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AIM: To determine the role of the transforming growth factor (TGF)- beta 3 inthe anterior cruciate ligament (ACL) reconstruction in a rabbit model. METHODS: The scaffold made up of silk fibroin mesh and type I collagen composite was filled with adipose-derived stem cells (ADSCs) inserted of transforming growth factor-beta 3 (TGF-beta 3) gene to obtain a construct for anterior cruciate ligament (ACL) reconstruction in vivo. For the ACL reconstruction surgery, ACL was damaged at first .The scaffold filled with ADSCs incorporating TGF- beta 3 was applied to a tunnel prepared in the femur and tibia of rabbits. As controls, the scaffold without TGF-beta 3 gene or ADSCs, or Autologous ligament was applied.3 months later, bone regeneration in the tunnel and ACL tissue regeneration were histologically evaluated, and the mechanical strength of the regenerated ACL were assessed. RESULTS: Contrast to the control groups, Combination of TGF-beta 3 release resulted in enhanced mechanical strength of the regenerated ACL tissue and the surrounding the scaffold results in a better biological regeneration of ligament-like tissue. Additionally, significant bone regeneration around the scaffold was observed in the bone tunnel. CONCLUSION: Our results indicate that TGF-beta 3 may contribute to the ACL reconstruction in a rabbit model.