The Research On The Conditioned Medium Derived From Stem Cells Of Infrapatellar Fat Pad To Inhibit The Degradation Of Cartilage Extracellular Matrix And Delay The Progression Of Knee Osteoarthritis In Rats

Osteoarthritis(OA)is a highly prevalent joint site disease worldwide,and its incidence is mainly concentrated in middle-aged and elderly people.Knee joint,hip joint,shoulder joint and hand joint are the most common sites.It is characterized by degenerative changes and gradual destruction of articular cartilage,inflammatory hyperplasia of synovial tissue,sclerotic and cystic changes of subchondral bone and periarticular osteophyte formation.The main symptoms of OA patients are joint pain,swelling,and limited mobility,and joint stiffness,deformity,and progressive loss of joint function in advanced patients.On the one hand,the disease of OA reduces the quality of daily life of patients.At the same time,the diagnosis and treatment of OA brings great psychological and economic burden to society,family and individuals.At present,there are various forms of treatment for OA.In the early and middle stage of non-surgical treatment,oral and joint injection drugs and traditional Chinese medicine physiotherapy are mainly used,while in the late stage,surgical treatment is mainly used.However,the two treatment methods are still mainly to relieve the temporary symptoms of patients,and the long-term efficacy of patients is still not optimistic.So far,the exact pathogenic mechanism of OA is still not completely clear.In general,under the stimulation of various conditions,degenerative injury occurred in cartilage tissue,chondrocytes showed inflammation,aging and apoptosis,and the synthesis of chondrocyte extracellular matrix was weakened.With the further development of the disease,the expression of related degradation enzymes in the extracellular matrix is further increased,which leads to the destruction and degradation of cartilage tissue,and eventually causes irreversible damage.In conclusion,the increased degradation of chondrocyte extracellular matrix and the increased level of chondrocyte inflammation and apoptosis are important links leading to the development of OA.In recent years,the effective treatment of mesenchymal stem cells(MSCs)for degenerative diseases has attracted more and more attention.However,some studies have shown that the therapeutic effect of MSCs is mainly attributable to their derived secretion set,which includes all the cell protective factors produced by MSCs.Therefore,the conditioned culture(CM)of MSCs gradually appeared in people’s field of vision.Mscs-cm contains all the paracrine substances of MSCs,such as cytokines,cell growth factors,extracellular microvesicles,exosome components,etc.Studies have confirmed the therapeutic effects of MSCs-CM on OA,angiogenesis,liver fibrosis and motor neurons in mice.However,we know little about the efficacy of MSCs-CM in OA and the specific treatment mechanism,and no clear conclusions have been drawn so far.Therefore,based on the above theories,this study intends to explore the efficacy and possible mechanism of MSCs-CM in vivo and in vitro,and provide a new treatment method for OA.Part Ⅰ:In Vitro study of IPFSCs-CM delaying the progression of OAObjective:To explore the changes of the effect of IPFSCs-CM on OA chondrocytes in vitro experimentMethods:(1)Human chondrocytes were extracted from the discarded cartilage of knee joint replacement.(2)Alcian blue staining and immunofluorescence staining were used to identify chondrocytes,and the staining was observed under a microscope.(3)Human primary IPFSCs were isolated,extracted and cultured from the discarded subpatellar fat pad tissue of knee replacement,and the conditioned medium(CM)was obtained.(4)IPFSCs were identified by flow cytometry and stem cell three-line differentiation test kit.(5)CCK-8 method was used to calculate the effect of IPFSCs-CM on the vitality of human OA chondrocytes.(6)using Western Blot method,quantitative reverse transcription polymerase chain reaction(reversetranscriptionquantitativepolymerasechainreaction,RT-qPCR)and immunofluorescence method to explore the intervention IPFSCs-CM stimuliEffect on expression of extracellular matrix related proteins in cartilage.(7)Western-Blot,RT-qPCR,flow cytometry and immunofluorescence staining were used to detect the effect of IPFSCs-CM on the expression of chondrocyte apoptosis-related genes.(8)Western-Blot,RT-qPCR,enzyme-linked immunosorbent assay(ELISA)and immunofluorescence staining were used to detect the effect of IPFSCs-CM on the expression of genes related to inflammation in chondrocytes.