Integrin Gene Methods Promotes Creation Of Tissue-engineered Cartilage Under Periodic Mechanical Stress In Rats

Objective To investigate the effect of integrin up-regulation on periodic mechanical stress-induced chondrocytes,with a view to seeking good methods for constructing tissue-engineered cartilage in vitro.Methods1. Isolated 6-8 day old Sprague-Dawley(SD) rat articular chondrocytes, the third-generation cells were seeded in 6-well plates, the chondrocytes were transfected with Lv-integrin vector, Lv-GFP vector and Lv-sh RNA–integrin β1.The chondrocytes were cultured under periodic mechanical stress or static condition for 1h before Western-blotting analysis.2. The chondrocytes were cultured under periodic mechanical stress or static condition for 8h before Real-time PCR analysis.The chondrocytes were cultured under periodic mechanical stress or static condition 8h per day for 3d prior to CCK-8assay, respectively.3. Three groups of cells were seeded on PLGA scaffold, 6 weeks after culture in vitro, the tissue-engineered cartilage of the scaffolds were analysed via gross morphology, histological and immunohistochemical.Results1.We employed lentivirus vector Lv-integrin β1 to restore integrin β1 expression and constructed lentivirus vector Lv-sh RNA- integrin β1 to suppress integrin β1expression, while the empty vector Lv-GFP served as a control. Three days after transfection, we evaluated GFP expression in each group by fluorescence microscopy.More than 80% of chondrocytes were effectively transfected suggesting a high transfection efficiency. The protein expression levels of integrin β1 in each group were analyzedby Western blot. We found that compared with the Lv-GFP group, integrin β1 expression was significantly increased in the Lv-integrin β1 group(p<0.05) while Lv-sh RNA-integrinβ1 transfection markedly abolished integrin β1expression in the Lv-sh RNA- integrin β1 group.2.The proliferation and extracellular matrix synthesis of chondrocytes under periodic mechanical stress increased significantly, the expression and phosphorylation levels of ERK1/2 increased(P <0.05) with integrin up-regulation.3.The wet weight of integrin overexpression group cartilage were significantly increased than the other two groups(P<0.05), glycosaminoglycan(GAG), type II collagen(Collagen II) were significantly increased than the other two groups(P<0.05).Conclusion Integrin overexpression can improve the chondrocytes proliferation and matrix synthesis under periodic mechanical stress and improve the quality of tissue-engineered cartilage.