Mechanisms Of The Repair Of Articular Cartilage Defects Through TGF-β1 Regulating TGF-β/Smad And Hippo Signaling Pathway

Objective Bone marrow mesenchymal stem cells(BMSCs),which were extracted and cultivated from rats,were infected by lentivirus with transfection transforming growth factor 1(TGF-β1)and planted in calcium alginate gel stent to formation of TGF-β1-BMSCs-calcium alginate,which implanted into the articular cartilage defect in rats.To observe effects of repairing articular cartilage defects by complex treatment in the rats.Focusing on TGF-β-Smad and Hippo signal transduction pathway and explore the mechanism of TGF-β1-BMSCscalcium alginate gel in the treatment of articular cartilage defects.Methods Bone marrow was obtained from rat femoral shaft,and the BMSCs were cultured by bone marrow adherent method.P3 generation of BMSCs were infected by lentivirus with transforming growth factor-β1(TGF-β1)for 3days under the conditions of MOI=100,and compound culture with calcium alginate gel to gain the TGF-β1-BMSCs-calcium alginate.Fullthickness defects animal model were created over the articular surface of bilateral femoral condyles through drilling technique.45 rats were randomly divided into three groups including model group,BMSCs-calcium alginate group,TGF-β1-BMSCs-calcium alginate group.All rats in different groups were respectively killed in the 4th week and 8th week.Get the Specimens of the knee joint and detected with morphological observation and International Cartilage Repair Society(ICRS)Cartilage repair score,histopathology alcian blue hematoxylin(ABH)staining and Mankin cartilage repair score,immunohistochemical analysis the Col2a1、phosphorylation Smad2、YAP-1、Runx2、Col10 and RT-PCR detection Col2alexpression level.Results BMSCs grow well and the form of cell was normal,meet the experimental needs.The high infection rate and green fluorescent expression strong of BMSCs were infected by lentivirus with transforming growth factor-β1,this indicates that the lentivirus transfection TGF-β1 gene is successful.(1)Morphological observation shows:in the model group,there was obvious defect in the cartilage of the knee joint,and there was obvious boundary between the normal cartilage,and locally was seen small amount of subchondral bone tissue;The cartilage defect of BMSCs group was obvious,and there was a certain boundary between the normal cartilage,partial like hyaline cartilage tissue was found in the defect and locally visible white tissue fills;No obvious cartilage defects were found in TGF-β1+BMSCs group,the defect repair tissue was integrated with normal cartilage and no clear boundary,the surface of defect repair tissue is smooth,like hyaline cartilage tissue was found in the defect.The ICRS score was shown,the repair scores of cartilage defects in TGF-β1+BMSCs group were significantly higher than the model group,there are significant difference(P<0.01),compare to the BMSCs group,the difference was statistically significant(P<0.05).(2)Histopathology ABH staining shown:in the model group,cartilage defect repair tissue was mainly composed of tissue exudation,the locally defect irregularity,the repair surface rough,abnormal cell morphology and irregular arrangement,the defect is mainly filled with fibrous tissue.No obvious chondrogenic proteoglycans were found.in the BMSCs group,the cartilage defects of the knee were observed to be filled with newborn tissue on the defect,no obvious fibrosis tissue observed and the thickness of repair tissue relatively thin,the surface of defect relatively integrity,no obvious abnormalities were observed in the subchondral bone.Deficiency of protein polysaccharide in repair area of cartilage defect regeneration tissue.In the TGF-β1+BMSCs group,articular cartilage regeneration tissue was filled in the defect,articular cartilage repair surface smooth,the cell morphology grown well and the trabeculae arranged normally,the tissue of cartilage defect repair is reasonable,the structure clear,similar to normal cartilage structure.There is sufficient protein polysaccharide in the repair area of damaged cartilage.The result of Mankin score shown,in the 4 weeks after surgery,the model group score(8.3)significantly higher than TGF-β1+BMSCs group(4.5),there were significant differences between the two groups(P<0.01),the BMSCs group score(7.5)higher than TGF-β1+BMSCs group(4.5),the difference was statistically significant(P<0.05).In the 8 weeks after surgery,the model group score(10.7)significantly higher than TGF-β1+BMSCs group(3.5),there were significant differences between the two groups(P<0.01),the BMSCs group score(6.3)higher than TGF-β1+BMSCs group(3.5),the difference was statistically significant(P<0.05).(3)Immunohistochemistry result shows:the positive expressions of Col2a1,p-smad2 and YAP-1 in the TGF-β1+BMSCs group were significantly higher than the model group and the BMSCs group,the expression of Runx2 and Col 10 was significantly lower than that of the model group and the BMSCs group.The expression of TGF-β1+BMSCs in cartilage repair was improved by enhancing the expression of Col2a1,p-smad2 and YAP-1 to promote cartilage repair,and inhibition expression of Runx2 and Col10 to reduces the chondrocyte hypertrophy.The expression rate of p-Smad2,YAP-1 and Runx2 positive cells was consistent with the immunohistochemical results.(4)The mRNA results of Col2a1 were detected by Real-time PCR.In the 4 weeks after surgery,the expression of Col2a1 in the TGF-β1+BMSCs group was significantly higher than the model group and the BMSCs group(P<0.01);In the 8 weeks after surgery,the expression of Col2a1 in the TGF-β1+BMSCs group and BMSCs group was significantly higher than that in the model group,the difference was statistically significant(P<0.01).However,there was no significant difference between TGF-β1+BMSCs group and BMSCs group.Conclusions 1.BMSCs were cultured in combination with calcium alginate gel after TGFβ1 was transfected with lentivirus.In vivo,it was found to differentiate into chondrocytes and promote the early repair of articular cartilage defects.2.TGF-β1 is beneficial to chondrogenic differentiation of BMSCs via canonical phosphorylation Smad2/3 increased proteoglycan and collagen type Ⅱ expression to promote early-repairing of cartilage defect.3.TGF-β1 inhibits chondrocyte hypertrophy by increasing Yes-associated protein-1(YAP-1)and decreasing hypertrophy marker gene Runx2 and Col10 expression via Hippo signaling.