Effect And Mechanism Research Of Exosomes From Adipose-derived Stem Cells In The Treatment Of Knee Osteoarthritis

Background Osteoarthritis（OA）is the most common bone and joint disease in the elderly in the world.It is the main cause of knee pain and disability,but there is currently no treatment for preventing or curing knee osteoarthritis.Due to mesenchymal stem cells（MSCs）have the potential for immunoregulation and differentiation,emerging studies have applied MSCs to regeneration of defective tissues.Adipose tissue-derived stem cells（ADSCs）are one of the types of MSCs.It has similar surface markers and differentiation potential to MSCs from other tissues.But compared with MSCs from other tissues,ADSCs have the advantages of large quantity and easy access.It have been successfully applied to the treatment of OA animal models.Exosomes are kind of vesicles secreted by cells.They contain lots of biologically active substances,include proteins,DNA,RNA and lipids.Exosomes can be absorbed by recipient cells through paracrine,adjacent secretion and endocytosis,and influence the biological processes of recipient cells.Studies have found that MSCs-derived exosomes can repair damaged cartilage.The Wnt signaling pathway is very important in maintaining the integrity of joints.Excessive and inadequate β-catenin levels can disrupt the balance of articular chondrocytes by enhancing pathological maturation and apoptosis respectively.There is an upregulation of β-catenin levels in OA cartilage.Autophagy is an evolutionary and conservative process.The maintenance of cells depends on the role of continuous autophagy.Studies showed that autophagy can protect cartilage in OA,and in other diseases and cells,it has been found that the Wnt/β-catenin signaling pathway can negatively regulates autophagy response.At present,there is limited research on the efficacy and related mechanisms of ADSCs-derived exosomes in the treatment of OA.Whether ADSCs-derived exosomes can regulate β-catenin and autophagy in chondrocytes and thus affect the performance of chondrocytes has not been studied.Therefore,this study will study the following three aspects:（1）Whether the cell performance of ADSCs obtained by liposuction and direct removal of adipose tissue is different;（2）In vivo,the therapeutic effect of ADSCs and ADSCs-derived exosomes on synovial tissue and cartilage tissue of OA model rats will be observed and compare the difference in efficacy between them;（3）Effects of ADSCs derived exosomes on synovial fibroblasts and chondrocytes derived from OA patients and their effects on β-catenin and autophagy in chondrocytes.Part I Extraction and identification of adipose stem cells and their exosomesObjective ADSCs were extracted from adipose tissue obtained from liposuction and hip joint replacement,and exosomes were extracted from ADSCs culture medium.The surface markers,differentiation ability of ADSCs and morphology,expression marker proteins of ADSCs derived exosomes were identified.Methods ADSCs were extracted from the adipose tissue obtained by liposuction and hip replacement surgery using combined digestion of trypsin and collagenase.The surface markers of the cells were analyzed by flow cytometry.ADSCs were induced into osteogenesis,adipogenesis,and chondrogenesis,and the potentials of osteogenesis,adipogenesis,and chondrogenesis were identified by staining with Alizarin Red,Oil Red O and Alisin Blue.The OCN,PPARγ and COL2A1 mRNA expression were detected after induction by fluorescence quantitative PCR.CCK8 was used to detect the proliferation ability of ADSCs.Compared the potential of three direction differentiation of ADSCs and proliferation between two derived ADSCs.ADSCs-derived exosomes were obtained by ultracentrifugation and identified by transmission electron microscopy and Western blot.Results Cells obtained from liposuction and hip replacement surgery adipose tissue were fibroblasts with consistent morphology,and the surface markers CD31,CD34 and CD45 were negatively expressed,while CD73,CD90 and CD105 were positively expressed,which were consistent with the expression of ADSCs markers.ADSCs derived from liposuction and hip replacement surgery have similar proliferative capacity and tri-lineage differentiation potential.The exosomes with a diameter between 50 nm and 100 nm can be obtained from ADSCs medium by ultracentrifugation,and the specific marker proteins CD9 and CD63 are positive.Conclusion ADSCs obtained from liposuction and hip replacement surgery adipose tissue have similar surface markers,tertiary differentiation potential,and proliferative capacity,and exosomes can be obtained from both medium by ultracentrifugation.Part II The effect of ADSCs and its exosomes on OA model ratsObjective To observe the effects of ADSCs and their exosomes on ethology,synovial tissue and cartilage tissue of OA model rats.Methods The OA model rat was estalished by the Hulth method.Twenty four SD rats were selected after successfully establishing apoplexy model for 4 weeks and divided into 3 groups according to the random number table method,8 rats in each group.Group A was a control group,which was injected intraarticularly with 60 μL normal saline;Group B was an ADSCs group,1×10⁶ ADSCs were resuspended in 60 μL normal saline and injected into the knee joint of OA rats;Group C was the exosomes group,which was treated with ADSCs-derived exosomes by intra-articular injection.All rats were treated for 8 weeks,twice a week.The behavioral indicators,synovial and articular surface sections and the expression of related genes and proteins were observed after intervention in rats.Results No complications occurred in all rats during treatment.After successful modeling,ADSCs and ADSCs-derived exosomes were used for treat OA rats 8 weeks.The results showed that the pain stimulation response,gait,activity and swelling of the rats in group B and C were milder than those in group A,the difference was statistically significant（P<0.05）;there