Extracellular Vesicles Enhance Osteogenic Differentiation of Mesenchymal Stem Cells without any Chemical Agents

The use of extracellular vesicles (EVs) in bone tissue engineering is emerging as a promising alternative strategy to stem cells. For clinical application, EVs must be biomanufactured from suitable source cells, systematically characterized, and validated for their efficacy in bone regeneration. This study focuses on the possibility of this translation using in vitro methods with rat bone marrow stem cells (rBMSCs). Bone marrow was harvested from Wistar rats and cultured using the direct adherence method. The rBMSCs were characterized through trilineage differentiation, flow cytometry, immunofluorescence, and real-time PCR. The optimal isolation method of EVs derived from the rBMSC was investigated. EVs isolated through ultracentrifugation yielded homogeneous EVs with good quality and quantity. The EVs derived from rBMSCs were characterized through Nanoparticle Tracking Analysis, Dynamic Light Scattering, Transmission Electron Microscopy, and Western Blot analysis. The osteogenic differentiation of rBMSCs was evaluated using the isolated EVs, confirmed by the MTT Cell Proliferation Assay and In vitro Osteogenesis Assays. The concentration- and time-dependent enhancement of osteogenic differentiation by rBMSC-derived EVs was also examined. Most importantly, EVs promoted osteogenic differentiation without adding any conventional chemical agents in the culture media. These findings will pave the way for further investigations to link EVs' therapeutic benefits in bone tissue engineering and related applications.