The Effect And Mechanism Of IPFP-MSC Derived Exosomes On Protection Of Articular Cartilage

Background:Osteoarthritis?OA?is one of the most common degenerative joint diseases presented in aged population,which is mainly characterized by cartilage degradation,synovial inflammation and subchondral bone remodeling.The impaired homeostasis of chondrocytes is a prominent event that contributes to OA progression.The present treatments for OA are limited and insufficient to prevent the initiation and progression of the disease.Therefore,it is needed to explore new strategies for the prevention and treatment of OA.Mesenchymal stem cells?MSCs?have hold promising potential to regenerate cartilage in animal and pre-clinical studies.In recent years,more and more studies have revealed that the efficacy of MSC-based therapies on the cartilage regeneration has been attributed to the paracrine activity of exosomes,which deliver specific cargoes to the recipient cells.Exosome,a membrane-bound vesicle secreted by many cells,could be a critical messenger for cell-cell communication.With the size ranging from 30-150 nm in diameter,exosomes are thought to transfer bioactive lipids,nucleic acids and proteins among cells.MSCs-derived exosomes are more stable than stem cells under various physiological conditions and are to a certain degree immune privileged,making them suited for therapeutic interventions.MCSs-derived exosomes have been reported to be effective on cartilage protection.For instance,exosomes derived from human embryonic MSCs promoted cartilage regeneration.The bone marrow-MSCs-derived exosomes protected cartilage from degeneration in vivo and in vitro.Exosomes derived from miR-140-5p-overexpressing human synovial mesenchymal stem cells could promote cartilage regeneration and delay knee OA progression in a rat model.Taken together,MSCs-derived exosomes would be a promising therapeutic strategy for OA patients,which could be further optimized for its clinical application.Recently,adipose-derived MSCs present promising therapeutic prospect in regeneration medicine including the treatment of OA.Moreover,our group previously found that infrapatellar fat pad?IPFP?-derived MSC(MSC^IPFP)combined with chitosan/hyaluronic acid nanoparticles promoted chondrogenic differentiation.However,it is still unknown whether IPFP-derived MSCs could protect the cartilage in an exosome-dependent manner.As it is relatively feasible to obtain human IPFP from OA patients by arthroscopic operation in clinic,we anticipated that IPFP-MSCs derived exosomes would be a new potential strategy for OA treatment.Autophagy is a highly conserved degradation process in all eukaryotic cells.Previous studies have demonstrated that autophagy closely participates in cartilage biology and apparently delays the pathological progress of OA.Autophagy increases COL2A1 and aggrecan expression,while it decreases cartilage-degrading enzymes MMP13 and ADAMTS5 level in IL-1?-treated chondrocytes.Deletion of Atg5?a key autophagy-related gene?in chondrocytes promotes age-related osteoarthritis in mice.In contrast,cartilage-specific deletion of mTOR could protect mice from osteoarthritis via enhancing autophagy activity.Moreover,pharmacological inhibition of mTOR by intra-articular injection of rapamycin can activate autophagy and effectively relieve the pathological phenotypes of OA.In addition,REDD1 deficiency exacerbates the severity of injury-induced OA through regulation of autophagy and some strategies alleviate cartilage lesions mainly through activating autophagy.In brief,autophagy plays a key role in OA progression and targeting autophagy could be a promising strategy for OA treatment.Purpose:To investigate the role and underlying mechanism of infrapatellar fat pad?IPFP?MSCs-derived exosomes(MSC^IPFP-Exos)on OA in vitro and in vivo.Methods:1.Isolation of MSCs^IPFPThe infrapatellar fat pad was harvested and washed in phosphate-buffered saline?PBS?,and then finely diced into small pieces by using a surgical scissor.The diced tissues were digested in 0.1%type I collagenase for 10 h,and the cell suspension was filtered throu gh a40-?m cell strainer.The released cells were centrifuged at 400 g for 5 min and resuspended in Red Cell Lysis Buffer at room temperature for 10 min.The cells were then centrifuged again,and resuspended in Dulbecco's modified Eagle's medium?DMEM?