was no significant difference between group B and group C（P>0.05）.Synovial HE staining showed a large number of lymphocyte infiltration in synovial tissue of group A,synovial cell proliferation and stratification.The synovial cells and interstitial lymphocytes in the synovial tissues of group B and group C were reduced,the inflammatory response was weakened,and the state of the synovial cells became normal.Saffron O-fixing green staining showed that the cartilage structure in Group A was disordered,chondrocytes contracted,Saffron O-fixing green staining was weakened and the tide line was damaged.While in group B and group C,the cartilage structure was relatively complete,and the chondrocytes were normal.Saffron O-fast green dyeing is dark and the tide line is relatively complete.The average modified Mankin’s score in group A is 8.6 points,group B and group C were 3.4 points and 3.6 points respectively.The modified Mankin’s score of group A was significantly higher than group B and group C,the difference was statistically significant（P<0.05）,while there was no significant difference between group B and group C（P>0.05）.These indicated that cartilage destruction in group A was more severe than that in group B and group C.The expression of TNF-α mRNA and protein in synovial tissue of group A was significantly higher than those of group B and group C,and the expression of IL-10 mRNA and protein was lower than those of group B and group C.The detection of cartilage tissue marker gene and proteins showed that the expression of type II collagen mRNA and proteins in group B and group C was higher than those in group A.Conclusion The efficacy of ADSCs-derived exosomes in treating OA model rats is similar to that of ADSCs.Both can reduce the inflammatory response of synovial tissue and have a repairing effect on cartilage tissue.Part III Effect and Mechanism of ADSCs-derived Exosomes on Synovial Fibroblasts and ChondrocytesObjective To explore the effects of ADSCs-derived exosomes on the secretion of inflammatory factors and anti-apoptosis and migration of chondrocytes from synovial fibroblasts derived from OA patients.Methods Isolation and culture of synovial fibroblasts and chondrocytes from patients with OA.Synovial fibroblasts was intervented with ADSCs-derived exosomes.The expression of inflammatory factors after intervention was detected.ADSCs-derived exosomes and H₂O₂ intervention chondrocytes were co-cultured to observe their effects on chondrocyte apoptosis.Chondrocytes cultured with activated synovial fibroblast conditioned medium and ADSCs-derived exosomes.The migration capacity,β-catenin and autophagy indicators were observed.Results ADSCs-derived exosomes co-cultured with activated fibroblast synovial cells derived from OA patients.The results showed that activated fibroblast synovial cells can significantly reduce the level of pro-inflammatory factors IL6,TNF-α and NF-κB after exosome treatment,while increase the level of the anti-inflammatory factors IL-10.After ADSCs-derived exosomes can reduce inflammation of activated synovial fibroblasts were observed,this study continued to study the effect of ADSCs-derived exosomes on the apoptosis of articular chondrocytes under oxidative stress.In this study,articular chondrocytes were cultured with H₂O₂ to simulate the oxidative stress response induced by M1 macrophages in joint synovium.After H₂O₂ intervention,the apoptosis of chondrocytes was significantly increased.After added ADSCs-derived exosomes,the result showed that with the increase of exosomes concentration,the number of chondrocyte apoptosis decreased significantly.According to the results of ADSCs-derived exosomes on chondrocytes apoptosis,the concentration of 10 × 10¹⁰ particles/m L exosomes had the most obvious effect on chondrocytes.We use this concentration for subsequent experiments.In order to observe the effect of activated synovial fibroblasts conditioned medium on chondrocyte migration and the intervention of ADSCs-derived exosomes,we were divided into control group,conditioned medium group,and exosomes+conditioned medium group.This study showed that chondrocytes reduced MAP1LC3 B mRNA expression in conditioned medium,while CTNNB1,P62 mRNA expression increased,protein detection showed that LC3II/I ratio decreased,β-catenin and P62 protein expression increased.After exosomes intervention,MAP1LC3 B mRNA expression increased,while CTNNB1,P62 mRNA expression decreased;detection of the corresponding protein showed that LC3II/I ratio increased,and β-catenin and P62 protein expression decreased.Previous studies have shown that β-catenin can regulate the degradation of autophagy.The results in this study showed that exosomes increased autophagy and decrease the expression of β-catenin,which suggests that exosomes may increase autophagy by reducing β-catenin,so that promoted chondrocytes migration.In order to confirm the effect of autophagy on chondrocyte migration,we used 3-MA（20μM）,an autophagy-inhibiting small molecule drug intervention,and the result showed that the chondrocyte migration ability was significantly reduced after 3-MA intervention,and gene testing showed that P62 mRNA after 3-MA intervention was increased,the expression of MAP1LC3 B mRNA was decreased,and the expression of β-catenin mRNA was not significantly different from that of exosome intervention.Compared with the control group,the protein detection also showed that the LC3II/I ratio was reduced and the expression of P62 protein was increased.There was no significant change in β-catenin.Conclusion ADSCs-derived exosomes can reduce the release of proinflammatory factors and increase the expression of anti-inflammatory factors in active synovial fibroblast.It reduces the apoptosis of chondrocytes under oxidative stress.ADSCs-derived exosomes increases the autophagy of chondrocytes by β-catenin/autophagy pathway and promotes migration ability of chondrocyte,so as to plays a role in cartilage repair.