Squid pen and cuttlebone-derived β-chitin composite scaffolds with gradient mineralization for functional ACL reconstruction and bone-tendon Interface regeneration.

Artificial ligaments are widely used in anterior cruciate ligament (ACL) reconstruction. However, their clinical performance is often limited by insufficient graft-bone integration and the inability to recapitulate the graded microenvironment of the native bone-tendon interface (BTI). To address this challenge, this study developed a marine-derived biomimetic composite scaffold for BTI repair. High-purity, anisotropically aligned β-chitin fibers derived from squid pens were used to fabricate the artificial ligament component, exhibiting a tensile stress of 11.50 ± 0.62 MPa and an elastic modulus of 194.52 ± 26.00 MPa, which were within the reported physiological range of native ACL tissue. For BTI reconstruction, a gradient adapter layer was engineered using the natural lamellar structure of cuttlebone. The outer layer was mineralized with hydroxyapatite (HA) to support osteointegration, whereas the inner layer retained an aragonite-rich structure that may facilitate interfacial association with the artificial ligament component. In a rat ACL reconstruction model (n = 3 per group per time point for Micro-CT and histological analyses; n = 3 per group for biomechanical testing), the C-H-A group (composite scaffold combined with autologous tendon reinforcement) showed enhanced BTI healing at 12 weeks post-operation, supported by collagen-rich matrix deposition, tendon-related marker remodeling, and improved bone tunnel regeneration. At 12 weeks, the BV/TV inside the bone tunnel reached 30.50% in the C-H-A group, compared with 21.18% in the CH group and 15.92% in the Control group. Biomechanical testing further showed that the C-H-A group achieved the greatest functional recovery among the reconstructed groups, with a failure load of 17.46 N and stiffness of 8.51 N/mm; however, these values remained lower than the native Normal baseline (35.6 ± 4.1 N and 16.8 ± 2.2 N/mm). Taken together, these findings support the potential of this biomimetic system as a promising preclinical strategy for soft-to-hard tissue repair, while further mechanistic investigation and validation remain necessary.