Bioengineering Developmentally Inspired Matrix Vesicles as Designer Nanotherapeutics for Bone Regeneration

Extracellular vesicles (EVs) are emerging as promising acellular nanotherapeutics for musculoskeletal repair. Matrix vesicles, a matrix-bound subset of EVs, are essential mediators of endochondral ossification in bone development and fracture repair. This study aims to design bioengineered matrix vesicles from hypertrophic cartilage microtissues to drive endochondral ossification for bone repair. Human mesenchymal stromal cell (hBMSC) microtissues were differentiated with/without BMP2 in chondrogenic or hypertrophic medium. Isolated matrix vesicles were characterized for physiochemical properties and biological functionality. BMP2 and hypertrophic conditioning significantly increased vesicle yield (1.5-fold), alkaline phosphatase activity (3.24-fold), calcium binding capacity (8.82-fold), and growth factor content (BMP2, VEGF). These vesicles promoted proliferation, migration, and mineralization of hBMSCs and enhanced angiogenesis in human endothelial colony forming cells (hECFCs), with BMP2 and hypertrophically conditioned vesicles showing the most pronounced effects. Proteomics analysis confirmed the enrichment of proteins involved in extracellular matrix remodelling, mineral deposition and vascularization within these hypertrophically engineered vesicles. These findings demonstrate that hypertrophic induction of cartilaginous microtissues substantially improves the yield and therapeutic potential of matrix vesicles. Taken together, this research unveils a powerful strategy to bioengineer developmentally inspired vesicles that not only recapitulate key cues of endochondral ossification but offers a tailorable, multifunctional nanotherapeutic platform for improved bone regeneration strategies.