A Preliminary Study On The Mechanism Of Mechanotransduction Of Integrin α5 And β1 Of HGFs On PLGA Scaffolds

Objective:To investigate the effects of compressive force and Integrinα5β1 inhibited on expression of Integrinα5 andβ1,focal adhesion kinase(FAK),phospho-FAK(p-FAK)and Collagen-Ⅰ(COL-Ⅰ)of human gingival fibroblasts(HGFs)cultured on PLGA-collagen scaffold,and to study the mechanism of Integrinα5β1 on the mechanotransduction of HGFs.Methods:1.HGFs were cultured by the method of tissue adhering and plated on the PLGA scaffolds from the 4th to 6th generation.2.Using loading force applied compressive force of 25g/cm~2 to the PLGA-HGFs.The mRNA and protein expression of Integrinα5β1,FAK,p-FAK and COL-Ⅰof HGFs were detected by RT-qPCR and Western blot after 0,3,6,12,24,48 hours.3.Various concentrations(0,10nM,100nM,1μM,10μM,100μM,1mM)of Integrinα5β1 inhibitor(ATN-161)were apllied to the HGFs cultured with PLGA scaffold.The proliferation of HGFs was detected by CCK8 after 0,24,48,72 hours.And then various concentrations(0,10nM,100nM,1μM,10μM)of ATN-161 were apllied to the PLGA-HGFs.The mRNA expression of Integrinα5β1 was detected by RT-qPCR after 48 hours.4.The ATN-161(concentrations of 1μM)individual or the ATN-161(1μM)with compressive force of 25g/cm~2was apllied to the PLGA-HGFs.And the mRNA and protein expression of Integrinα5,Integrinβ1,FAK(p-FAK)and COL-Ⅰof HGFs were detected by RT-qPCR and Western blot after 0,3,6,12,24,48 hours.Results:1.HGFs were successfully cultured by the method of tissue adhering.The cells from the 4th to 6th generation were pure and stable in shape and plated on the PLGA scaffolds for the next experiments.2.The mRNA and protein expressions of Integrinα5β1,FAK,p-FAK and COL-Ⅰof HGFs under compressive force were all up-regulated.The mRNA and protein expressions of Integrinα5β1 and FAK reached the highest level at 3-6h and decreased slowly after 6h.The protein expression of p-FAK reached the peak at 12h,and decreased gradually to the normal level after 12h.The mRNA and protein expressions of COL-Ⅰwere both increased,with the highest expression level at 24h,and decreased slowly after 24h.Compared with integrinα5β1 and FAK,COL-Ⅰshowed slower response to the compressive force.3.As the concentration less than or equal to 10μM,the ATN-161 had no effect on the proliferation of HGFs for 72h.As the concentration over 10μM,the ATN-161 had negative effect on the proliferation of HGFs.And it increased by time.The ATN-161 had the most significant inhibiting effect on integrinα5β1 at concentration of 1μM.4.The mRNA and protein expressions of Integrinα5β1,FAK,p-FAK and COL-Ⅰof HGFs with ATN-161(0.1μM)were all decreased.The mRNA expressions of Integrinα5β1,FAK and COL-Ⅰof HGFs with ATN-161 and compressive force were slightly increased at 3h and decreased after 3h.The protein expression of integrinα5β1,FAK and COL-Ⅰwere also decreased generally.The protein expression of COL-Ⅰincreased significantly at 3-6h and decreased gradually after 6h.Conclusions:The three-dimensional culture and mechanical loading model of compressive force were successfully established.The compressive force can promote the mRNA and protein expression of Integrinα5β1,FAK,p-FAK and COL-Ⅰof HGFs,indicating that they might play important roles in the mechanotransduction and extracellular matrix fibrosis of HGFs.HGFs accept mechanical signal by Integrinα5β1,regulate the expression and phosphorylation of FAK,and affect the synthesis of COL-Ⅰfinally.