Conditioned Extracellular Vesicles Derived from Dedifferentiated Fat Cells Promote Bone Regeneration by Altering MicroRNAs

The identification of suitable seed cells represents a critical scientific challenge in the field of oral and maxillofacial bone tissue regeneration. Dedifferentiated fat (DFAT) cells have biological characteristics similar to adipose-derived stem cells (ASCs), but possess the advantages of minimally invasive acquisition, strong proliferation and high homogeneity. Compared to cell therapy, extracellular vesicles (EVs) have lower immunogenicity, better stability and more convenient transportation and storage, which makes EV-based cell-free therapy to be a promising alternative strategy. It is not clear whether EVs derived from DFAT cells can be used as an effective therapy for bone regeneration. The purpose of this study was to verify the effect of EVs derived from DFAT cells on promoting osteogenic differentiation of bone marrow derived mesenchymal stem cells (BMSCs) to provide a reference for the treatment of alveolar bone loss. DFAT cells from Sprague-Dawley (SD) rats were isolated and cultured by ceiling culture. DFAT cells were conditioned for 14 days using an osteogenic induction medium. EVs derived from conditioned DFAT cells were extracted by ultracentrifugation and then identified and characterized by Western blot, transmission electron micro-scope (TEM) and nanoparticle tracking analysis (NTA). After labeling the conditioned EVs with the green fluorescent dye PKH-67, the EVs were cocultured with BMSCs for 12 hours and then the cells were observed using an laser scanning confocal microscopy (LSCM). CCK-8 kit assay was used to explore the effect of conditioned EVs on the proliferation of BMSCs. Alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining and real-time quantitative polymerase chain reaction (qRT-PCR) were used to verify the effect of conditioned EVs on osteogenic differentiation of BMSCs.