TGIF1 Gene Silencing In Tendon-derived Stem Cells Improves The Tendon-to-bone Insertion Site Regeneration

Background/Aims Tendon-to-bone insertion site is the special form of connective tissues derivatives of common connective progenitors, fibrocartiliage loss at the site leads to poor healing outcome. TGF-β plays bidirectional effects(chondrogenic or fibrogenic) through different signaling pathways at different stages. TGIF1 is a key factor that represses the gene expression induced by TGF-β. Tendon stem cells as a newly discovered stem cells exhibited higher multilineage differentiation potential. The slow healing process of tendon-to-bone junctions can be accelerated via implanted tendon-derived stem cells(TDSCs) with silenced transforming growth interacting factor 1(TGIF1) gene.Methods The TDSCs of rat were harvested by achilles tendon, then identified and multilineage differentiation. For chondrogeneic differentiation, a pellet culture system was used to compare the chondrogenic differentiation of TDSCs transfected with TGIF1 si RNA and over-expression of TGIF1. The chondrogenic differentiation was tested to evaluate the effects of gene modification on rat tendon derived stem cells. At days 3, 7 and 21 from micro-mass culture, q RT-PCR and Western blotting were used to detect the expression of chondrogenic and tendon markers. Sixty-four SD rats were randomly divided into four groups: fibrin glue +TGIF1 silencing group, fibrin glue + TGIF1 overexpressing group, fibrin glue +TDSC(without TGIF1) group and fibrin glue only group. Normal supraspinatus tendon were transected from the foot point. We injected 1×106 TDSCs+ fibrin glue into the tendon-bone interface on the fibrin glue +TDSC(without TGIF1) group, fibrin glue on fibrin glue only group, 1×106 TDSC silencing TGIF1 + fibrin glue on TGIF1 silencing group and 1×106 TGIF1 overexpressing TDSC+ fibringlue on TGIF1 overexpressing group. At weeks 2 and 4 post surgery, eight animals in each group were sacrificed and the supraspinatus tendon from the insertion site was harvested. The insertion site was processed for histological analysis.Results In this study, isolated rat tendon-derived stem cells were strong proliferative capacity. TDSCs were positive for CD90, CD73, and were negative for CD34. Induced differentiation test showed that rat tendon-derived stem cells have the ability to differentiate into osteoblast, chondrogenic and adipocyte. The silencing of TGIF1 significantly unregulated the chondrogenic genes and markers. Similarly, TGIF1 inhibited TDSC differentiation into cartilage via interactions with TGF-β-activated Smad2 and suppressed the phosphorylation of Smad2. The area of fibrocartilage at the tendon-bone interface was significantly increased in the TGIF1 silencing group compared with the control and the TGIF1-overexpressing groups in the early stages of the animal model. The interface between the tendon and bone showed a increase of new bone and fibrocartilage in the TGIF1 silencing group at 4 weeks. Fibrovascular scar tissue was observed in the TGIF1-overexpressing group and the fibrin glue only group. Low levels of fibrocartilage and fibrovascular scar tissue were found in the TDSCs group. In the TGIF1 silencing group, western blotting showed significant differences in the protein levels of chondrogenic markers compared with the control groups and the TGIF1 overexpressing group.Conclusion Collectively, this study shows that the tendon-derived stem cell modified with TGIF1 gene silencing has promising effects on tendon-to-bone healing which can be further explored as a therapeutic tool in regenerative medicine.