrial transfer effects, we used LPS for the purpose of simulating TMJOA damage in vitro. To compare the amount of DCs maturation, a variety of detection methods we used (Fig. [119]4G). JC-1 fluorescence staining of mitochondrial membrane potential showed that EVs could increase the membrane potential reduced by LPS, while no effect was observed with R-EVs (Fig. [120]4H). Surface markers expression was detected using flow cytometry. Findings indicated that the expressions of CD80, CD86 as well as MHC-II in DCs were increased after LPS treatment, but decreased after EVs treatment (Fig. [121]4I-J). ELISA analysis of TNF-α and IL-6 in supernatants confirmed EVs treatment reduced DCs inflammation, whereas R-EVs did not (Fig. [122]4K). The diagram shows the changes in surface antibody expression on DCs between the activation state and the tolerance state after treatment (Fig. [123]4L). The etiological factors of TMJOA typically do not include bacterial infections, hence we employ IL-1β induced inflammation model as a supplement. The concentration of IL-1β was assessed using the relative mRNA expression levels for Il-6 and Tnf-α (Fig. [124]S6). This anti-inflammatory effect was similarly observed in an in vitro model of IL-1β (Fig. [125]S7). These findings underscore that EVs can change the activation state of DCs to reduce injury via mitochondrial transfer, offering insights into potential therapeutic strategies for managing inflammatory conditions. Fig. 4. [126]Fig. 4 [127]Open in a new tab EVs change the state of DCs to reduce injury via functional mitochondria transfer. (A) GO analysis of variably expressed messenger RNA associated signaling pathways in EVs vs. LPS groups. (B) Electron microscopic image of mitochondria structure in EVs, yellow arrows indicate mitochondrial structure. (C) Representative Western Blot image for TOMM20 of EVs and R-EVs. (D) Particle size analysis of EVs (left) and R-EVs (right). (E) Protein changes in EVs and R-EVs. (F) Schematic diagram illustrating the trace of EVs and mitochondria in EVs, fluorescence images showing in vitro tracer of EVs. (G) Representative confocal laser scanning microscope images showing in vitro tracer of mitochondria transferred by EVs. (H) JC-1 fluorescent images showing mitochondrial membrane potential in DCs treated with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (I) Flow cytometry analysis of surface antibodies CD80, CD86 and MHC-II in DCs treated with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (J) Quantification of flow cytometry analysis data across different groups. (K) ELISA quantification of IL-6 and TNF-α in supernatants of DCs treated with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (L) Schematic diagram illustrating the transition of DCs between different states Mitochondria are essential for the treatment of EVs in rat TMJOA Initially, we induced TMJOA in rats using intra-articular injections of CFA. We established short-term (10 days) and long-term inflammation (4 weeks) models in rats and administered treatments accordingly (Fig. [128]5A). Results revealed that R-EVs proved ineffective in reducing inflammatory damage and the swelling of the joints in both short-term inflammation and long-term inflammation models (Fig. [129]5B). Compare to EVs group, Real-time PCR analysis of temporomandibular joint discs showed that Mmp-9, Mmp-3, Mmp-13, Tnf-α and Il-6 exhibited upregulated expression in the R-EVs group (Fig. [130]5C-D). Furthermore, the reduction of FC/CC was observed in both H&E and Safranin O/Fast Green Staining in the R-EVs group compared to the EVs group (Fig. [131]5E-F). Immunofluorescence staining indicated an upregulation regarding to MMP-13, TNF-α and IL-6 compared to the EVs group (Fig. [132]5G). Fluorescence density and intensity were quantitatively analyzed (Fig. [133]5H). The results highlight the essential role of mitochondria function in the therapeutic mechanism of EV in TMJOA. Fig. 5. [134]Fig. 5 [135]Open in a new tab Mitochondria are essential for the treatment of EVs. (A) Grouping of the rat TMJOA short-term inflammation model: NC, PBS, EVs and R-EVs (n = 6 in all groups); Rat TMJOA long-term inflammation model: NC, PBS, EVs and R-EVs (n = 6 in all groups). (B) Representative images of condyles from the short-term inflammation and long-term inflammation rat models from the NC, PBS, EVs and R-EVs groups. (C-D) Expression levels of Mmp9, Il-6, Mmp3, Tnf-α and Mmp13 mRNA in relation to