/F12?Invitrogen,America?supplemented with 10%fetal bovine serum?FBS;Gibco,America?and 1%P/S.2.Culture of primary chondrocytesCartilage tissues were isolated by digesting the matrix overnight in high-glucose DMEM supplemented with 0.2%type II collagenase and 1%P/S.The cell suspension was filtered by a 40-?m cell strainer;and the collected cells were centrifuged at 400 g for 5 min,and then resuspended in high glucose DMEM supplemented with 10%FBS and 1%P/S.The medium was replaced every other day.Cells were used at passage 1.Surface antigens of MSCs^IPFP were characterized by using flow cytometry and the Human MSC Analysis Kit?BD Biosciences?.3.Isolation of exosomesAfter reaching 80%confluency,MSCs^IPFP were washed with PBS twice and the medium was replaced with DMEM/F12?Invitrogen,America?supplemented with 10%exosome-depleted FBS?SBI?and 1%P/S.After two days,the conditioned medium of MSCs^IPFP was collected for isolation of exosomes.Two methods were used in this study:ExoQuick??EQ?reagent kit?SBI?and ultrafiltration.Exosomes collected from different methods were stored in aliquots at–80°C for further use.4.The identification of exosomesThe concentration and size distribution of MSC^IPFP-Exos were measured using a NanoSight LM10 instrument?Malvern,UK?.Transmission electron microscopy?TEM?was used to identify exosome morphology.Exosomal surface marker proteins including CD81,CD9 and CD63 were analyzed by western blotting.DiO-labelled exosomes were characterized on single exosome level by imaging flow cytometer?ImageStream^X Mark II,Merck?.5.The establishment of DMM-induced OA model in micethe DMM surgery was performed by surgical sectioning of the medial meniscotibial ligament and the sham operation was performed by incision of the cutaneous and muscular planes at baseline6.Effects of MSC^IPFP-Exos on cartilage protection and pain behaviors in DMM-induced OA miceAll mice were treated with MSC^IPFP-Exos or PBS at indicated time.After 8 weeks,the knee joints were harvest for histologic analysis and immunohistochemical analysis.Mice were treated with MSC^IPFP-Exos or PBS at indicated time.After 10 weeks,then gait parameters of freely moving mice were measured by using the video-based Catwalk gait analysis system.7.Effects of MSC^IPFP-Exos on IL-1?-induced chondrocytesChondrocytes were incubated with DiO-labelled MSC^IPFP-Exos,and then the green fluorescence signal of DiO in chondrocytes were observed under fluorescence microscope.Cell viability was determined by using CCK-8 assay.Annexin V-FITC/PI staining was taken to detect the effect of MSC^IPFP-Exos on apoptosis in IL-1?-treated chondrocytes.Cell migration was measured by wound scratch assay.Protein levels of anabolism-related gene and catabolism-related gene were detected by western blot assay and immunofluorescence assay.8.Effects of MSC^IPFP-Exos on autophagy of chondrocytesProtein levels of autophagy-related markers were detected by western blot assay.Transmission electron microscope was used to measure the number of autophagosomes in chondrocytes.The mRNA levels of 84 autophagy-related genes were analyzed by high throughput-qPCR?HT-qPCR?assay.Protein levels of mTOR and phosphorylation of P70S6Kinase?P-p70S6K?in chondrocytes were measured by western blot assay.9.The miRNA expression profiles of MSC^IPFP-ExosThe miRNA expression profiles of exosomes derived from MSC^IPFP were detected by using high-throughput sequencing?miRNA-seq?.SW1353 cells?a human chondrocyte-like cell line?were co-transfected with the indicated RNA oligonucleotides and the reporter plasmids.After incubation for 24 hours,SW1353 cells were treated with the MSC^IPFP-Exos.The luciferase activities were measured with the Dual-Glo Luciferase System?Promega?according to the manufacturer's protocol.10.Intra-articular injection of antagomir-100-5pMSC^IPFP-Exos with or without antagomir-100-5p were respectively injected into the knee joint of mice with surgical induced OA as indicated.After 8 weeks of DMM surgery,knee joints of mice were harvested for histological analysis and immunohistochemical analysis.Results:1.MSC^IPFP-Exos significantly prevent the cartilage destructionArticular cartilage was obviously damaged after DMM surgery,while the exosome-treated group(PBS-Exo^IPFP)showed more complete integration of cartilage with a smooth surface