| Osteoarthritis(OA)is a degenerative joint disease that can not be effectively treated due to limited endogenous regenerative capacity.It is a common cause of disability in middle-aged and elderly people.With the aggravation of population aging,the incidence of OA has increased year by year,and has developed into a prominent social problem.It is predicted that by 2020,25%of the adult population in the United States will be affected by this disease,which will bring a serious burden to individuals,families and society.The etiology and pathogenesis of OA have yet to be elucidated,and the clinical treatment effect is not good.It is generally believed that the imbalance between the synthesis and degradation of chondrocytes,extracellular matrix and subchondral bone is an important cause of OA.At present,the clinical treatment methods of OA mainly include the use of analgesics,non-steroidal anti-inflammatory drugs,the addition of lubricating supplements in joint injuries,and the use of joint replacement surgery in the late stage of osteoarthritis.However,these methods can only alleviate the pain of patients with osteoarthritis and delay the progression of OA,and can not reverse the course of OA caused by irreversible articular cartilage.Mesenchymal stem cells(MSCs)have been extensively used to study the degeneration and injury of articular cartilage in osteoarthritis and other joint pathological processes in recent years.As a new type of seed cells,MSCs have strong ability of self-renewal,proliferation and multi-potential differentiation.MSCs can induce and differentiate into cartilage,osteogenesis,fat and other adult cells in vivo,which makes it possible for MSCs to treat OA.Human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)are an important cell source of MSCs.They are widely used because of their low immunogenicity,strong in vitro expansion ability and the ability to repair damaged cartilage tissue by differentiation into cartilage.Induced pluripotent stem cell-derived mesenchymal stem cells(iPSC-MSCs)are a new type of stem cells,which have more effective and reliable regeneration ability than traditional stem cells,and can produce a large number of MSCs with uniform functions through induction.The pluripotent stem cell-extract(iPSC-extract)is a cell extract of iPSC,which is a product obtained by cleavage of iPSC,including exosomes and cell contents.In this study,we established a rat model of experimental bone and joint to compare the therapeutic effects of mesenchymal stem cells from different sources on osteoarthritis.Objective:The experimental osteoarthritis model of rats was established by operation,and hUC-MSCs、iPSC-MSCs and iPSC-extract were injected into the articular cavity to explore the therapeutic effect of MSCs from different sources on OA.Methods:The rat OA model was established by Hulth method.The model animals were randomly divided into model group,hUC-MSCs(2.0×10~6,5.0×10~6cell/ml),iPSC-MSCs(2.0×10~6cell/ml),iPSC-extract 2.0×10~6cell/ml)),sodium hyaluronate(HA,10 mg/ml)positive control group,with 10 rats in each group and 10 rats in the control group.After12 weeks,the rats in the HA group were given 50 ml of drug in the articular cavity of the operation side,and the sham-operated group and the model group were given saline in the same way.Five weeks after administration,the difference of joint diameter between the operation side and the non-operation side was measured;the joint cavity structure of the operation side was examined by X-ray and Kellgren-Lawrence score was performed;the joint surface structure of the femoral condyle and tibial plateau on the operation side was observed by operation microscope and Pelletier score was performed;the knee joint pathology of the operation side was examined by HE staining and scored according to Mankin score standard;and the operation side was detected by immunohistochemistry.The expressions of MMP-13,Collagen II and ADAMTS-5 in the lateral knee joint were detected by protein chip method.Results:1.Effects of MSCs from different sources on the difference of knee joint diameter in OA ratsAfter the rats were sacrificed,the diameters of the knee joints on the surgical side and the non-surgical side of the rats were measured with a vernier caliper.The results showed that compared with the sham operation group,the difference in knee diameter of the model group was significantly increased,suggesting that the OA model was successfully induced;compared with the model group,hUC-MSCs(2.0×10~6,5.0×10~6cells/50μl)and iPSC-MSCs(2.0×10~6 cells/50μl)can significantly reduce the difference in knee joint diameter,iPSC-extract(2.0×10~6 cells/50μl)group was not statistically significant,there was no difference between the groups.2.Effects of MSCs from different sources on knee X-ray score of OA ratsBefore the rats were sacrificed,the rats were anesthetized with 3%pentobarbital sodium and X-ray examination was performed.The results showed that the X-ray scores of the model group were significantly higher than those of the sham-operated group.Compared with the model group,iPSC-MSCs(2.0×106 cells/50μl)could reduce the knee joint X-ray score and severity.