Static Topographical Cue Combined with Dynamic Fluid Stimulation Enhances the Macrophage Extracellular Vesicle Yield and Therapeutic Potential for Bone Defects.

Extracellular vesicles (EVs) hold promise for tissue regeneration, but their low yield and limited therapeutic efficacy hinder clinical translation. Bioreactors provide a larger culture surface area and stable environment for large-scale EV production, yet their ability to enhance EV therapeutic efficacy is limited. Physical stimulation, by inducing cell differentiation and modulating EV cargo composition, offers a more efficient, cost-effective, and reproducible approach compared to the cargo loading of EVs and biochemical priming of parental cells. Herein, the effects of a 3D-printed perfusion bioreactor with a topographical cue on the macrophage EV yield and bioactivity were assessed. The results indicate that the bioreactor increased the EV yield 12.5-fold and enhanced bioactivity in promoting osteogenic differentiation and angiogenesis via upregulated miR-210-3p. Mechanistically, fluid shear stress activates Piezo1, triggering Ca2+ influx and Yes-associated protein (YAP) nuclear translocation, promoting EV secretion and enhancing macrophage M2 polarization in conjunction with morphological changes guided by aligned topography. Moreover, a porous electrospun membrane-hydrogel composite scaffold loaded with bioreactor-derived EVs exhibited outstanding efficacy in promoting osteogenic differentiation and angiogenesis in a rat cranial defect model. This study presents a scalable, cost-effective, and stable platform for the production of therapeutic EVs, potentially overcoming key challenges in translating EV-based therapies to the clinic.