Functionalized osteogenic extracellular vesicles derived from BMP9-stimulated mesenchymal stem cells (MSCs) effectively induce bone regeneration.

Effective healing of large bone defects is a clinical challenge. Extracellular vesicle (EV)-mediated cell-free therapy has great potential in bone defect repair; however, unmodified EVs generally exhibit limited osteogenic potential. Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic cytokines reported to date. Here, we modified the EVs from BMP9-stimulated adipose-derived mesenchymal stem cells (iMAD), designated as B9-EVs. Subcutaneous injections of B9-EVs in immune-competent mice elicited no detectable host immune response. B9-EVs effectively induced in vitro osteogenic markers and promoted subcutaneous bone formation and cranial defect repair in vivo, similar to the direct use of recombinant adenovirus-mediated BMP9 overexpressing (AdR-B9) transduced iMAD cells. The miRNA-seq analysis of B9-EVs identified a distinct set of osteogenesis-related miRNAs. RNA-seq analysis revealed that osteogenesis-associated transcripts regulated in B9-EVs-stimulated MSCs were overlapping with but also distinct from those in AdR-B9-stimulated MSCs. Further bioinformatic analysis established an miRNA-mRNA network and revealed that the functionalized B9-EVs may regulate 10 miRNAs and 11 mRNA transcripts to induce osteogenesis through mechanisms that are unique and distinct from those associated with direct BMP9 stimulation. Collectively, given their cell-free and non-immunogenic nature, the functionalized osteogenic B9-EVs hold great potential for bone tissue engineering by providing effective osteogenic factors through EV-mediated paracrine signaling mechanisms.