Mesenchymal stem cell-derived exosomes (MSC-Exos) have emerged as promising therapeutic agents for bone regeneration due to their regenerative and immunomodulatory properties. This study investigates the effects of hypoxic and Photobiomodulation (PBM) preconditioning on the characteristics and functional properties of exosomes derived from human adipose-derived mesenchymal stem cells (hAdMSCs), with a focus on bone regeneration. hAdMSCs were characterized by their spindle-shaped morphology and high expression of mesenchymal markers (CD73: 99.6%, CD105: 99.9%) and minimal expression of hematopoietic and endothelial markers (CD45: 0.2%, CD31: 2.25%). Exosomes were isolated from hAdMSCs cultured under normal (N-EX), hypoxic (H-EX), PBM (PBM-EX), and combined PBM + hypoxia (PBM + H-EX) conditions. Exosomal markers (CD9, CD63, and CD81) and size distribution (72.2–88.4 nm) confirmed successful isolation, with no significant differences in size between different groups. Functional assays revealed that a 200 μg exosome dose significantly enhanced cell proliferation and metabolic activity (p < 0.0001). Exosome-treated groups exhibited significantly enhanced cell viability, maintained stable glucose uptake, and accelerated migration compared to control groups. Notably, the PBM-EX group demonstrated the most remarkable outcomes, achieving complete scratch closure within just 20 h. Osteogenic differentiation potential, assessed via alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining, revealed that the PBM-EX group exhibited the highest ALP activity and mineralization levels (p < 0.0001), followed by the PBM + H-EX, H-EX, and N-EX groups. These findings suggest that PBM preconditioning enhances the regenerative potential of hAdMSC-derived exosomes, making it a promising strategy for bone tissue engineering and regenerative medicine applications.

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