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.