| Osteoarthritis(OA)is a common joint disease,characterized by degenerative changes in articular cartilage,reactive proliferation of bone and cartilage around the joint.With age,the morbidity of OA is significantly increased,and the prevalence of women is prominently higher than that of men.In the world,with the aging population,OA will become the fourth most devastating disease by 2020,even related to all-cause mortality.Due to complex pathogenesis and genetic tendency,it is generally believed that the interaction of mechanics and biological factors result in the imbalance of synthesis and degradation for chondrocytes,extracellular matrix and subchondral bone.The clinical therapies of OA include non-pharmacological treatment,pharmacological treatment and surgical treatment.Non-steroidal anti-inflammatory drugs(NSAIDs)and corticosteroid injections are largely used since many years but the current treatment strategies have no impact on the progressive degeneration of joint tissues.Therefor novel therapeutic approaches for OA include cell-based therapies that have become thriving areas of research and development.Mesenchymal stem cells(MSCs)are characterized by potential of self-renewal and differentiation.The important property of MSCs are their capacity to repair cartilage tissue,to inhibit the secretion of inflammatory cytokines by chondrocytes and to differentiate into chondrocytes in vivo.Recent data confirming the role of MSCs as a potential therapeutic strategy in OA.The studies demonstrate that the cell content and proliferation ability of human umbilical cord mesenchymal stem cells(hUC-MSCs)are superior to bone marrow MSCs.There are many advantages of hUC-MSCs,such as low immunoregulation,the simple of culture method,no ethics controversy and so on.It is not yet clear that hUC-MSCs play therapeutic roles and mechanisms on experimental model of osteoarthritis.Objective:To study the therapeutic effect of hUC-MSCs on osteoarthritis model by Hulth method.Co-cultured chondrocytes and hUC-MSCs to explore the effect of hUC-MSCs on the chondrocytes function and some of its mechanisms,so as to provide an experimental basis for the clinical application of hUC-MSCs in the treatment of osteoarthritis.Methods:1.Osteoarthritis models of the single knee were established by Hulth method.OA rabbits were randomly divided into six groups:model group,hUC-MSCs(4×10~5,2×10~6,1×10~7cells/ml)groups,rUC-MSC(2×10~6 cells/ml)sodium hyaluronate(HA,10mg/ml)group.The sham rabbits were used as control group(7-8 in each group).Except for rabbits in HA group(5 times,once a week),other groups were administrated through a single intra-articular injection after four weeks.In addition,the same volume of saline was delivered in the sham operation group and model group animals.Six weeks later,compare the difference value of perimeter between the two joints of rabbits and evaluate joints X-ray of rabbits.After all rabbits were sacrificed,gross observations of articular cartilage,HE staining were evaluated.2.Osteoarthritis models of the single knee were established by Hulth method.OA rats were randomly divided into six groups:model group,hUC-MSCs(0.5×10~6,1×10~6,2×10~6cells/ml)groups,hUC-MSCs multi-injection group and HA(10mg/ml)group.The sham rabbits were used as control group(6-8 in each group).Except for rats in HA group(5 times,once a week)and in hUC-MSCs multi-injection group(3 times),other groups were administrated through a single intra-articular injection after four weeks.In addition,the same volume of saline was delivered in the sham operation group and model group animals.Six weeks later,evaluate joints X-ray of rats.After all rats were sacrificed,gross observations of articular cartilage,HE staining were evaluated.The expression of collageⅡ,MMP-13 and TIMP-1 in the knee joints of rats were detected by immunohistochemistry;The levels of IL-1β,IL-6,MMP-13 and TIMP-1 in rats synovial fluid were detected by ELISA.3.In vitro,the primary chondrocytes of OA patients were cultured in vitro by two-step enzymatic digestion.hUC-MSCs and chondrocytes were co-cultured for experiments.The mRNA levels of aggrecan,col-2 and sox-9 in chondrocytes were detected by RT-qPCR;Chondrocyte