Development of Functional Tendon Organoids: A 3D In Vitro Model to Unravel the Cellular and Molecular Mechanisms of Tendon Healing

Tendinopathies remain a significant clinical challenge due to the limited efficacy of current therapies, which often fail to restore full tissue function and lead to chronic pain and/or recurrence. Tendon healing is a multifaceted biological process involving the coordinated and modulated activity of inflammation, the immune and nervous systems (1). Despite its complexity, there is still no validated in vitro model that recapitulates the multicellular dynamics of tendon regeneration. In this context, organoid technology would offer a powerful approach to dissect cellular crosstalk in a controlled environment. Notably, unlike other tissues, no tendon organoids have been developed to date, representing a critical gap in the study of tendon biology and healing. However, a pioneering biological system developed by Barboni et al. (2) has shown that fetal tendon-derived factors, when co-cultured with amniotic epithelial stem cells (AECs), known for their teno-differentiative and immunomodulatory properties, can induce the formation of 3D tendon-like structures. Recent advances demonstrated that tendon biomimetic scaffolds engineered with AECs not only enhance their tenogenic commitment and immune modulation but also gain paracrine activity, inducing the formation of tendon-like structures from naïve AECs within 14-21days (3). This research aims to characterize both the 3D-engineered scaffolds and the derived tendon-like organoids. Notably, both the engineered scaffold itself and the structures formed in co-culture meet the criteria to be defined as tendon organoids, showing tissue-like architecture, tendon-specific gene expression (SCX, THBS4, COL1, TNMD), and proteins, and secreted extracellular COL1. Connexin expression and functional assays, as fluorescence recovery after photobleaching (FRAP) further confirmed intercellular communication. Functional gap junctions via connexins are essential for the structural and functional maturation of tendon-like organoids. These findings support the establishment of the first tendon organoid system, providing a novel platform to explore tendon biology and regeneration and to develop next-generation assembloids integrating vascular and neural components.