Therapeutic Effects And Mechanisms Of Human Umbilical Cord Mesenchymal Stem Cells-derived Exosomes In Osteoarthritis Of Rat

Ⅰ.Background of the studyOsteoarthritis(OA)is a chronic degenerative bone and joint disease caused by ageing,obesity,strain,trauma,congenital abnormalities and joint deformities,resulting in joint stiffness,deformity and dysfunction.The incidence of the disease continues to rise each year as the population ages and the proportion of obese people increases.The articular cartilage itself has no vascular structure and the chondrocyte regeneration capacity is very weak,so the disease is characterised by progressive exacerbation once it occurs.As the pathogenesis is unknown,there is no effective prevention or treatment available.Current research tends to suggest that OA is a chronic inflammatory condition in which multiple factors in the local microenvironment of the lesion are involved in chondrocyte damage,resulting in an imbalance between the extracellular matrix(ECM)of the chondrocytes and the remodelling of the regenerating tissue.The histopathological changes are degenerative necrosis of the chondrocytes,vascularisation of the necrotic area which in turn induces ossification,resulting in stiffness and deformation of the joint.Macrophages are thought to be the driving factor in the formation and progression of this lesion.Activated macrophages produce pro-inflammatory mediators as well as a variety of enzymes that degrade ECM,which exacerbate the inflammatory environment and lead to further chondrocyte destruction.However,little is known about the molecular mechanisms of macrophage involvement in cartilage injury and lesion formation.With the advancement of stem cell research in regenerative medicine,stem cells have shown good promise in the treatment of OA.Among them,mesenchymal stem cells(MSCs)have been shown to be effective in the treatment of this disease in various animal studies and clinical observations.In particular,human umbilical cord mesenchymal stem cells(hUC-MSCs)are favoured because they are derived from discarded umbilical cord tissue and have the advantages of high proliferation capacity and low immunogenicity.However,the application and scientific issues such as safety concerns,inconvenience of preservation and transportation,and differentiation for non-therapeutic purposes,which cannot be avoided in stem cell therapy,still need to be explored in depth.Meanwhile,the search for an alternative stem cell with good effect,high safety,easy collection and stable properties has become a hot topic of research in this field.The treatment of disease by stem cells is mainly achieved through paracrine action,and in particular the exosomes(Exos)in the secretions play an important role in this process.Exos are an extracellular vesicle secreted outside the cell by stem cells and contains a variety of proteins,nucleic acids and lipids involved in cell-to-cell communication.Compared to stem cells,Exos treatment allows for more precise dose control,are more stable under different pathophysiological conditions and causes little to no immune rejection by the body.These unique advantages have made stem cell Exos a hot topic in many scientific studies.In cartilage repair,it has been shown in recent years that stem cell-derived Exos can greatly enhance the repair capacity of cartilage and act as a cartilage protector.For example,Exos from subpatellar fat pad MSCs protected cartilage from injury and improved gait abnormalities in OA mice;Exos from embryonic MSCs had a therapeutic effect on OA mice by balancing the synthesis and degradation of ECM in chondrocytes;also,Exos from embryonic MSCs had immunomodulatory effects,reducing infiltration of M1 macrophages in OA mice and downregulating The expression of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)can induce infiltration of M2 macrophages and repair OA cartilage defects and synovial damage,thus inhibiting the inflammatory response in OA.MSCs-derived Exos contain a variety of small non-coding RNAs(microRNAs,miRNAs)that may facilitate intercellular communication and contribute to tissue healing.Many of these miRNAs are involved in signal transduction,cartilage metabolism and arthritis progression.miR-26a-5p in human bone marrow MSCsderived Exos reduce joint inflammation by promoting the proliferation and migration of synovial fibroblasts in the joint and inhibiting apoptosis.miR-100-5p in subpatellar adipose MSCs-derived Exos promote chondrocyte autophagy by regulating the mTOR signaling pathway.It was shown that autophagy in chondrocytes increased the expression of type Ⅱ collagen(COL2A1)and aggrecan(ACAN),while decreasing the expression of ECM degrading enzymes such as matrix metalloproteinase(MMP)13 and The expression levels of platelet-reactive protein recombinant a disintegrin and metalloproteinase with thrombospondin(ADAMTS)5 were also reduced in IL-1βtreated chondrocytes.Ⅱ.Objectives of the StudyThis paper intends to observe the therapeutic effects of hUC-MSCs-Exos on OA through in vivo experiments on rat knee OA and in vitro experiments on IL-1βinduced OA chondrocyte model;to study the effects of hUC-MSCs-Exos on the polarization of M0 and M1 macrophages;to investigate the mechanism of hUCMSCs-Exos inhibiting chondrocyte damage by applying sequencing technology combined with bioinformatics analysis,which provides new ideas for basic research on osteoarthritis and new methods for clinical treatment.