A bilayer scaffold was engineered using COL1 and hydroxyapatite (HAp) powders, each blended with 10% gelatin and stirred separately to form uniform mixtures. The HAp layer was first cast and set at 4 °C, followed by the addition of the COL1 layer. After sequential cooling, the scaffold underwent chemical cross-linking using either Genipin, EDC/NHS, or a dual cross-linking strategy to enhance structural integrity and biological performance.

The optimal scaffold—dual cross-linked COL1/HAp—was loaded with human amniotic mesenchymal stem cells (hAMSCs) and implanted in a rat rotator cuff injury model. Micro-CT, histological, and biomechanical analyses confirmed superior healing, including aligned collagen fibers, fibrocartilage formation, and improved mechanical strength at 12 weeks post-implantation.

This work highlights the critical role of COL1 in scaffold design and provides a viable strategy for enhancing tendon–bone interface healing through biomimetic materials combined with stem cell therapy.