Treatment Of Experimental Rat Knee Articular Cartilage Defects With EVs Secreted By BMSCs Loaded With MiRNA-27b

ObjectiveDefining the extracellular vesicles(EVs)secreted by Bone-marrow mesenchymal stem cells(BMSCs)to the chondrocytes in the early inflammatory environment caused by cartilage defects Protection and regeneration.And explore the main mechanism of action in the course of treatment.MethodsIn vitro: The chondrocyte inflammatory environment model was induced by 10 ng/ml of interleukin-1?(IL-1?).The extracellular vesicles were collected by ultracentrifugation and identified.The chondrocytes were divided into 4 groups in vitro(no treatment in the blank group,10 ng/ml IL-1? in the inflammation control group,and EVs-miR-27 b group in the inflammation control group).Based on the addition of 80 ?g/ml extracellular vesicles carrying miR-27b(EVs-miR-27b)and EVs-nor group,80 ?g/ml ordinary extracellular vesicles(EVs-nor)were added to the inflammatory control group.MTT colorimetric assay was used to detect the proliferation of chondrocytes in various groups.The apoptosis of chondrocytes was detected by flow cytometry.The mitochondrial membrane potential of each group was detected by JC-1 mitochondrial membrane potential.In addition,we also detected the expression levels of MMP-13,Casepase-9,cleave Casepase-9,Caspase-3 and cleave Caspase-3 in chondrocytes by Western blot.In vivo: An experimental SD rat knee articular cartilage defect model was established by surgically drilling the femoral condyle of the femoral condyle of the knee.After successful modeling,the rats were randomly divided into 3 groups and injected with 20 ?l of EVs-miR-.27 b,EVs-nor,saline,and sham operation group,6 weeks later,to observe the local regeneration of experimental cartilage defects;ELISA method to detect the content of IL-1? and TNF-? in joint fluid;immunostaining the expression of type II collagen in the articular cartilage of each group was measured.ResultsTransmission electron microscopy showed many tea-like vesicles with a clear membrane structure of 40-120 nm in the field of view.Specific surface markers CD63 and CD81 were detected by Western blot.The results of in vitro experiments showed that the proliferation of chondrocytes in EVs-miR-27 b group was stronger than that in EVs-nor group(P<0.05),which was significantly higher than that in the blank group(P<0.05),and the apoptosis of chondrocytes was obvious in the inflammatory environment.Attenuation(P<0.05),Western blot results showed that the activation levels of Casepase-9 and Casepase-3 in chondrocytes of EVs-miR-27 b group were decreased(P<0.05),and the relative content of MMP-13 was decreased.In vivo: In the experimental rat knee articular cartilage defect model,the EVs-miR-27 b group significantly repaired the cartilage defect relative to the EVs-nor group and the sham operation group.The levels of IL-1? and TNF-? in the joint fluid of EVs-miR-27 b group were slightly lower than those in the blank group,which was significantly lower than that in the EVs-nor group(P<0.05).ConclusionEVs-miR-27 b can inhibit the apoptosis of chondrocytes in the inflammatory environment of the joint cavity caused by cartilage defects,promote the proliferation of chondrocytes,and reduce the activation of Casepase-9 and Casepase-3 in chondrocytes,and reduce the expression of MMP-13.In the experimental rat cartilage injury model,intra-articular injection of EVs-miR-27 b has obvious anti-inflammatory effects and slows down the degeneration of articular cartilage.