Ⅲ.Methodology of the study1.hUC-MSCs culture and identificationUmbilical cord Huatong glue was isolated and tissue block culture was performed for 7-10 days to grow long spindle-like cells.Immunofluorescence and flow cytometry were applied to identify the expression of MSC markers,targeted induction to detect the multi-directional differentiation ability of the cells and to identify hUC-MSCs.2.hUC-MSCs-Exos extraction and identificationThe hUC-MSCs-Exos were obtained from the culture supernatant of hUC-MSCs by differential ultracentrifugation.hUC-MSCs-Exos were counted by ELISA,its shape and particle size were identified by transmission electron microscopy and particle size analyzer.hUC-MSCs-Exos were obtained in large quantities by flow cytometry and protein immunoblotting(western blot,WB)to identify the expression of its markers.3.hUC-MSCs-Exos in the treatment of osteoarthritis in ratsThe anterior cruciate ligament transection combined with medial meniscectomy(ACLT+pMMx)induced OA model in rats.hUC-MSCs-Exos were injected into the joint cavity,and the repair of cartilage damage on the articular surface was observed by HE,fen red-O solid green staining,and the expression of chondrocytes and macrophage markers were identified by immunohistochemical staining to evaluate the effect of hUC-MSCs-Exos in treating OA in rats.4.Effect of exosomes on the polarization of macrophage cellshUC-MSCs-Exos acted on M0 and M1 macrophages and their pro-inflammatory and anti-inflammatory factor gene expression levels were measured by quantitative real-time polymerase chain reaction(RT-qPCR).The expression of M1 cell markers and M2 cell markers after the action of hUC-MSCs-Exos on M0 and M1 macrophages was analysed by immunofluorescence to investigate the effect of hUCMSCs-Exos on macrophage polarisation.5.hUC-MSCs-Exos role M1 macrophage and chondrocyte co-culture systemA co-culture system of macrophages and chondrocytes was established,and the gene and protein expression levels of chondrocyte ECM markers and ECM catabolic enzyme markers were detected by RT-qPCR and immunofluorescence.TUNEL staining was used to analyze the apoptosis of chondrocytes and to study the effect of M1 macrophages treated with hUC-MSCs-Exos on chondrocytes.6.Effect of hUC-MSCs-Exos on the biological behaviour of chondrocytesThe effect of hUC-MSCs-Exos on chondrocyte proliferation was assessed using CCK-8 and EdU staining,the effect of hUC-MSCs-Exos on chondrocyte migration were examined using transwell cell migration assay,and the effect of hUC-MSCsExos on apoptotic chondrocytes was investigated using Annexin V-FITC/PI staining.7.hUC-MSCs-Exos acting chondrocyte model of OAChondrocytes were induced with 10ng/mL IL-1β for 24h and chondrocyte models of OA were replicated.Gene and protein expression levels of chondrocyte ECM markers and ECM catabolic enzyme markers were measured by RT-qPCR,immunofluorescence and WB.8.Bioinformatics analysisThe miRNAs of hUC-MSCs-Exos were analysed using high-throughput sequencing technology,together with the transcriptome of normal chondrocytes,chondrocytes from OA models and chondrocytes from hUC-MSCs-Exos-acting OA models.Combined with bioinformatics,the possible mechanisms of hUC-MSCsExos-acting OA model chondrocytes were analysed and preliminary validation was performed by WB.Ⅲ.Findings1.hUC-MSCs and their Exo isolation,identificationThe hUC-MSCs were successfully obtained by culturing umbilical cord Huatong gum in tissue block culture.hUC-MSCs highly express the markers of MSCs CD44,CD73,CD90,CD 105 and can be directed to differentiate towards chondrocytes,osteoblasts and adipocytes.Using differential ultracentrifugation,5.2×109 hUCMSCs-Exos were isolated per mL of hUC-MSCs culture supernatant.hUC-MSCsExos were shown to be spherical in shape with a typical bilayer membrane structure,with a diameter of approximately 30-150 nm.hUC-MSCs-Exos expressed the Exos marker proteins CD63 and CD81.The above results suggest that hUC-MSCs-Exos can be obtained in large quantities to support the subsequent studies of this experiment.2.Protective effect of hUC-MSCs-Exos on knee cartilage in a rat OA modelIn the ACLT+pMMx induced OA model,pathological observations showed narrowing of the lumen and roughness of the joint surface in the knee joints of the experimental animals.The articular cartilage showed homogeneous powder-stained degenerative necrotic changes and disorganized subchondral bone trabeculae.There was partial loss of the extracellular matrix of the articular surface cartilage.Immunohistochemical staining showed a decrease in the number of COL2A1-positive cells and an increase in MMP13-positive cells in the articular surface chondrocytes.In the OA model treated with hUC-MSCs-Exos,the pathology showed smooth articular cartilage surfaces,intact cartilage tissue,near-normal articular lumen,regular bone trabeculae arrangement and uniform extracellular matrix staining of cartilage.Compared to the OA model group,the OARSI score was lower.Immunohistochemical staining showed an increase in the number of COL2A1positive cells and a decrease in MMP 13-positive cells