Pretreatment Of Kartogenin In Infrapatellar Fat Pad Mesenchymal Stem Cells Promotes Their Exosomes Mediated Articular Cartilage Repair

Objectives and background: The pathological changes of osteoarthritis usually manifest as irregular defects of articular cartilage.Due to the lack of vessels and nerves in the articular cartilage,the body’s own repair ability is limited when it is injured.The emergence and development of tissue engineering of regenerative medicine has brought us hope of repairing the diseased articular cartilage.The purpose of this study was to evaluate the role of exosomes extracted from infrapatellar fat pad mesenchymal stem cells pretreated with KGN,to clarify their ability and feasibility to promote cartilage repair,and to explore which contents of the exosomes play a major role in cartilage repair and the related mechanisms.Methods and results: Part I: The effect of exosomes derived from infrapatellar fat pad mesenchymal stem cells pretreated with Kartogenin on promoting chondrogenesis in vitro Methods:(1)Isolation of infrapatellar fat pad mesenchymal stem cells from rabbit infrapatellar fat pad.(2)Using flow cytometry to detect surface markers of mesenchymal stem cells and identifing its multi-directional differentiation potential.(3)Pretreatment of mesenchymal stem cells with Kartogenin,and collection of supernatants from pre-treated and untreated mesenchymal stem cells were performed,and ultracentrifugation was used to collect exosomes.(4)Western blot was used to detect the expression of specific surface proteins in the two exosomes.The protein concentration of the extracted exosomes was detected by BCA method,and the exosome concentration was detected using Nanosight.TEM was used to detect the morphology of exosomes.(5)Isolation and identification of chondrocytes from rabbit knee articular cartilage.(6)Adding exosomes in chondrocytes culture and detecting the effects of proliferative capacity.(7)After adding exosomes in chondrocytes culture,the expression of cartilage-specific proteins and genes was detected by Western blot and qRT-PCR.Results:(1)We successfully isolated rabbit infrapatellar fat pad mesenchymal stem cells.(2)Flow cytometry and three-line differentiation experiments demonstrated that the mesenchymal stem cells conformed to the standard and had multi-directional differentiation potential.(3)Exo and KGN-exo were extracted by ultracentrifugation,and the exosome-specific surface proteins CD9 and TSG101 were positive confirmed by Western blot.The flat discshaped biconcave was observed under transmission electron microscope.Nanosight test showed that the particle size was concentrated between 40-100 nm,suggesting that the high purity of extracted exosomes.(4)The rabbit chondrocytes were successfully isolated,and the results showed that exo and KGN-exo can significantly promote the proliferation of chondrocytes.(5)After treatment of chondrocytes with exo and KGN-exo,Western blot and qRT-PCR showed that KGN-exo can significantly promote cartilage-specific protein and gene expression.Conclusion: We successfully isolated rabbit infrapatellar fat pad mesenchymal stem cells,and identified that they meet the international common standards,and extracted exosomes from the supernatant of stem cell culture.The identification also meets the experimental requirements.We demonstrated that exosomes can enhance the proliferation of chondrocytes and promote the expression of cartilage-specific proteins and genes in vitro.Part II: The effect of exosomes derived from infrapatellar fat pad mesenchymal stem cells pretreated with Kartogenin on cartilage repair in vivo Methods:(1)The usage of surgical methods in the manufacture of cartilage defect animal models.(2)Four different methods to treat experimental animals: control,infrapatellar fat pad mesenchymal stem cells,exo,KGN-exo(3)The experimental animals were sacrificed in batches at 4 and 12 weeks after surgery,and the specimens were processed for macroscopic evaluation and histological staining and evaluation.Results:(1)Knee articular cartilage defects models were successfully established.(2)The experimental animals were killed in batches at 4 and 12 weeks after surgery.Macroscopic evaluation results showed that the surface of the repaired tissue was smooth in KGN-exo treatment group.The defect site was almost covered by the regenerated cartilage tissue,and there was almost no crack in the repaired tissue which was well integrated with surrounding normal cartilage,and it was difficult to see the boundary.The scores of the KGN-exo group were significantly higher than the other three groups.