Electrospun Scaffold-Enabled Bioreactor for Scalable Manufacturing of Mechanically Programmed Macrophage-Derived Extracellular Vesicles Targeting Aged Bone Defects

Extracellular vesicles (EVs) represent promising cell-free therapeutics for the regeneration of aged bone defects. Mechanical stimulation is critical in bone regeneration and significantly influences both EV secretion and cargo composition. Despite this, translating mechanically programmed EVs to clinically relevant doses remains a significant challenge. Most current scalable bioreactor systems has achieved high yields through high-density cell culture and fluid flow, yet they do not provide programmable or spatially consistent mechanical stimulation. In this study, an electrospun scaffold-enabled bioreactor was developed to integrate high-density three-dimensional macrophage culture with programmable cyclic stretching, enabling simultaneous EV scale-up and cargo programming. The interconnected fiber junction network transmits boundary-applied strain with enhanced spatial consistency, while the highly porous scaffold supports dense cell seeding and efficient EV release and collection. This approach increases EV yield by 13.8-fold compared to conventional two-dimensional culture. Fiber-mediated mechanical conditioning activates Piezo1-dependent Ca 2+ signaling and integrin-associated mechanotransduction, promotes Yes-associated protein (YAP) nuclear translocation, and remodels EV cargo composition. The resulting mechanically stimulated EVs (ms-EVs) demonstrate concurrent pro-regenerative activities, including cellular rejuvenation, vascular repair, and osteogenic enhancement, and perform favorably compared to canonical interleukin-4 (IL-4)-polarized M2 macrophage-derived extracellular vesicles (M2-EVs). In aged rat femoral defects, ms-EV-functionalized scaffolds increase bone volume fraction by approximately 1.7-fold at 8 weeks, reduce p16 + senescent cells, and enhance CD31 + neovascularization. These findings establish electrospun scaffolds as scalable mechanobiological manufacturing substrates for the production of functionally programmed EV therapeutics. Graphical