compared to the PBS injection group?PBS?in DMM-induced OA mice.DMM-induced decrease of collagen II in chondrocyte could be largely reversed by PBS-Exo^IPFP,while the increased expression of ADAMTS5 and MMP13 in DMM model were dramatically down-regulated after the treatment of PBS-Exo^IPFP.2.MSC^IPFP-Exos partially improve the gait abnormality in DMM mice modelQuantitative data demonstrated the print area,the mean intensity and swing speed of right hind foot?right:surgical side?were not statistically different between PBS-Exo^IPFPPFP group and PBS group 10 weeks after DMM surgery.However,we found that the duty cycle ratio of RH to LH?RH,right hind foot;LH,left hind foot?was obviously increased and closer to the value“1”in mice with PBS-Exo^IPFPPFP treatment compared to mice with PBS treatment,indicating an improved percentage of stance phase during the step cycle.3.MSC^IPFP-Exos inhibit cell apoptosis and promote anabolism in IL-1?-induced chondrocytesMSC^IPFP-Exos have no significant effect on the cell viability in chondrocytes without IL-1?treatment,but it could partially reverse IL-1?-decreased cell viability.The percentage of apoptotic cells was significantly increased in IL-1?-treated cells,which could be partially reversed by MSC^IPFP-Exos.IL-1?treatment significantly decreased Collagen II expression,while it up-regulated the protein levels of MMP13 and ADAMTS5.Interestingly,the supplement of MSC^IPFP-Exos in chondrocytes dramatically reversed the effect of IL-1?on Collagen II,MMP13 and ADAMTS5.4.MSC^IPFP-Exos enhance the level of autophagy via inhibition of mTOR signaling pathwayMSC^IPFP-Exos significantly increased the level of LC3-II and promoted the degradation of SQSTM1/P62 in chondrocytes,while MSC^IPFP-Exos-mediated increase of LC3-II were abolished by knockdown of the crucial autophagy-related gene ATG7suggesting an enhanced effect on autophagy by MSC^IPFP-Exos in this model.Moreover,autophagy inhibitor Baf A1 abolished MSC^IPFP-Exos-mediated decrease of MMP13 in IL-1?-treated cells.Among these differentially expressed genes in high HT-qPCR assay,the mRNA of mTOR was dramatically decreased in IL-1?+MSC^IPFP-Exos-treated chondrocytes.MSC^IPFP-Exos could dose-dependently decrease the protein level of mTOR.5.miR-100-5p enriched in MSC^IPFP-Exos decrease the expression of mTORFive most abundant miRNAs?miR-100-5p,let-7i-5p,miR-221-3p,hsa-miR-21-5p and miR-148a-3p?accounted for 43.9%of the total miRNA reads.Luciferase reporter assay showed that the MSC^IPFP-Exos caused a significant reduction of luciferase activity in the cells transfected with pmir-mTOR,but not with pmir-mTOR-mu.6.Intra-articular injection of antagomir-100-5p demolishes the remedial effect of MSC^IPFP-Exos on damaged cartilage in DMM modelSafranin O&fast green staining showed that intra-articular injection of antagomir-100-5p remarkably reduced the effect of MSC^IPFP-Exos-mediated cartilage protection in DMM-induced OA mice.Moreover,our results demonstrated that MSC^IPFP-Exos mediated decrease of MMP13 and increase of collagen II were reversed by intra-articular injection of antagomir-100-5p.In addition,MSC^IPFP-Exos decreased the expression of mTOR and p62 and increased the level of LC3-II,while the antagomir-100-5p weakened this effect of MSC^IPFP-Exos on the indicated protein levels in vivo.Conclusions:In this study,we showed that MSC^IPFP-derived exosomes could protect the cartilage from damage and ameliorate gait abnormality in OA mice.To the mechanisms,MSC^IPFP-Exos promoted cell proliferation of chondrocytes,enhanced matrix synthesis and reduced expression of catabolic factors in vitro.Moreover,MSC^IPFP-Exos could significantly enhance the level of autophagy in chondrocytes via inhibition of mTOR signaling pathway.The data from exosomal RNA-seq showed that the level of miR-100-5p that could bind to the 3'-untranslated region?3'UTR?of mTOR was the highest among microRNAs in MSC^IPFP-Exos.In addition,MSC^IPFP-Exos decreased the luciferase activity of mTOR 3'UTR,while the miR-100-5p inhibitor reversed the MSC^IPFP-Exos-decreased mTOR signaling pathway.Furthermore,intra-articular injection of antagomir-miR-100-5p could partially attenuate MSC^IPFP-exos-mediated protective effect on articular cartilage in vivo.Collectively,our findings may provide a new potential therapeutic strategy for OA in the future.