apoptosis were detected by apoptosis kit;Chondrocyte proliferation was detected by CCK-8 assay;GAGs in the chondrocytes were detected by DMMB;The expression and localization of Wnt andβ-catenin in chondrocytes were detected by immunofluorescence;The expression of Wnt,β-catenin and phosphorylatedβ-catenin in chondrocytes were detected by western blot.Results1.Therapeutic effect of hUC-MSCs on knee OA model(1)Therapeutic effect of hUC-MSCs on rabbits knee OA modelThe rabbit knee established by Hulth method was swollen obviously.Each group of hUC-MSCs could significantly reduce the Pelletier score of knee joints;hUC-MSCs(2×10~6,1×10~7cells/ml)could significantly reduce the swelling index of joint and improve score of joint pathology;hUC-MSCs(1×10~7cells/ml)could significantly reduce the score of knee joint X-ray.(2)Therapeutic effect of hUC-MSCs on rats knee OA modelThe rat model of knee osteoarthritis showed that hUC-MSCs(1×10~6,2×10~6cells/ml)and hUC-MSCs multi-injection could significantly reduce X-ray score,reduce knee Pelletier score,improve cartilage injury,improve the knee joint pathology and reduce the pathological score.2.Effect of hUC-MSCs on the level of inflammatory cytokines in the rat model of osteoarthritisThe result of immunohistochemistry showed that hUC-MSCs(1×10~6,2×10~6cells/ml)and hUC-MSCs multi-injection could significantly increased the expression of CollageⅡ,decreased the expression of MMP-13 and restored MMP-13/TIMP-1 ratio in rats knee joint.The results of ELISA showed that hUC-MSCs(1×10~6,2×10~6cells/ml)and hUC-MSCs multi-injection could decrease the levels of IL-1βand IL-6 in restore MMP-13/TIMP-1 ratio in rats synovial fluid.3.Effect of hUC-MSCs on chondrocyte function in OA patients The stimulation of chondrocytes with L-1β(10ng/ml)could reduce the mRNA levels of aggrecan,col-2 and sox-9,promote the apoptosis of chondrocytes and inhibite the proliferation of chondrocytes;Co-cultured hUC-MSCs with chondrocytes for 48 hours,which could increase the mRNA levels of aggrecan,col-2 and sox-9,inhibit chondrocyte apoptosis and promote chondrocyte proliferation.4.Protective effect of hUC-MSCs on the degradation of cartilage matrix in OA patientsCo-cultured chondrocytes and hUC-MSCs by transwell for 48 hours to test the content of GAGs in chondrocytes.The results showed that the chondrocytes were induced by IL-1β(10ng/ml)for 48 hours,which could reduce the content of GAGs.Co-cultured chondrocytes and hUC-MSCs could significantly increase the content of GAGs.It is may demonstrated that hUC-MSCs have protective effect for the degradation of cartilage matrix.5.Regulation effect of hUC-MSCs on chondrocytes function by participating in Wnt/β-catenin signaling pathwayImmunofluorescence results showed that Wnt was expressed in cell membrane,cytoplasm and nucleus of chondrocytes,andβ-catenin wes mainly expressed in the cytoplasm of chondrocytes.In western blot,chondrocytes induced IL-β(10ng/ml),the expression of Wnt andβ-catenin is lower and the the expression of phosphorylationβ-catenin is higher.hUC-MSCs could significantly increased the Wnt andβ-catenin expression and inhibitβ-catenin to phosphorylate in chondrocyte.GSK-3β,the inhibitor of Wnt/β-catenin signaling pathway has same role as hUC-MSCs.These results suggest that h UC-MSCs may regulate the function of chondrocytes by activating the Wnt/β-catenin signaling pathway.Conclusion:1.The model of rabbits and rats osteoarthritis were successfully established by Hulth method.Intra-articular injection of hUC-MSCs could improve joint injury,and the therapeutic effect is proportional to the number of injections of hUC-MSCs.2.Intra-articular injection of hUC-MSCs can effectively reduce the level of inflammatory cytokines,restore the balance of MMPs/TIMPs and promote the regeneration of CollagenⅡin OA rats.3.Co-cultured hUC-MSCs and chondrocytes could significantly inhibit chondrocyts apoptosis,promote chondrocytes proliferation and inhibit the degradation of cartilage matrix.4.hUC-MSCs may promote the expression of aggrecan,col-2 and sox-9 mRNA and regulate the function of chondrocytes by activating the Wnt/β-catenin signaling pathway. |
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