each other from articular disc treated with NC, PBS, EVs and R-EVs. (E) Representative H&E and safranin O-fast green staining of condyles from the different groups. (F) FC/CC ratio derived from H&E and safranin O-fast green staining in the different groups. (G-H) Representative immunofluorescence pictures (G) and quantification of fluorescence intensity (H) for IL-6, TNF-α and MMP13 in condyles from the NC, PBS, EVs and R-EVs groups in both short-term and long-term inflammation Transferred functional mitochondria activate the ERK1/2/FoxO1/autophagy pathway To elucidate the mechanisms by which EV operate upon entering DCs, we conducted a comprehensive analysis of sequencing data. GO enrichment analysis of RNA-seq revealed significant enrichment of genes participating in “positive regulation of MAPK cascade” and “autophagy” in the EVs group compared to the LPS group (Fig. [136]6A). Meanwhile, the “MAPK signaling pathway” was consistently identified in both RNA-seq and metabolomics analyses (Fig. [137]6B). Immunofluorescence staining of DCs further supported these results, showing increased LC-3 levels following treatment with EVs (Fig. [138]6C and E), accompanied by a reduction in intracellular MitoSOX content (Fig. [139]6D and E). Conversely, the R-EVs group exhibited an opposite trend. Proteins extracted from DCs were subjected to various treatments and assessed using Western blot. We observed that the change of MAPK/P38 pathway was not significant. In the MAPK/JNK pathway, although the change was significant in the LPS group, the change was not significant between the EVs and the R-EVs group. In the MAPK/ERK1/2 pathway, the results showed increased levels of ERK1/2 and p-ERK1/2 levels in the EVs group, while their levels were found decreased in the R-EVs group. As for autophagy, no noticeable alteration was detected in FoxO1. However, a significant increase in p-FoxO1 levels can observed, and elevated contents of LC-3 and p62, while their levels were found to be decreased in the R-EVs group. As for the expression levels of ERK1/2 and FoxO1 in the R-EVs group were lower than those in the LPS group, which may be due to enhanced autophagy in the R-EVs group (Fig. [140]6F-G). Western blot results showed that there was not much difference in FoxO1 expression after treatment with the FoxO1 inhibitor, while the expression levels of P-FoxO1, LC3, and p62 all decreased (Fig. [141]6H-I). To confirm, we subsequently labeled EVs with PKH26 and administered them into the rat joint. Immunofluorescence analysis exhibited phagocytic activity towards these EVs in vivo (Fig. [142]S8A). The co-localization analysis of fluorescence images revealed the co-expression of PKH26^+ EVs and CD11c^+ DCs (Fig. [143]S8B). Immunofluorescence analysis revealed increased LC-3 expression after EVs treatment in DCs, and this change was reduced in the R-EVs group. (Fig. [144]6J). Fluorescence colocalization analysis showed the ratio of LC-3 and CD11c co-expression to the expression of CD11c alone (Fig. [145]6K). Together, these findings indicate that DCs internalized transferred functional mitochondria from EVs, which activated the ERK1/2/FoxO1/autophagy pathway (Fig. [146]6L). Fig. 6. [147]Fig. 6 [148]Open in a new tab Transferred functional mitochondria activate the ERK1/2/FoxO1/autophagy pathway. (A) GO analysis of variably expressed messenger RNA associated signaling pathways in EVs vs. LPS groups. (B) Top 20 common pathways from RNA-seq and Metabolomics analyses between EVs vs. LPS groups. (C) Representative immunofluorescence images of LC-3 in the DC, LPS, EVs and R-EVs groups. (D) Representative immunofluorescence images of mitoSOX in the DC, LPS, EVs and R-EVs groups. (E) Quantification of LC-3 and mitoSOX in the DC, LPS, EVs and R-EVs groups. (F-G) Illustrative Western Blots (F) and quantification (G) for JNK, P-JNK, P38, P-P38, ERK1/2, P-ERK1/2, FoxO1, P-FoxO1, LC-3, p62 and Actin in LPS, EVs and R-EVs groups. (H-I) Illustrative Western Blots (H) and quantification (I) for FoxO1, P-FoxO1, LC-3, p62 and Actin in LPS, EVs, AS and DMSO groups. (J) Representative immunofluorescence images of CD11c and LC-3 expression in different groups. (K) Quantification of colocalization between CD11c and LC-3. (L) Schematic diagram illustrating the autophagy activation pathway Transfer of functional mitochondria leads to metabolic reprogramming of DCs To explore specific changes in DCs. RNA-seq data revealed gene enrichment in the “response to fatty acid” and “glucose metabolic process” in the LPS vs. the Control comparison (Fig. [149]S9A). Additionally, GO analysis of the EVs group also demonstrated gene enrichment specifically in the “fatty acid metabolic process” (Fig. [150]7A). In terms of KEGG pathway analysis, we observed that LPS treatment significantly enriched pathways related to “Fatty acid biosynthesis”, “Glycolysis/Gluconeogenesis”, and “Oxidative phosphorylation” (Fig. [151]S9B). Similarly, there was significant enrichment observed in pathways associated with “Oxidative phosphorylation” and “Fatty acid degradation” in the EVs group (Fig. [152]7B). GSEA analysis showed that “Oxidative phosphorylation” and “Beta-oxidation” were down-regulated and “inflammatory response” were up-regulated in the LPS group. However, “Fatty acid biosynthes” decreased in the EVs group (Fig. [153]S9C). Findings presented above suggest that the alterations primarily occur in “glycolysis” and “fatty acid metabolic process”. We have compiled a heat map to visually represent the differentially expressed genes involved in these two processes (Fig. [154]7C and Fig. [155]S9D). In metabolomics, the proportion of “Fatty Acyls” in the classification of differential metabolites in the EVs group was 11.13% (Fig. [156]7D). Lactic Acid levels increased after LPS treatment and decreased following EVs treatment (Fig. [157]7E). Meanwhile, “Glycolysis/Gluconeogenesis” and “TCA cycle” were also present in the common pathway of metabolomics and RNA-seq (Fig. [158]7F). The enrichment pathways of core metabolites were determined using KEGG analysis, revealing the predominant involvement of the EVs group were “alpha-Linolenic acid metabolism”, “Linoleic acid metabolism” and “Arachidonic acid metabolism” (Fig. [159]7G). The group receiving LPS treatment exhibited a higher abundance of pathways related to the metabolism of fatty acids (Fig. [160]S9E). The results of OCR experiment showed that EVs could restore the cell respiration reduced by LPS, while R-EVs did not have this effect (Fig. [161]7H). At the same time, the trend of cellular basal respiration, maximum respiration, ATP production were the same (Fig. [162]7I). We analyzed the levels of lactic acid in the cell supernatant. The results demonstrated an increase in both levels following LPS treatment, and these levels were subsequently significantly reduced after EVs treatment. Furthermore, the decline was observed to be inhibited following R-EVs treatment (Fig. [163]7J). Ldha, Hk2, and Facilitative glucose transporter 1 (Glut1) were assessed through Real-time PCR. The findings indicated that Ldha, Hk2, and Glut1 were upregulated following LPS treatment. Conversely, after EVs treatment, there was minimal change in Hk2 expression while significant downregulation was observed for Glut1 and Ldha (Fig. [164]7K). We also analyzed the levels of free fatty acids in the cell supernatant. The results demonstrated an increase in both levels following LPS treatment, and these levels were subsequently significantly reduced after EVs treatment. Furthermore, the decline was observed inhibited following R-EVs treatment (Fig. [165]7L). As for fatty acid metabolism, Cpt1b did not change significantly after EVs treatment. However, the levels of Carnitine O-Octanoyltransferase (Crot) and Carnitine Acetyltransferase (Crat) were increased, and the changes were not significant after R-EVs treatment (Fig. [166]7M). These findings suggest that LPS enhances the glycolysis and fatty acid synthesis (FAS) in DCs, while suppressing fatty acid oxidation (FAO). Conversely, treatment with EVs reduces glycolysis and FAS, while promoting FAO (Fig. [167]7N). The Real-time PCR experimental results showed that when glycolysis (2-DG) and fatty acid synthesis (C75) were inhibited, the expression of inflammatory cytokines Il-6 and Tnf-α in chondrocytes decreased, as did the expression of catabolism-related molecules Mmp13 and Adamts5, while the expression of Col II increased. In contrast, when oxidative phosphorylation (FCCP) was inhibited, the levels of Il-6, Tnf-α, Mmp13, and Adamts5 increased, and Col II expression was reduced (Fig. [168]S10). In summary, the findings imply that mitochondrial transfer can modulate the glycolysis and fatty acid metabolism of DCs, thereby influencing chondrocyte recovery. Fig. 7. [169]Fig. 7 [170]Open in a new tab Transfer of functional mitochondria leads to metabolic reprogramming of DCs. (A) GO analysis