(3)HE staining and Safranin O/Fast Green staining showed that the defect of KGNexo group was almost completely repaired,no obvious cracks were observed.Strong positive staining of Safranin O suggests that it contains a large amount of proteoglycan,the repair tissue is completely integrated with the surrounding normal cartilage,and it is difficult to see the boundary.The histological score of the KGN-exo group was also significantly higher than the other three groups.Conclusion: We successfully created a rabbit knee articular cartilage defect model.After treatment with 4 different methods,macroscopic evaluation and histological staining and evaluation results indicate that the exosomes extracted from the KGN treated infrapatellar fat pad mesenchymal stem cells have the strongest ability to promote the repair of cartilage defects,which is obviously better than the other three groups.Part III: Mechanism of exosomes derived from infrapatellar fat pad mesenchymal stem cells pretreated with Kartogenin on cartilage repairMethods:(1)Using proteases and nucleases to cleave proteins and nucleic acids in KGN-exo,respectively,achieving KGN-exo-RNAse and KGN-exoPROnase.(2)Divided into 4 groups for testing exosomes promoted the phenotypic changes of chondrocytes.Western blot and qRT-PCR were used to detect the Sox-9,Aggrecan,Col-II changes in protein and gene expression in chondrocytes treated with exosomes.(3)Divided into 4 groups for testing exosomes promoted the ability of chondrocyte proliferation.(4)Extracting miRNAs from two exosomes and detecting differences in miRNA expression between the two exosomes by high-throughput sequencing.(5)Detecting the expression of related miRNAs after treatment with two exosomes in chondrocytes.(6)To verify the effect of exosomes overexpressing miR-140-5p,miR-127-5p and miR-125 b on chondrogenesis.(7)To verify the effect of exosomes inhibiting miR-140-5p,miR-127-5p and miR-125 b on chondrogenesis.Results:(1)Protease and nuclease were used to obtain KGN-exo-RNAse with only protein and KGN-exo-PROnase with only nucleic acid.(2)qRT-PCR results showed that the expression of cartilage-specific genes in KGN-exoPROnase was significantly higher than that in KGN-exo-RNAse group,and the results of Western blot were consistent with qRT-PCR results.(3)In the study of exosome-promoting cell proliferation,the proliferation rate of chondrocytes in KGN-exo-PROnase group was significantly decreased,which was significantly lower than that in KGN-exo-RNAse group.(4)miRNA expression in two exosomes was detected by high-throughput sequencing,and the top three miRNAs with different expression were selected by comparison with each other: miR-140-5p,miR-127-5p,miR-125 b.(5)After adding exosomes in chondrocytes cultured,qRT-PCR results indicated that the expression levels of miR-140-5p,miR-127-5p and miR-125 b in KGN-exo group increased significantly.(6)After treatment with miRNA agonist,the contents of miR-140-5p,miR-127-5p and miR-125 b in the exosomes were significantly increased.After treatment with ago-miR-140-5p-exo,ago-miR-127-5p-exo and ago-miR-125b-exo,the expression of Sox-9,Aggrecan and Col-II in chondrocytes was significantly increased,among which the expression of agomiR-140-5p-exo group particularly high.(7)The content of miR-140-5p,miR-127-5p,and miR-125 b in KGN-exo was significantly reduced after treatment with miRNA inhibitors.At the same time,after treatment with inhibition of miR-140-5p,miR-127-5p and miR-125 b,the expression of Sox-9,Aggrecan,and Col-II in chondrocytes was significantly reduced.Conclusion: We used protease and nuclease to treat KGN-exo separately and the results suggest that mesenchymal stem cell-derived exosomes promote cartilage repair mainly rely on nucleic acid components in exosomes.For promoting chondrocyte proliferation,it mainly depends on the protein components in exosomes.High-throughput sequencing results showed differentially expressed miRNAs in KGN-exo and exo,suggesting that a significant increase of capacity in exosomes-promoting cartilage repair after KGN pretreatment may be associated with these miRNAs.Of course,the specific mechanism of exosomes in promoting the repair of cartilage defects still needs further investigation.We will focus on this issue in our future study.Summary 1.This study used KGN to pretreat infrapatellar fat pad mesenchymal stem cells and extracted exosomes,which can significantly enhance the ability of exosomes to promote cartilage repair and regeneration in vitro and in vivo.2.The ability of exosomes after KGN pretreatment to promote chondrogenic differentiation mainly depends on the action of nucleic acid components in the exosomes,while in promoting the proliferation of chondrocytes,it mainly depends on the protein components in the exosomes.3.KGN pretreatment of mesenchymal stem cells mainly changed the expression levels of miR-140-5p,miR-127-5p,miR-125 b in the secreted exosomes,resulting difference in promoting cartilage repair ability.Of course,the conclusions of this study are only preliminary and one-sided.The detailed mechanism of exosomes in promoting the repair of cartilage defects is far from clear.In future research,we will explore the targets of related miRNAs and specific signal passes.