BMP‐2/TGF‐β1 gene insertion into ligament‐derived stem cells sheet promotes tendon–bone healing in a mouse

Bone morphogenetic protein‐2 (BMP‐2) and transforming growth factor‐β1 (TGF‐β1) reportedly induce the osteogenic and tenogenic differentiation of anterior cruciate ligament (ACL)‐derived stem cells (LDSCs), respectively. However, few studies have investigated the effect of BMP‐2/TGF‐β1 on the differentiation of LDSC. We developed a BMP‐2/TGF‐β1 gene insertion into an LDSC cell sheet that promotes tendon–bone healing in a mouse ACL reconstruction (ACLR) model. CD34+ LDSCs were isolated from human ACL stump tissues, virally transduced to express BMP‐2 or TGF‐β1, and then embedded within cell sheets. All mice underwent ACLR using an autograft wrapped with a cell sheet and were randomly divided into three groups: BMP‐2‐, TGF‐β1‐, and BMP‐2/TGF‐β1‐transduced. At 4 and 8 weeks, tendon–bone healing was evaluated by micro‐CT, biomechanical test, and histological analysis. BMP‐2 and TGF‐β1 promoted the osteogenic and tenogenic differentiation of LDSC in vitro. BMP‐2/TGF‐β1‐transduced LDSC sheet application contributed to early improvement in mean failure load and graft stiffness, accelerated maturation of the tendon–bone junction, and inhibited bone tunnel widening. Furthermore, reduced M1 macrophage infiltration and a higher M2 macrophage percentage were observed in the BMP‐2/TGF‐β1‐transduced LDSC group. This work demonstrated that BMP‐2 and TGF‐β1 promoted CD34+ LDSCs osteogenic and tenogenic differentiation in vitro and in vivo, which accelerated the tendon–bone healing after ACLR using autografts wrapped with cell sheets in a mouse model. Graphical and Lay Summary Bone morphogenetic protein‐2 (BMP‐2) and transforming growth factor‐β1 (TGF‐β1) regulate the osteogenic and tenogenic differentiation of anterior cruciate ligament (ACL)‐derived stem cells (LDSCs), respectively. In this study, the authors demonstrate that both BMP‐2 and TGF‐β1‐transduced LDSC sheet wrapped around the tendon graft accelerated the tendon–bone healing. This work is the potential treatment for gene and stem cells in ACL reconstruction surgery.