Collagen binding properties separate two functionally distinct subpopulations of milk extracellular vesicles regarding bone regenerative capacity

Extracellular vesicles (EVs) are heterogeneous in their composition. The proteins on their surface determine their binding properties to the meshwork of extracellular matrix (ECM). Here, we report that type I collagen-binding property separates two subpopulations of EVs from cow milk (mEVs): collagen-binding mEVs ( cb + mEVs) and non-collagen binding mEVs ( cb- mEVs). cb + mEVs showed noticeable uptake by human bone marrow mesenchymal stromal cells (hBMSCs) and osteogenic functionality in vitro (1.2-fold increase in mineralization). By proteomics profiling we identified Annexin V (AnxV) and confirmed its enrichment on CD9 positive cb + mEVs using immunomagnetic separation. By implanting a hydrogel construct enriched with cb + mEVs into a femoral condyle defect in osteoporotic rats, we demonstrated their superior bone regenerative capacity in vivo (2.4-fold increase in bone formation). Our study suggests that EV binding to the ECM protein type I collagen can be used to isolate a functional mEV subpopulation for bone tissue regeneration. This approach represents an important step forward in relating EV properties to their functionality, which will promote clinical translation.