(9)After 2 weeks of IPFSCs-CM intervention,the contents of type II collagen(COL2A1)and proteoglycan(ACAN)in human cartilage were detected by immunohistochemical staining,saffron solid green staining and alcian blue staining.Results:(1)Under the microscope,it was observed that the extracted chondrocytes were generally in the shape of long fusiform.ACAN in chondrocytes could be dyed blue by Alcian dye solution,while COLⅡ was dyed green by immunofluorescence.Combined with the cell morphology and the two staining results,it could be determined that the extracted cells were chondrocytes.(2)The extracted IPFSCs showed long fusiform growth under ordinary light microscopy,and flow cytometry showed that the extracted IPFSCs highly expressed mesenchymal stem cell related markers,namely CD44,CD75,CD90 and CD105.The identification of three-line differentiation ability showed that the extracted cells had the ability of three-line differentiation,and combined with the identification of cell morphology,stem cell surface markers and three-line differentiation ability,we could determine that the extracted cells were IPFSCs.(3)The results of CCK8 activity assay showed that the proliferative activity of chondrocytes was significantly decreased after IL-1βstimulation,but the activity was enhanced after IPFSCs-CM intervention.(4)We found that the expressions of extracellular matrix related genes COL2A1 and ACAN were increased and the expressions of MMP13 and ADAMTS5 were decreased in OA chondrocytes treated with IPFSCs-CM.(5)We found that the expression levels of anti-apoptosis-related genes BCL-2 were up-regulated and the expression levels of apoptosis-related genes BAX,PARP,cleaved PARP,and caspase3 were decreased in OA chondrocytes treated with IPFSCs-CM.(6)In human OA chondrocytes,we found that the expression levels of inflammation-related genes COX2,iNOS,TNF-α and IL-6 in chondrocytes decreased after treatment with IPFSCs-CM.(6)The expression of ACAN and COL2A1 in chondrocytes increased after IPFSCs-CM treatment.Conclusion:(1)the intervention of IPFSCs-CM effectively inhibited the degradation of ECM in OA chondrocytes.(2)the intervention of IPFSCs-CM can inhibit the inflammatory process of chondrocytes.(3)the intervention of IPFSCs-CM can inhibit the apoptosis of chondrocytes.Part Ⅱ:In vivo study on delay of OA progression by IPFSCs-CMObjective:To explore the therapeutic effect of IPFSCs-CM injection on knee OA in rats.Method:In the in vivo,we by destroying the stability of the medial meniscus of knee joint of rats(destabilizationofmedialmeniscus,DMM)and fracture of anterior cruciate ligament(anteriorcruciateligamenttransection ACLT)method to establish the model of OA.The specific random groups were as follows:Sham operation group(Sham group),modeling saline intervention group(Vehicle group),modeling IPFSCs-CM intervention group(CM group).After 4 weeks of intervention,the knee tibia and femur were observed and pathologic scores were obtained.hematoxylineosin(HE),coniferine solid green staining and alcian blue staining were used to evaluate the condition of cartilage tissue of rat knee joint.Knee joint pathological grading of rat we by the international association of osteoarthritis(OsteoarthritisResearchSocietyInternational,OARSI)classification rating system;Immunohistochemical staining was used to detect the expression differences of extracellular matrix related genes Col-Ⅱ and MMP13 and apoptosis related genes PARP and caspase-3 in cartilage.The apoptotic changes of cartilage tissue were evaluated by TUNEL staining.Results:(1)There were no adverse conditions such as accidental death and wound suppuration infection in all the rats.(2)Gross observation of the tibia and femur of the knee joint showed that the articular surface of the sham group was round without wear,and the color was light red.The articular surface of Vehicle group was seriously worn,the surface was uneven,and the cartilage tissue was dark yellow;Compared with Vehicle group,IPFSCs-CM group improved the above conditions.(3)HE,saffron solid green and Alcian blue staining showed that the articular facial cartilages in the Vehicle group presented a degenerative shape,and the cartilage degeneration was alleviated after the intervention of IPFSCs-CM,and the OARSI score also supported the above conclusions.(4)Immunohistochemical results showed that after the intervention of IPFSCs-CM,the expression of COL2A1 in the extracellular matrix of chondrocytes increased,while the expression of MMP13,PARP and caspase-3 decreased.(5)TUNEL staining showed that the apoptosis rate of cartilage tissue decreased after the intervention of IPFSCs-CM.Conclusion:(1)intra-articular injection of IPFSCs-CM can effectively reduce the degradation of ECM in rat OA chondrocytes(2)IPFSCs-CM knee joint injection can effectively reduce the apoptosis rate of OA chondrocytes in rats.