| Backgroud:Osteoarthritis(OA)is one of the most common chronic degenerative diseases,which seriously affects the life quality of patients.This disease is much more common for middle-aged and elderly people.The incidence of the disease increased with age.OA has become an important issue affecting the quality of life in human society,which is an important reason for the long-term dysfunction of adults.Knee osteoarthritis(KOA)is the most common form of OA,the incidence rate ranged from12%to 12%in middle-aged and old people.It has been an important disease that caused knee pain and now has become the degenerative disease needs attention.Cartilage defect is one of the common joint changes.The repair and regeneration of articular cartilage is very weak.Bone marrow mesenchymal stem cells(MSCs)is the stem cells derived from bone marrow.It has the characteristics of good amplification effect and high differentiation potential.BMSCs has good chondrogenic potential,and produces cartilage matrix through differentiation,and promotes growth of cartilage cells with some growth factors,so as to achieve the effect of cartilage regeneration.The DKK-3 protein belongs to the DKK protein family,which includes DKK-1,DKK-2 and DKK-4,and DKK-3 has reported that its content in the OA synovial fluid is elevated.DKK-3 protein is a blocker of Wnt/beta signaling pathway in DKK-3 family,and Wnt/beta signaling pathway is an important factor affecting the proliferation and differentiation of chondrocytes.In addition,DKK-3 protein can regulate the TGF beta signaling pathway to regulate the activity of inflammatory cells and maintain the integrity of tissues,suggesting that DKK-3 may play a protective role in cartilage.Objectives:First,to observe the role of DKK-3 protein in the progression of KOA disease and to understand whether it has a protective effect on cartilage in KOA.Secondly,we know the effect of DKK-3 protein on the differentiation of cartilage induced by BMSCs.Finally,it was observed in vitro that DKK-3 could promote the repair of cartilage in BMSCs.The experiment is divided into three parts,which are as follows:Part 1:The Role of DKK-3 in the progress of KOAMethods:1.Using Bama miniature pigs as experimental animals,8 were divided into two groups(DKK-3 group and control group).Two groups of Bama miniature pigs underwent bilateral knee surgery.Anterior cruciate ligament and medial meniscus was cut off.2.Intraoperative injection was started after operation.DKK-3 group was injected with DKK-3 protein(250ng/ml,5ml).The control group was injected with PBS solution(5ml),once every two weeks,until three months after operation.3.Three months after the operation,the animals were sacrificed to the specimen for examination.The International Cartilage Repair Association(ICRS)was applied to assessment the condition of KOA,HE/Toluidine blue/Safranin O staining was applied,and the expression of Sox-9 and COL-II was test by PCR and Western Blot.Results:The results of ICRS grading score were as follows:the ICRS score of group DKK-3 was 2.6 + 0.70,and the control group was 3.4 + 0.97.The ICRS scores of the two groups were statistically different,p=0.048.HE staining and safranin O staining and toluidine blue staining of cartilage defects observed in DKK-3 group were average depth,area and extent than the control group,DKK-3 group KOA severity was relatively mild.The expression level of Col-II and Sox-9 of cartilage markers and protein in DKK-3 group was significantly higher than that in the control group,and the difference was statistically significant,p<0.05.Part 2:the role of DKK-3 in promoting the cartilage differentiation of BMSCsMethods:1.The BMSCs was cultured,and the expression of cell surface antigen was detected by flow cytometry.2.BMSCs cartilage differentiation induction,in the induction process,the DKK-3 group added DKK-3 protein in proportion(maintaining the concentration of 250ng/ml),and the control group did not add.3.Samples were cultured for fourteenth days for type ? collagen and Sox-9 gene expression level;28 days from specimen:alcian blue staining and type II collagen and Sox-9 expression.Results:1.The surface markers of BMSCs were detected by flow cytometry.The positive expression markers included CD105(98.57%),CD29(99.79%),CD73(99.91%),and negative markers,including CD34(0.62%),CD45(0.29%),CD 11b(0.44%).2.The specimens were induced to be cultured for 28 days,and the normal dewaxing section was observed under the microscope.The degree of cartilage staining and cartilage density in group DKK-3 was better than that of the control group.3.The gene and protein expression of Col-? and Sox-9 in DKK-3 and control group in twenty-eighth days were higher than fourteenth days,p<0.05.The expression level of Col-? and Sox-9 of cartilage marker gene in group DKK-3 was significantly higher than that of control group at fourteenth days and 28 days,and the difference was statistically significant.Part ?:the role of DKK-3 in the repair of Bama miniature pig cartilage defect Methods:1.Using Bama miniature pig as experimental animal,4 pigs were divided into two groups(DKK-3 group and control group),two groups of Bama miniature pigs underwent femoral trochlear cartilage defect preparation operation(diameter 1cm,full-thickness cartilage defect).At the same time in the bottom,a hole to the bone marrow cavity was drilled and let the bone marrow blood flow.2.DKK-3 group was treated with hydroxybutyl chitosan combined DKK-3 protein(250ng/ml)to the cartilage defect.The control group only used hydroxybutyl chitosan to add the carftilage defect,and then sutured after the bone marrow blood was fully infiltrated through the stent.3.Three months after the operation were to animal specimen respectively:The International Cartilage Repair Association(ICRS)was applied to assessment the condition of KOA,HE/Toluidine blue/Safranin O staining was applied,and the expression of Sox-9 and COL-? was test by PCR and Western Blot.Results:The results of ICRS cartilage repair score were 9.25 + 0.96 in group DKK-3 and 4.50 +1.73 in the control group.The ICRS scores in the two groups were statistically different,p=0.005.HE staining and safranin O staining and toluidine blue staining results of repairing cartilage defect in DKK-3 group specimen thickness,area,scope is better than that of control group.The expression level of cartilage marker gene and protein Col-?and Sox-9 in cartilage repair area of DKK-3 group was significantly higher than that of control group,and the difference was statistically significant(p<0.05).Conclusions:1 · DKK-3 has a protective effect on cartilage during the process of KOA.2.DKK-3 can promote the differentiation of BMSCs into cartilage.3.DKK-3 can promote the repair of BMSCs cartilage defect. |
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