TGF-β1/smad Signaling Pathway Regulates The Healing Of Hamstring Tendon After Rabbit’s Anterior Cruciate Ligament Reconstruction

Objective To explore TGF-β1/Smad signaling pathway in the healing of Hamstring tendon after rabbit’s ACL reconstructionMethods To establish the rabbit model of autogenous Hamstring tendon reconstruction of ACL.The rabbits were killed at 1 week after operation,and the ligament tissue of tendon-bone interface is taken for primary culture of ligament fibroblasts.The third to tenth generation of cells are divided into two groups A and B.Group A is cultured with DMEM medium containing 10g/ml TGFβ1,and divided into 4h,8h,12 h,24h,48 h,and the blank control group;The group B is cultured with DMEM medium containing 10g/ml TGFβ1 and different concentrations of SB431542(0μmol/L,0.5μmol/L,5μmol/L,10μmol/L,50μmol/L)for 48 h.The technology of Western blot and immunofluorescence is applied to evaluate the expression of P-smad2,P-smad3 and collagen typeⅠ.Results 1.Primary culture of ligament fibroblasts.Ligament fibroblasts cells are cultivated successfully in the method of collagenase digestion.Continuing to cultivate the cells with 10%FBS/DMEM,the cells adhered to the wall,and they were spindle shaped,similar to those of typical fibroblasts.2.The expression of P-Smad2,P-Smad3 and collagen type I were detected by Western blot The relative expression levels of P-Smad2,P-Smad3 protein and type I collagen in the A group were higher than those in the control group at different time points,and the difference was significant(P<0.05).Where there was no significant difference between the expression of P-Smad2 protein in different time group(P>0.05),the expression of P-Smad3 and type I collagen was time dependent.with the gradual increase of stimulating time of TGF beta 1,the expression of P-Smad3 into the platform at 24 h,there was no significant difference of expression(P>0.05).Type I collagen expression was no significant difference in the stimulation of 4-8h(P>0.05),each group expression difference was significant after 12-48h(P<0.05);In the group B,the expression of P-Smad2,P-Smad3 protein and collagen type I decreased with the increase of SB431542 concentration,except 0.5 mol/L group and 0 mol/L group(control group)had no significant difference(P>0.05),other groups of P-Smad2,P-Smad3 protein and collagen type I had significant difference(P<0.05).3.Cell immunofluorescence The expression of P-Smad2,P-Smad3 protein and type I collagen were detected by immunofluorescence assay in each group of cells cultured in 48 h.The fluorescence intensity of A group was significantly higher than that of B group and control group.Conclusion TGFβ1 can promote the expression of collagen type I in the healing of autogenous Hamstring tendon reconstruction of ACL by activating the TGF-β1/Smad signaling pathway...