Extracellular vesicles secreted by LRP1⁺ ligament-derived stem cells promote tendon-bone healing after ACL reconstruction via miR-708-5p/Bambi axis.

Abstract

Anterior cruciate ligament (ACL) rupture often requires reconstruction surgery, with a key postoperative challenge being robust integration between the graft and bone tunnel. Studies have shown that extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSCs) can enhance tendon-bone healing strength. As tissue-resident stem cells in the ACL, ligament-derived stem cells (LDSCs) are among the first activated after injury, migrating to the damaged site to participate in regenerative repair. In this study, single-cell RNA sequencing (scRNA-seq) of human ACL remnant samples revealed that LRP1-positive LDSCs (LRP1⁺LDSCs) have significantly stronger stemness and chondrogenic differentiation potential than LRP1-negative ones. Based on this, EVs from LRP1⁺LDSCs (LRP1⁺LDSCs-EVs) were isolated and delivered into bone tunnels of a rat ACL reconstruction (ACLR) model using gelatin methacryloyl (GelMA) hydrogel as a carrier. Results showed that LRP1⁺LDSCs-EVs effectively promoted cartilage regeneration at the tendon-bone interface (TBI) post-ACLR and significantly improved biomechanical function recovery. MiRNA sequencing characterized the miRNA cargo in LRP1⁺LDSCs-EVs, identifying highly enriched miR-708-5p as a key mediator. This miRNA directly binds to and suppresses Bambi mRNA expression, reducing Bambi protein's competitive inhibition of BMP signaling (Bambi acts as a decoy receptor for the TGF-β pathway). This mechanism activates chondrogenic differentiation and promotes TBI regenerative healing. Overall, EVs from the LRP1-positive LDSCs subpopulation significantly enhance graft-bone tunnel integration efficiency after ACLR, providing a novel targeted and efficient cell-free therapeutic strategy for enhancing tendon-bone integration in orthopedic surgery.