(Grade 3,4)The number of OA animals improved the joint cavity structure,reduced osteophyte formation and osteosclerosis.Other drug-administered groups had no significant difference in reducing the X-ray score.3.Effects of different sources of MSCs on the Pelletier score of knee joint in OA ratsAfter the rats were sacrificed,the knee joints of the surgical side of the rats were dissected.The results showed that the Pelletier score of the knee joint surface of the model group was significantly higher than that of the hypothetical surgery group;compared with the model group,the Pelletier score was significantly reduced in each of the administration groups,and iPSC-MSCs(2.0×10~6 cells/50μl)The group’s reduced Pelletier score was significantly different from that of the hUC-MSCs(2.0 x 10~6 cells/50μl).4.Effects of different sources of MSCs on the pathology of knee joint in OA ratsAfter the rats were sacrificed,the knee joint femoral condyle of the knee joint was taken for pathological examination.The results showed that the skeletal tissue of the sham-operated group was normal and the pathological score was low.The cartilage margin of the model group was defective,the cartilage arrangement was disordered,and the pathological score was significantly higher than that of the model group.Compared with the model group,each drug-administered group was compared.It can significantly improve the structure of joint disease and reduce the grade of pathological score.The pathological scores of iPSC-MSCs(2.0×10~6cell/50μl)group were significantly different from those of hUC-MSCs(2.0×10~6cell/50μl).5.Effects of different sources of MSCs on the expression of Collagen II,MMP-13 and ADAMTS-5 in knee joints of OA ratsThe results of immunohistochemistry showed that the loss of type II collagen in the cartilage layer of the model group was severe and the expression was significantly reduced compared with the sham operation group.Compared with the model group,each cell treatment group could significantly increase the expression of type II collagen in chondrocytes.Among them,high-dose hUC-MSCs(5.0×10~6cell/50μl)showed significant differences in the administration of hUC-MSCs(2.0×10~6cell/50μl)in the lower dose group of type II collagen in chondrocytes,suggesting that hUC-MSCs were administered.There was a dose difference;with the same number of cells,iPSC-MSCs administration showed a significant difference in the expression of type II collagen in chondrocytes compared to the hUC-MSCs and iPSC-extract administration groups.The results of MMP-13 immunohistochemical staining showed that the expression of articular cartilage in the model group was significantly increased,and the expression of MMP-13 was significantly decreased in each drug-administered group.Compared with low-dose hUC-MSCs,the high-dose hUC-MSCs group had significant differences in the reduction of MMP-13 expression levels;iPSC-MSCs were compared to hUC-MSCs and iPSC-extract groups using the same number of cells.Administration significantly reduced the expression of MMP-13 in chondrocytes;hUC-MSCs(2.0×10~6cell/50μl)down-regulated the expression of MMP-13 in chondrocytes significantly compared with iPSC-extract(2.0×10~6cell/50μl)。The results of immunohistochemical staining of ADAMTS-5 showed that the expression of ADAMTS-5 in the articular chondrocytes of the model group was significantly increased compared with the sham operation group;compared with the model group,except for the iPSC-extract(2.0×10~6 cells/50μl)group.In addition,the other cell-administered groups significantly reduced the expression of ADAMTS-5 in rat articular cartilage;high dose of hUC-MSCs reduced the level of ADAMTS-5 in rat cartilage significantly better than the low-dose hUC-MSCs group.Using the same number of cells,iPSC-MSCs administration significantly reduced the expression of ADAMTS-5 in the down-regulated articular chondrocytes compared with the hUC-MSCs and iPSC-extract administration