3D Tendon Organoid: A Functional In Vitro Model for Studying Tendon Regeneration and Intercellular Communication

Tendons are specialized connective tissues essential for musculoskeletal function by transmitting mechanical forces from muscles to bones. Tendon disorders remain a major clinical burden, particularly in aging individuals, athletes, and subjects exposed to lifestyle-related risk factors (1). However, current tendon treatments remain limited, and advanced in vitro models capable of reproducing the tendon microenvironment are still lacking. In this context, tendon organoid systems offer a promising platform for investigating tendon biology, tissue maturation, and regenerative mechanisms under controlled experimental conditions. This study aimed to validate a 3D co-culture system that mimics tendons in vitro. Different cultural conditions, including ovine amniotic epithelial cells (oAECs) control (CTR), AECs-engineered 3D scaffolds (3D), and AECs co-cultured with 3D (AECs-co 3D) (2), were maintained for up to 21 days and evaluated at structural, molecular, and functional levels. After 21 days, AECs cultured in both 3D and AECs-co 3D differentiated toward the tenogenic lineage, and AECs-co 3D formed organized tendon-like structures (TLS), as confirmed by the expression of mature tendon-specific proteins tenomodulin (TNMD) and collagen type 1 (COL1) compared to CTR. Differentiated AECs not only expressed COL1 intracellularly but also deposited it extracellularly and showed significant TNMD protein expression (p<0.05). To evaluate intercellular communication, Fluorescence Recovery After Photobleaching (FRAP) analysis (3) showed that both 3D and TLS cultures exhibited greater fluorescence recovery than CTR, indicating enhanced molecular mobility and intracellular exchange dynamics. TLS showed the strongest effect, with enrichment of cells with >50% recovery and a reduction in cells with <30% recovery (p<0.0001). Moreover, connexin 43 and connexin 32 expression at 48h and 21 days confirmed the establishment of gap junction-mediated intercellular communication. Overall, these findings indicate that the validated co-culture system promotes AEC organization into TLS and supports connexin-mediated communication, representing a first step toward tendon organoids and future tendon assembloids that incorporate vascular and neural compartments.