Cell trafficking and regulation of osteoblastogenesis by extracellular vesicle associated bone morphogenetic protein 2

Extracellular vesicles (EVs) are characterized by complex cargo composition and carry a wide array of signalling cargo, including growth factors (GFs). Beyond surface‐associated GFs, it is unclear if EV intralumenal growth factors are biologically active. Here, bone morphogenetic protein‐2 (BMP2), loaded directly into the lumen of EVs designated engineered BMP2‐EVs (eBMP2‐EVs), was comprehensively characterized including its regulation of osteoblastogenesis. eBMP2‐EVs and non‐EV ‘free’ BMP2 were observed to similarly regulate osteoblastogenesis. Furthermore, cell trafficking experiments suggest rapid BMP2 recycling and its extracellular release as ‘free’ BMP2 and natural occurring BMP2‐EVs (nBMP2‐EVs), with both being osteogenic. Interestingly, BMP2 occurs on the EV surface of nBMP2‐EVs and is susceptible to proteolysis, inhibition by noggin and complete dissociation from nBMP2‐EVs over 3 days. Whereas, within the eBMP2‐EVs, BMP2 is protected from proteolysis, inhibition by noggin and is retained in EV lumen at 100% for the first 24 h and ∼80% after 10 days. Similar to ‘free’ BMP2, bioprinted eBMP2‐EV microenvironments induced osteogenesis in vitro and in vivo in spatial registration to the printed patterns. Taken together, BMP2 signalling involves dynamic BMP2 cell trafficking in and out of the cell involving EVs, with distinct differences between these nBMP2‐EVs and eBMP2‐EVs attributable to the BMP2 cargo location with EVs. Lastly, eBMP2‐EVs appear to deliver BMP2 directly into the cytoplasm, initiating BMP2 signalling within the cell, bypassing its cell surface receptors.