The Research Of Postoperative Knee Arthrofibrosis Inhibited By Exosomes Derived From Human Infrapatellar Fat Pad Mesenchymal Stem Cells

Objective:To explore the inhibitory effect and possible mechanism of exosomes derived from human infrapatellar fat pad mesenchymal stem cells（IPFSCs-Exo）on postoperative knee arthrofibrosis.Methods:（1）The human infrapatellar fat pad mesenchymal stem cells（IPFSCs）and their derived exosomes（IPFSCs-Exo）were extracted.（2）The model of knee arthrofibrosis in rats was constructed.After anesthesia,the knee joint capsule was opened,the medial femoral condyle was exposed,and the cortical bone area of about 3 x3mm was removed with a special drill for animals,and the incision was sutured after washing.At the beginning of the first week after surgery,PBS and 1010 particles/ml IPFSCs-Exo were injected into the surgical modeling areas of rats twice a week for 4 weeks.After 4 weeks,histological sections were prepared,HE staining was used to evaluate the degree of knee arthrofibrosis in the surgical modeling areas,and Masson staining and immunohistochemistry were used to evaluate the collagen content in the fibrotic tissues.（3）The cellular inflammatory environment of fibroblasts was simulated by IL-1β.The effect of IPFSCs-Exo on the proliferation of fibroblasts in inflammatory environment was detected by CCK-8 assay.（4）After IPFSCs-Exo intervened in fibroblasts in inflammatory environment,the possible molecular targets for inhibiting knee arthrofibrosis were searched by RNA-seq.（5）After IPFSCs-Exo intervened in fibroblasts in inflammatory environment,the effects of IPFSCs-Exo on the cell cycle of fibroblasts were detected by flow cytometry,the DNA synthesis of fibroblasts was detected by EdU cell proliferation assay,and the effects of IPFSCs-Exo on the mRNA and protein expression levels of cell proliferationrelated genes（Cyclin D1,PCNA）were detected by RT-PCR and western blot.（6）In order to verify whether MT2A is the molecular target of IPFSCs-Exo,MT2A was silenced through the construction and introduction of lentiviral vector.MT2A-silenced human fibroblasts were treated with IPFSCs-Exo,and the effect of IPFSCs-Exo on the proliferation of fibroblasts in inflammatory environment was detected.Results:（1）The isolated and cultured cells derived from the infrapatellar fat pad showed a long spindle-like shape with adherent growth under the optical microscope.The extracted cells were strongly positive for CD44,CD73,CD90,and CD 105 detected by flow cytometry.And the extracted cells could differentiate into adipogenic,osteogenic and chondrogenic cells.Collecting the culture supernatant of IPFSCs,and extracting exosomes-like vesicles from the supernatant by ultracentrifugation,which had a disc-shaped bilayer membrane structure under transmission electron microscope.Nanosight analysis showed that the size of vesicles ranged from 50 to 200 nm,and the concentration of exosome-1ike vesicles was about 1010 particles/ml.Western blot showed that the extracted exosome-like vesicles highly expressed exosomal surface markers CD63 and CD81.（2）The results of histological sections showed that HE staining confirmed that the degree of knee arthrofibrosis in the surgical modeling areas in IPFSCs-Exo group was significantly lower than that in PBS group.Masson staining showed that the collagen content in the surgical modeling areas in IPFSCs-Exo group was significantly lower than that of PBS group.Immunohistochemical results showed that the expression of Collagen I and Collagen III in the surgical modeling areas of IPFSCs-Exo group was significantly lower than that of PBS group.（3）RT-PCR results showed that low concentration of IL-1β（10 ng/ml）could promote the expression of inflammatory cytokines and the proliferation of fibroblasts.CCK8 assay showed that IPFSCs-Exo could inhibit the proliferation of fibroblasts in the inflammatory environment in a concentration-dependent manner,but had no significant effect on the proliferation of fibroblasts in the noninflammatory environment.（4）MT2A gene that was significantly up-regulated after IPFSCsExo intervened in fibroblasts in the inflammatory environment was screened by RNA-seq,which may be related to knee arthrofibrosis.（5）Flow cytometry analysis showed that IPFSCsExo could block fibroblasts in G1 phase in the inflammatory environment;EdU cell proliferation assay showed that IPFSCs-Exo could inhibit the DNA synthesis of fibroblasts in the inflammatory environment;RT-PCR and Western blot showed that IPFSCs-Exo could down-regulate the mRNA and protein expression levels of proliferation-related genes（Cyclin D1,PCNA）of fibroblasts in the inflammatory environment.The above results indicated that IPFSCs-Exo could inhibit the proliferation of fibroblasts in the inflammatory environment.（6）After MT2A was silenced by lentivirus transfection,EdU cell proliferation assay showed that the proportion of EdU-positive cells was partially reversed,while RT-PCR and Western blot showed that the mRNA and protein expression levels of proliferation-related genes（Cyclin Dl,PCNA）were also partially reversed.Conclusion:IPFSCs-Exo could effectively inhibit the degree of knee arthrofibrosis in the surgical modeling areas.The mechanism may be that IPFSCs-Exo inhibited the proliferation of fibroblasts in the inflammatory environment,and MT2A may be its potential intervention target.