groups.6.Effects of different sources of MSCs on the levels of inflammatory factors in rat knee osteoarthritis modelThe detection of inflammatory factor TNF-αin rat knee joint showed that the level of TNF-αin the joint cavity of the model group was significantly higher than that of the sham operation group.Compared with the model group,each cell administration group The expression of inflammatory factor TNF-αwas significantly decreased.The high-dose hUC-MSCs group had significant difference in the lower dose group of inflammatory factor TNF-α.The inflammatory factor TNF-αwas decreased between different source cells.There was no significant difference in the expression level of TNF-α;the detection of inflammatory factor IL-6 in the knee joint of rats showed that compared with the sham operation group,the IL-6 level in the joint cavity of the model group was significantly increased compared with the model group.Except for the low-dose hUC-MSCs group,the cell-administered group significantly reduced the expression of the inflammatory factor IL-6,and the iPSC-MSCs administration decreased the IL-in the hUC-MSCs group compared with the hUC-MSCs group.There was a significant difference in the level of 6;the expression of inflammatory factor L-selectin in the joint cavity fluid showed that the expression of L-selectin in the joint cavity of the model group was significantly higher than that of the sham operation group.In comparison,each cell-administered group can significantly reduce the expression level of L-selectin,among which The expression of inflammatory factor L-selectin in hUC-MSCs was significantly lower than that in iPSC-MSCs and iPSC-extract group,and there was no significant difference between different doses of hUC-MSCs in the cell-administered group.The detection of TIMP-1 expression level in cytokines showed that compared with the sham operation group,the level of TIMP-1 in the joint cavity of the model group was significantly higher than that of the sham operation group,compared with the model group,except for the low dose hUC-MSCs.Outside the group,the cell-administered group significantly reduced the expression of the inflammatory factor TIMP-1.The iPSC-extract group between the different cell-administered groups showed significant differences in TIMP-1 levels compared with hUC-MSCs and iPSC-MSCs.Compared with iPSC-MSCs group,hUC-MSCs decreased TIMP-1 level significantly less than iPSC-MSCs group;anti-inflammatory factor IL-10 expression level in joint cavity fluid showed that IL in model group compared with sham operation group The level of IL-10 was significantly lower.Compared with the sham operation group,except for the high dose of hUC-MSCs,the IL-10 expression level was significantly increased in each cell administration group,and there was no significant difference between the groups.7.Effects of hUC-MSCs on the expression of aggrecan and sox-9 mRNA in chondrocytes of patients with OAAfter stimulation of chondrocytes of OA patients for 48 hours,IL-1β(10ng/ml)can inhibit the expression of aggrecan and sox-9 mRNA in OA patients and promote chondrocyte apoptosis.After 48 hours of co-culture with hUC-MSCs,the expression levels of aggrecan and sox-9 mRNA of chondrocytes could be significantly increased,suggesting that hUC-MSCs can regenerate endogenous cartilage in patients with OA.8.Effects of hUC-MSCs on the changes of GAGs content in chondrocytes of patients with OAIL-1β(10ng/ml)stimulated the chondrocytes of OA patients for 48h,and significantly increased the content of GAGs released into the culture medium.After co-cultured with hUC-MSCs for 48h,the content of GAGs in culture medium decreased significantly,suggesting that hUC-MSCs Chondrocytes can be protected by inhibiting degradation of the matrix.Conclusion:1.An experimental rat osteoarthritis model was successfully established by Hulth surgery.Intra-articular injection of MSCs from different sources can effectively improve articular cartilage damage and joint pathology in rats.2.Different MSCs have different effects on OA rats:iPSC-MSCs have certain advantages in reducing Pelletier score,joint swelling and regulating cytokine levels compared with hUC-MSCs;hUC-MSCs promote cartilage matrix synthesis and inhibit it The degradation is dose dependent.3.hUC-MSCs inhibited the degradation of chondrocyte matrix,and protected chondrocytes were associated with increased expression of chondrocyte marker genes SOX-9,aggrecan mRNA,and promotion of type II collagen regeneration. |
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