Mechanism Of Adipose Mesenchymal Stem Cells-derived Extracellular Vesicles Binding With Chitosan Oligosaccharides In Cartilage Injury Repair

Objective: Articular cartilage is a non-self-repairing tissue,and cartilage damage is a common health disorder worldwide.The clinical manifestations are movement dysfunction,joint pain and,in severe cases,the occurrence of osteoarthritis.At present,there is no cure for cartilage injury in clinical practice.Only drug analgesia,physiotherapy,surgery,chondrocyte and tissue transplantation,and joint replacement can be used to reduce the impact of the injury and relieve symptoms in the short term.Therefore,there is an urgent need for effective therapeutic strategies for reparative therapy.Mesenchymal stem cells(MSCs)are pluripotent stem cells capable of self-renewal and can differentiate into a variety of cell lineages,such as osteoblasts and chondrocytes.Studies have shown that MSCs can promote the repair and regeneration of articular cartilage.Compared with stem cells from other sources,adipose-derived MSCs are widely used in the field of biomedicine due to their relatively easy isolation,high yield,and great potential for proliferation and differentiation.Extracellular vesicles are released by a variety of cells and serve as tools for intercellular communication.Extracellular vesicles can select functional biologically active substances such as protein molecules,genes(RNA and DNA),cytokines,and deliver them to the extracellular environment or other target cells.Mesenchymal stem cell-derived extracellular vesicles are considered to be an important part of cell-free regenerative medicine because they can carry special biologically active substances and retain the characteristics of mesenchymal stem cells.Chitooligosaccharide is a natural polysaccharide,which is biodegradable,has excellent biocompatibility and non-toxic properties,and has been widely used in biomedical fields,such as tissue engineering,and drug preparation.Studies have shown that chitosan oligosaccharide can promote osteoblast differentiation and new bone formation/maturity,and promote the proliferation of bone marrow mesenchymal stem cells and the differentiation of chondrocytes.The purpose of this study was to investigate the role of extracellular vesicles-chitosan oligosaccharide conjugates formed by adipose-derived mesenchymal stem cell-derived extracellular vesicles coupled with chitosan oligosaccharide in the repair of chondrocyte injury,and to provide new insights for the treatment of cartilage injury.The evidence also lays the foundation for the clinical application of extracellular vesicles.Methods: 1.Primary chondrocytes were isolated from rat cartilage tissue in this study,and the expression of collagen II was detected by immunohistochemistry.EVs were extracted from rat adipocytes.Then,morphological characteristics,particle size were observed by transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).In addition,western blot was used to assess the levels of CD63,TSG101,CD9 and Calnexin.Rat chondrocytes were cultured with different concentrations of EVs or COS for 48 hours,and then the cell viability was detected by CCK-8 assay.EVs were coated with COS to obtain EVs-chito-oligosaccharide conjugates(EVs-COS).The morphological characteristics and particle size of EVs-COS were observed by TEM and NTA.EVs and EVs-COS were labeled with PKH-67 and co-cultured with rat chondrocytes,respectively.Then,the distribution in chondrocytes was observed by inverted fluorescence microscope.2.IL-1β was used to establish chondrocytes injury model in vitro.Then,COS,EVs or EVs-COS were treated to IL-1β induced-chondrocytes.CCK-8,flow cytometry,transwell and wound healing assays were performed to assess cell proliferation,apoptosis and migration.Besides,the expression levels of chondrocyte specific genes(COL1A1,COL2A1),osteogenesis related genes(OCN,OPN,RUNX2),apoptosis related genes(C-MYC,p53,Bcl2)and PI3K/Akt pathway were detected by qRT-PCR and western blot assays.3.Rat full-thickness cartilage defect model was constructed,and then COS,EVs or EVs-COS were injected into the articular cavity for treatment.Histological changes of rat cartilage were observed by HE and ABH&E staining,and expression changes of COL1A1 and COL2A1 were detected by immunohistochemical staining.4.Transcriptome sequencing was used to screen the gene expression profiles of COS group,EVs group and EVs-COS group in osteoarticular cartilage injury model.GO annotation and KEGG pathway enrichment analysis were performed for the differentially expressed genes(DEGs).The DEGs were further verified by qRT-PCR and western blot assay in vitro and in vivo models.Results: 1.Immunohistochemical staining showed that the expression of collagen II was positive in the extracted rat chondrocytes.TEM,NTA and western blot analysis suggested that the EVs derived from adipocytes were cup-shaped or nearly round,about 133.9 nm in size,positive for CD63,TSG101 and CD9,and negative for calnexin.All the above results were consistent with the characteristics of EVs.320μg/m L COS or 20 μg/ m L EVs had the highest cell viability.TEM and NTA showed that the EVs-COS was also cup-shaped or nearly round,with a size of about 183.4 nm.Inverted fluorescence microscope observed EVs and EVs-COS could be taken up by rat chondrocytes.2.Results of CCK-8,flow cytometry,transwell and wound healing assays showed that EVs,COS and EVs-COS promoted chondrocytes proliferation and migration,and inhibited chondrocytes apoptosis,and the effect of EVs-COS was superior to COS.qRT-PCR and western blot assays suggested that EVs,COS and EVs-COS regulated chondrocyte specific genes(COL1A1,COL2A1),osteogenesis related genes(OCN,OPN,RUNX2),apoptosis related genes(C-MYC,p53,Bcl2)and PI3K/Akt pathway expression,and the effect of EVs-COS was superior to COS.3.HE and ABH&E staining showed that EVs,COS and EVs-COS could promote chondrocyte injury repair,and the repair effect of EVs-COS was better than that of COS.Immunohistochemical staining showed that EVs,COS and EVs-COS increased the expression of COL1A1 and COL2A1 proteins,and the effect of EVs-COS was superior to COS.4.In vivo model,sequencing results showed that a total of 2091 DEGs,503 DEGs and 412 DEGs were respectively identified between EVs-COS and model groups.EVs-COS regulated PI3K/Akt signaling pathway(COL1A1,COL3A1,ITGA4,BAD,FOXO3),Wnt signaling pathway(SFRP5,SOX9,GLI1),AMPK signaling pathway(PPP2R3A,CPT1 B,ACACB),MAPK signaling pathway(CACNA1S,CACNG1,MAPK1).The consistency rate of sequencing findings and RT-q PCR/western blot results was 80%,a relatively high reliability of the sequencing result.Sequencing results showed five differentially expressed genes Vps13 a,Itga1,Birc6,Ifi27l2 b and Gli1.Conclusion:1.The EVs-COS can be absorbed by rat chondrocytes.2.In the in vitro model,EVs-COS promoted chondrocytes proliferation and migration,inhibited apoptosis,regulated chondrocyte specific genes(COL1A1,COL2A1),osteogenesis related genes(OCN,OPN,RUNX2),apoptosis related genes(C-MYC,p53,Bcl2)and PI3K/Akt pathway expression.3.In the in vivo model,EVs-COS increased COL1A1 and COL2A1 protein expression,promoted chondrocytes injury repair.4.The negative regulation of Wnt signaling pathway,PI3K-Akt signaling pathway,AMPK signaling pathway and MAPK signaling pathway was associated with cartilages injury repair.Vps13 a,Itga1,Birc6,Ifi27l2 b and Gli1 may be potential targets for the treatment EVs-COS in cartilages injury repair.Above all,our results shed light on the underlying pathogenesis of osteoarthritis and lay the foundation for EVs-COS as a new potential drug for the treatment of cartilages injury and traumatic osteoarthritis.