Recapitulating Native‐Like Strain Distributions in a Tissue‐Engineered Enthesis by Creating Structural, Biochemical, and Mineral Gradients

ABSTRACT The incorporation of robust meniscus‐to‐bone interfaces into tissue‐engineered menisci is critical for their clinical translation. Generating gradients in collagen fiber organization and mineral content for tissue‐engineered entheses is essential for achieving native tissue‐like mechanics; however, engineering such gradients remains challenging. This study presents a tissue‐engineered enthesis model consisting of a fibrochondrocyte‐seeded cylinder of type I collagen gel with trabecular bone plugs on both ends. Using a tri‐chamber bioreactor, spatially controlled biochemical (e.g., TGF‐β1 and glucose) and biomechanical stimuli were applied, generating native‐like collagen fiber structure and mechanics within tissue‐engineered enthesis constructs. Confocal elastography revealed a more uniform local strain distribution and reduced peak strain in the enthesis constructs cultured in a tri‐chamber bioreactor compared to those in a single chamber bioreactor, likely attributed to the enhanced collagen fiber organization. To further improve the integration at the collagen–bone plug interface, we introduced partially demineralized bone plugs to the constructs. Partial demineralization improved the mechanical performance of enthesis constructs, decreasing peak strain by > 30% and strain gradients by 50%, while increasing toughness and strain at failure by 50% and 40%, respectively. Overall, these findings highlight the importance of zone‐specific biochemical and biomechanical stimuli and biomimetic scaffold materials to improve tissue‐engineered implants.