Synovium-Derived Mesenchymal Stem Cell-Based Scaffold-Free Fibrocartilage Engineering for Bone–Tendon Interface Healing in an Anterior Cruciate Ligament Reconstruction Model

Background: Current tendon and ligament reconstruction surgeries rely on scar tissue healing which differs from native bone-to-tendon interface (BTI) tissue. We aimed to engineer Synovium-derived mesenchymal stem cells (Sy-MSCs) based scaffold-free fibrocartilage constructs and investigate in vivo bone–tendon interface (BTI) healing efficacy in a rat anterior cruciate ligament (ACL) reconstruction model. Methods: Sy-MSCs were isolated from knee joint of rats. Scaffold-free sy-MSC constructs were fabricated and cultured in differentiation media including TGF-β-only, CTGF-only, and TGF-β + CTGF. Collagenase treatment on tendon grafts was optimized to improve cell-to-graft integration. The effects of fibrocartilage differentiation and collagenase treatment on BTI integration was assessed by conducting histological staining, cell adhesion assay, and tensile testing. Finally, histological and biomechanical analyses were used to evaluate in vivo efficacy of fibrocartilage construct in a rat ACL reconstruction model. Results: Fibrocartilage-like features were observed with in the scaffold-free sy-MSC constructs when applying TGF-β and CTGF concurrently. Fifteen minutes collagenase treatment increased cellular attachment 1.9-fold compared to the Control group without affecting tensile strength. The failure stress was highest in the Col + D + group (22.494 ± 13.74 Kpa) compared to other groups at integration analysis in vitro . The ACL Recon + FC group exhibited a significant 88% increase in estimated stiffness ( p = 0.0102) compared to the ACL Recon group at the 4-week postoperative period. Conclusion: Scaffold-free, fibrocartilage engineering together with tendon collagenase treatment enhanced fibrocartilaginous BTI healing in ACL reconstruction.