Harnessing extracellular vesicles for endochondral bone regeneration: Mechanisms and applications

Developmentally inspired strategies that recapitulate endochondral ossification (EO), the physiological mechanism underlying secondary fracture repair, are emerging as promising approaches for bone regeneration. Extracellular vesicles (EVs), nanoscale carriers of bioactive molecular cargo secreted by diverse cell types, have recently gained prominence as pivotal mediators of EO, orchestrating processes from mesenchymal condensation and cartilage template formation to callus maturation and remodeling. Owing to their intrinsic biocompatibility, stability, and signaling specificity, EVs represent a new class of bioactive agents for cell-free bone repair. This review integrates current understanding of the native roles of EVs in EO-driven bone healing with advances in parental cell priming, scalable production, and cargo modulation aimed at enhancing therapeutic potency. Particular emphasis is given to biomaterial-based administration strategies that enable spatiotemporal control of EV retention, release, and activity within bone defects. We also discuss manufacturing standardization, storage, and regulatory frameworks aligned with ISEV guidelines, which are essential for clinical translation. By synthesizing insights from EV biology, bioprocess engineering, and biomaterial design, this review provides a comprehensive framework for advancing cell-free, EO-inspired regenerative therapies that bridge developmental mechanisms with scalable, clinically viable bone repair solutions. STATEMENT OF SIGNIFICANCE: Bone regeneration is increasingly guided by developmentally inspired strategies that mimic endochondral ossification (EO). Extracellular vesicles (EVs) are promising cell-free mediators of EO-based repair, but an integrated framework linking EO biology with engineering, delivery, and manufacturing is lacking. This review synthesizes evidence for EV functions across fracture-healing stages (inflammation, soft callus formation, hard callus development, and remodeling) and highlights how parental cell priming and cargo modulation can strengthen immunomodulatory, angiogenic, chondrogenic, and osteogenic effects. We also outline biomaterial delivery design principles to achieve spatial and temporal control of EV activity within bone defects. Finally, we summarize scalable, GMP-compliant production, purification, storage, and regulatory considerations needed for clinical translation.