Optimized human dedifferentiated fat cells from the buccal fat pad-derived osteoinductive extracellular vesicles promote osteoblast differentiation

Bone reconstruction in the maxillofacial region typically relies on autologous bone grafting, which presents challenges, including donor site complications and graft limitations. Recent advances in tissue engineering have identified highly pure and proliferative dedifferentiated fat cells (DFATs) as promising alternatives. Herein, we explored the capacity for osteoblast differentiation and the osteoinductive characteristics of extracellular vesicles derived from DFATs (DFAT-EVs). DFATs were isolated from human buccal fat pads, cultured to confluency, and placed in either a standard or osteogenic induction medium. After culturing for 3 days, the conditioned medium was used to generate EVs using the size-exclusion chromatography and concentration filter method. Characterization of DFAT-EVs revealed typical EV morphology and positive markers (CD9 and CD63), with no differences between the two groups. assays demonstrated that EVs derived from the osteogenic induction medium (OI-EVs) significantly increased alkaline phosphatase activity and osteogenesis-related genes (Runx2 and collagen type I) compared to control EVs. Next-generation sequencing identified differentially expressed miRNAs, and gene ontology analysis suggested pathways involved in osteoblast differentiation. Isolating DFATs from buccal fat pads under osteogenic induction conditions offers a procedure confined to the oral cavity, eliminating the need for harvesting from other sites. Thus, DFAT-EVs hold promise for promoting bone regeneration in maxillofacial applications.