of signaling pathways associated with variably expressed messenger RNA between EVs vs. LPS groups. (B) KEGG pathway analysis of correlated signaling pathways for differentially expressed mRNA between EVs vs. LPS groups. (C) Heat maps revealing distinct differences in gene expression related to glycolysis and fatty acid metabolic between EVs vs. LPS groups. (D) Pie chart illustrating the classification of differential metabolites between EVs vs. LPS groups. (E) Box plot showing lactic acid content between LPS vs. Control and EVs vs. LPS groups. (F) Top 20 common pathways identified by RNA-seq and metabolomics analysis between LPS vs. Control groups. (G) Sankey bubble diagram depicting the top pathway from the green-yellow module. (H) OCR measurement depicting mitochondrial oxidative phosphorylation in DCs treated with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (I) Quantification of OCR, ATP-Production, Basal respiration and Maximal respiration in DCs treated with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (J) Lactic acid content in DCs supernatant treated with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (K) Levels of gene expression about Glut1, Hk2 and Ldha mRNA in DCs following treatment with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (L) Free fatty acid content in DCs supernatant treated with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (M) Expression levels of Cpt1b, Crot and Crat mRNA in DCs following treatment with LPS alone, LPS combined with EVs, and LPS combined with R-EVs. (N) Schematic diagram illustrating metabolic reprogramming in activation and tolerance states Discussion Current treatments for TMJOA focus on symptom management rather than restoring condylar lesions [[171]28]. MSC therapy has emerged as a challenging area in the treatment of osteoarthritis [[172]29]. Studies have indicated that EVs contribute to the mitigation of inflammatory responses across multiple disease scenarios [[173]14, [174]15]. This conclusion is also consistent with our research findings. Our research has revealed that ASCs-EVs have notable therapeutic benefits in reducing symptoms of TMJOA in rats. This effect is mediated through the delivery of functional mitochondria to DCs, initiating a cascade of intracellular responses. We’ve demonstrated that DCs are capable of internalizing both ASCs-EVs and their mitochondria both in vivo and in vitro, a step that is vital for the therapeutic outcome. Previous studies have shown that exosomes obtained from embryonic-derived MSCs can effectively alleviate pain and promote the repair of TMJOA in rat models [[175]2]. Mitochondrial transfer has been increasingly recognized as an important mechanism by which MSCs exert their immunomodulatory and regenerative functions [[176]30–[177]33]. Suppressing the function of mitochondria in ASCs resulted in a marked decrease in the therapeutic effectiveness of EVs for treating TMJOA, underscoring the indispensable nature of operational mitochondria. Furthermore, the impaired mitochondrial function in DCs during inflammation was observed to be reversed following the transfer of mitochondria through ASCs-EVs. Studies have shown that MSCs pretreated with Rhodamine 6G irreversibly bind to the mitochondrial ATP synthase complex, resulting in the production of EVs with mitochondrial functional defects, and the use of these ASCs-EVs leads to a loss of their therapeutic efficacy [[178]34]. Previous studies have shown that the mitochondrial function of DCs in the state of inflammation is reduced [[179]35]. This is also consistent with our findings. Together, these findings underscore the significant impact of functional mitochondrial transfer via ASCs-EVs in TMJOA therapy. Interestingly, we observed that engulfed functional mitochondria from ASCs-EVs can increase autophagy in DCs by activating FoxO1 through the ERK1/2 pathway. Activation of the ERK1/2 pathway has been shown to induce FoxO1 activation through elevated Ca^2+ levels [[180]36]. The FoxO1 pathway serves as an upstream regulator of autophagy, promoting its enhancement upon activation [[181]37, [182]38]. Autophagy helps maintain cellular homeostasis and respond to stress. In TMJOA, increased autophagy in DCs may remove damaged organelles and proteins, reduce oxidative stress, and modulate the immune response. This is consistent with previous studies on exosomes enhancing autophagy in recipient cells [[183]39, [184]40]. Collectively, these