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

Abstract 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.