Seminiferous tubule-inspired coaxial bioprinting-derived extracellular vesicles restore Leydig cell steroidogenesis through modulation of Wnt4/β-catenin signaling.

Chemotherapy-associated gonadotoxicity compromises Leydig cell steroidogenesis and leads to testosterone deficiency, yet repair is hindered by the testicular barrier-immune niche and by limited manufacturability of biologics. Inspired by the tubular architecture of seminiferous tubules, we develop a coaxial bioprinting-enabled encapsulation culture that generates continuous core-shell hydrogel microfibers for high-density 3D culture of human umbilical cord MSCs (hUMSCs) and continuous conditioned-medium harvesting, producing seminiferous tubule-inspired coaxial bioprinting-derived extracellular vesicles (STi-EVs). We directly benchmark STi-EVs against conventional 2D culture-derived EVs (2D-EVs) in a 4-hydroperoxycyclophosphamide (4-HC)-injured TM3 model and a cyclophosphamide-induced mouse model. Across cellular fitness, re-engagement of steroidogenic programs, and testosterone output, STi-EVs consistently outperform 2D-EVs, while more effectively alleviating testicular histopathology and restoring serum testosterone in vivo. Transcriptomics prioritize Wnt signaling and nominate Wnt4; orthogonal validation and Wnt4 gain- and loss-of-function perturbations, and β-catenin stabilization experiments support that modulation of Wnt4/β-catenin signaling functionally contributes to STi-EV-mediated steroidogenic restoration. Collectively, this work couples a scalable extracellular vesicles (EV) manufacturing paradigm with a mechanism-informed repair strategy for endocrine sequelae after alkylating chemotherapy.