Bone-Targeting Microspheres Enable Sustained Release of CD301b+ Macrophage-Derived Small Extracellular Vesicles to Promote Bone Repair

Rationale Large bone defects often exceed the body’s intrinsic capacity for self-repair. Successful bone healing depends on a coordinated immune transition alongside tightly coupled angiogenesis and osteogenesis, in which macrophages play a key regulatory role. Therefore, ideal bone-regenerative materials should integrate immunomodulatory, pro-angiogenic, and osteogenic functions. Methods and Results Small extracellular vesicles (sEVs) from CD301b + macrophages (CD301b⁺-sEVs) are isolated by ultracentrifugation for inclusion in a bone-targeting microsphere system fabricated by microfluidic technology through dynamic Schiff-base cross-linking of alendronate-functionalized succinylated chitosan with oxidized sodium alginate allowing pH-responsive release of CD301b⁺-sEVs. Transcriptome sequencing and comprehensive in vitro studies reveal that CD301b⁺-sEVs possess the ability to modulate immune homeostasis by promoting macrophage polarization toward the pro-repair M2 phenotype, upregulate angiogenic factors that can enhance vascularization as seen by HUVEC sprouting assay, and contain RNA that stimulate osteogenic differentiation of BMSCs through activation of the Akt/GSK-3β/β-catenin signaling pathway. Preclinical studies with a rat calvaria model show that microspheres loaded with CD301b + -sEVs significantly promote bone regeneration in comparison to microsphere controls. Conclusion This study not only overcomes key limitations of conventional sEVs therapies such as poor retention and instability, but also provides an innovative “targeted localization, intelligent release, and multifunctional synergy” strategy, offering a minimally invasive and highly effective therapeutic platform for bone repair.