Next-Generation 3D Tendon Organoid: A Functional In Vitro Model for Studying Tendon Regeneration and Healing Mechanisms

Tendinopathy is a common term used to define a variety of conditions arising from tendon overuse, and it is generally characterized by persistent pain and inflammation, defective repair and, often, scar tissue formation, which frequently results in incomplete restoration of biomechanical function and recurrence of the symptoms, therapeutic interventions often have limited efficacy and generally consist in anti-inflammatory pharmacological treatments, surgical interventions and movement therapy(1). This gap in clinical practice can be attributed to limitations in current knowledge of the molecular and cellular mechanisms behind tendinopathy and healing: tendon healing is a complex biological process involving a coordinated activity of inflammatory, immune and nervous systems (2). Despite its complexity, there is still no validated in vitro model that accurately recapitulates the multicellular dynamics of tendon regeneration. In this case, organoid technology shows a promising approach to dissect cellular crosstalk in a controlled environment. So far, unlike other tissues no tendon organoids have been developed to date, highlighting a serious gap in the study of tendon biology and regeneration. However, evidence has demonstrated that fetal tendon-derived factors, when co-cultured with amniotic epithelial stem cells (AECs), which possess teno-differentiative and immunomodulatory properties, can leads to the formation of 3D tendon-like structures (3). Recent advances have demonstrated that tendon biomimetic scaffolds engineered with AECs not only enhance their tenogenic commitment and immunomodulatory capacity but also acquire paracrine activity, inducing the formation of tendon-like structures from naïve AECs within 2 to 3 weeks (4). This research aims to characterize both the 3D-engineered scaffolds and the resulting tendon-like organoids. Notably, both the engineered scaffold and the co-culture derived structures meet the criteria for classification as tendon organoids, showing tissue-like architecture, tendon-specific gene expression (SCX, THBS4, COL1, TNMD), corresponding proteins production, and secretion of extracellular COL1. Connexin expression and fluorescence recovery after photobleaching (FRAP) further confirmed intercellular communication. Functional gap junctions mediated by connexins are fundamental for the structural and functional maturation of tendon-like organoids. These results support the establishment of the first tendon organoid system, providing a novel platform to investigate tendon biology and regeneration and to develop next-generation assembloids incorporating vascular and neural components.