in the articular surface chondrocytes.The above results indicate that the animal model of human OA was successfully replicated.hUC-MSCs-Exos had a protective and anti-inflammatory effect on the knee cartilage of the rat OA model.3.hUC-MSCs-Exos regulates macrophage polarizationTreatment of M0 and M1 macrophages with hUC-MSCs-Exos resulted in increased gene expression levels of the anti-inflammatory factors interleukin-10(IL10)and arginase 1(ARG1),and decreased gene expression levels of the proinflammatory factors IL-1β and TNF-α.hUC-MSCs-Exos treatment The number of CD 163-positive cells increased and the number of CD86-positive cells decreased in M0 and M1 macrophages,thus demonstrating that hUC-MSCs-Exos had a regulatory effect on macrophage polarization.4.hUC-MSCs-Exos inhibits M1 macrophage damage to chondrocytesA co-culture system of M1 macrophages and chondrocytes was established.The addition of hUC-MSCs-Exos to M1 macrophages in the upper chamber resulted in down-regulation of the expression of pro-inflammatory factors IL-1β and TNF-αgenes and up-regulation of anti-inflammatory factors IL-10 and ARG1 genes compared to untreated M1 macrophages.Chondrocytes co-cultured with M1 macrophages added to hUC-MSCs-Exos in the lower chamber showed upregulated expression of the chondrocyte ECM marker CO2A1 gene and protein and downregulated expression of the chondrocyte ECM catabolic enzyme marker MMP13 gene and protein compared to chondrocytes co-cultured with untreated M1 macrophages.M1 macrophages were able to induce apoptosis in chondrocytes.However,when hUC-MSCs-Exos was added,the number of apoptotic cells was significantly reduced.The results demonstrated that hUC-MSCs-Exos had a protective effect against M1 macrophage-induced chondrocyte damage and apoptosis.5.hUC-MSCs-Exos promotes chondrocyte proliferation and migration and inhibits chondrocyte apoptosisCCK-8 results showed that hUC-MSCs-Exos at 0.5,1,2 and 4×107 cells/mL promoted chondrocyte proliferation in a dose-dependent manner.hUC-MSCs-Exostreated chondrocytes showed significantly higher numbers of EdU-staining positive cells than the control group.The results of the Transwell assay showed that the number of chondrocytes migrating was significantly increased by the addition of hUC-MSCs-Exos compared to the control group.Annexin V-FITC/PI staining showed that the number of apoptotic chondrocytes was significantly reduced by the addition of hUC-MSCs-Exos compared to the model group.The results indicated that hUC-MSCs-Exos had a promotive effect on chondrocyte proliferation and migration and an inhibitory effect on chondrocyte apoptosis.6.hUC-MSCs-Exos reverses IL-1β-induced chondrocytes in OA modelThe results of RT-qPCR,immunofluorescence and WB assays showed that IL1β-induced chondrocytes in the OA model had reduced gene and protein expression levels of COL2A1 and ACAN,markers of chondrocyte ECM,reduced gene and protein expression levels of SOX9,a key chondrocyte transcription factor,upregulated gene and protein expression levels of MMP13 and ADAMTS5,markers of chondrocyte ECM catabolism,and upregulated gene and protein expression levels of COL1A2,markers of chondrocyte ossification.The addition of hUC-MSCs-Exos to OA model chondrocytes resulted in increased expression levels of COL2A1,ACAN and SOX9 genes and proteins,and down-regulated expression levels of MMP13,ADAMTS5 and COL1A2 genes and proteins relative to the OA model group.The results suggest that hUC-MSCs-Exos promotes ECM synthesis and inhibits catabolism in OA model chondrocytes.7.hUC-MSCs-Exos acts through miR-100-5p targeting and regulating FGFR3Five genes that may be regulated by miRNAs of hUC-MSCs-Exos were screened by high-throughput sequencing combined with bioinformatics analysis and verified by RT-qPCR.The expression of the five genes was consistent with the sequencing results.The most significantly differentially expressed FGFR3 gene was selected for study,and predicted results showed a binding site between FGFR3 and hUC-MSCsExos highly abundantly expressing miR-100-5p.WB results showed that hUC-MSCsExos-treated OA model chondrocytes relative to the OA model group had FGFR3,pPI3K,and p-mTOR protein expression levels were down-regulated.The results suggest that hUC-MSCs-Exos reverses IL-1β damage to chondrocytes,possibly by targeting the FGFR3/PI3K/mTOR signalling axis through miR-100-5p.Ⅳ.Conclusions of the study1.hUC-MSCs culture supernatant was collected and hUC-MSCs-Exos was obtained in large quantities by differential ultracentrifugation to meet the need for hUC-MSCs-Exos treatment for OA in vitro and in vivo experiments.2.An animal model close to human OA was surgically replicated.In the rat OA model,hUC-MSCs-Exos were able to protect articular chondrocytes,induced M2 macrophage infiltration and have therapeutic effects on rat OA.3.hUC-MSCs-Exos can regulate the polarization of macrophages to M2 type.In a co-culture system,hUC-MSCs-Exos inhibited the damage and apoptotic effects of M1 macrophages on chondrocytes.4.hUC-MSCs-Exos promoted chondrocyte proliferation and migration and inhibited their apoptosis,and exosomes successfully reversed IL-1β damage to chondrocytes by a mechanism that may function through miR-100-5p-targeted regulation of the FGFR3/PI3K/mTOR signaling axis.