results provide evidence that transferred functional mitochondria increase autophagy in DCs. In TMJOA, there is a complex interplay between the synovium and cartilage [[185]41]. Previous studies have shown that treating chondrocytes with immune cell supernatants can effectively simulate the TMJ microenvironment and promote cartilage regeneration [[186]42]. Similarly, in our experiments, the supernatant from DCs treated with EVs promoted the recovery of chondrocytes. Emerging evidence suggests that metabolism plays a pivotal role in regulating the responses of DCs. For instance, studies have indicated that EVs have the capacity to influence the behavior of immune cells [[187]13]. Recent experiments have indicated that hypoxic-treated EVs maintain a relatively stable metabolic state by transferring mitochondria into acinar cells [[188]33]. Regarding DCs metabolism, LPS treatment increased glycolysis and FAS while decreasing FAO in DCs, characteristic of activated DCs with enhanced immune responses. Prior research has indicated the significance of FAS in the transition of DCs [[189]18]. During the activation of DCs, LPS stimulation increases FAS following glycolysis [[190]43]. Additionally, we have observed an increase in the pentose phosphate pathway (PPP), providing components essential for FAS. Beyond that, FAO was decreased in LPS-cultured DCs [[191]44]. Consistently, our studies showed that activated DCs exhibit increased FAS and decreased FAO, associated with impaired immune priming due to elevated fatty acid levels in DCs [[192]45, [193]46]. In contrast, ASCs-EVs treatment reversed these metabolic changes, decreasing glycolysis and FAS and increasing FAO. Specifically, the expression of the Glut1 gene was markedly downregulated subsequent to EVs treatment. Glut1 plays a role in down-regulate glycolysis by reducing glucose transport. Regarding fatty acid metabolism, we found that although Cpt1b was down-regulated, which is crucial for transporting fatty acids exclusively in long-chain fatty acids. Whereas, short and medium-chain fatty acids are able to directly accessed or through the transporter Crat and Crot. The results of our study showed that Crat and Crot were up-regulated. Therefore, we suggest that DCs promoted cellular FAO by transporting short and medium-chain fatty acids after EVs treatment. Subsequently, we detected the decrease in free fatty acids in cell supernatant, indicating greater fatty acid decomposition than synthesis following EVs treatment. This result further supports the upregulation of FAO. This metabolic reprogramming was accompanied by reduced inflammatory cytokine production and a shift of DCs from an activation state to a tolerance state. Research has established that metabolism influences the functionality and immune reactions of DCs [[194]20, [195]47]. Our study aligns with the notion that activated DCs tend to engage in aerobic glycolysis, whereas tolerogenic DCs display a heightened oxidative metabolism. In addition to this, our findings demonstrate that alterations in DC metabolism can also facilitate the recovery of chondrocytes. Therefore, the comprehensive analysis of the above results reveals that metabolic reprogramming is a key mechanism by which ASCs-EVs regulate the function of DCs and enhance tissue repair in TMJOA. Conclusion In summary, our study provides evidence that ASCs-EVs can treat TMJOA by transferring functional mitochondria to DCs, activating the ERK1/2/FoxO1/autophagy pathway, and leading to metabolic reprogramming of DCs. However, there are constraints to our research. Firstly, the therapeutic potential of ASCs-EVs in other types of TMJOA models, sex-specific responses and their longer-term observations need further investigation. Secondly, subsequent studies focusing on optimizing the size specifications of EVs are required to enhance the accuracy and reliability of the research results. Moreover, further research is needed to clarify the mechanisms by which ASCs-EVs specifically interact with DCs in vivo, as well as to evaluate the long-term safety and efficacy of ASCs-EVs therapy. Nevertheless, this offering insights into modulating immune cell metabolism via ASCs and ASCs-EVs for managing inflammatory diseases such as TMJOA. Electronic supplementary material Below is the link to the electronic supplementary material. [196]Supplementary Material 1^ (59.5MB, avi) [197]Supplementary Material 2^ (7.1MB, docx) Acknowledgements