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

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.