Fractional carbon dioxide laser-induced photothermal activation of mesenchymal stem cell-derived exosomes accelerates diabetic wound healing by enhancing angiogenesis

Background: Exosomes (Exos) derived from mesenchymal stem cells (MSCs) have emerged as a promising therapeutic option for diabetic wound healing owing to their strong pro-angiogenic potential. Nevertheless, their relatively low bioactivity remains a major barrier to successful clinical application. Fractional CO₂ laser therapy offers a precise and controllable form of photothermal stimulation that may potentiate exosome activity without the need for additional exogenous agents, possibly promoting more effective diabetic wound repair.

Aim: To investigate the mechanisms through which low-energy fractional Exos derived from CO₂ laser-preconditioned adipose-derived MSCs (Ad-MSCs) (CO₂ laser-Exos) promote the healing of diabetic wounds.

Methods: Ad-MSCs were subjected to a single exposure of fractional CO₂ laser at energy densities of 30 mJ/cm², 40 mJ/cm², or 50 mJ/cm². Infrared thermography was employed to monitor temperature fluctuations in the culture medium. To determine the optimal energy level, western blotting was performed to assess heat shock protein 90 expression, while Apoptosis was analyzed by flow cytometry. Exos were subsequently isolated through ultracentrifugation, and sphingosine-1-phosphate (S1P) concentrations within the Exos were measured using enzyme-linked immunosorbent assay. The therapeutic efficacy and underlying mechanisms of CO₂ laser-Exos were further investigated through a series of in vitro and in vivo experiments.

Results: Following a single exposure to fractional CO₂ laser, the culture medium temperature increased rapidly and then gradually declined. Among the tested groups, Ad-MSCs treated with 40 mJ/cm² demonstrated the highest heat shock protein 90 expression and exhibited reduced Apoptosis. in vitro, CO₂ laser-Exos markedly promoted the proliferation, migration, and tube formation of human umbilical vein endothelial cells, while their S1P content was higher than that of unconditioned Exos. Under high-glucose conditions, human umbilical vein endothelial cells showed increased expression of S1P receptor 1 (S1PR1). Silencing S1PR1 significantly impaired the pro-angiogenic activity of CO₂ laser-Exos and suppressed the expression of phosphorylated protein kinase B, hypoxia-inducible factor 1 alpha, and vascular endothelial growth factor-A. In vivo, compared with Exos, CO₂ laser-Exos substantially accelerated diabetic wound healing by promoting neovascularization within the wound bed.

Conclusion: Low-energy fractional CO₂ laser irradiation augments the biological activity of MSC-derived Exos through photothermal stimulation. These Exos, in turn, enhance endothelial cell functions by activating the S1PR1/protein kinase B/hypoxia-inducible factor 1 alpha signaling pathway, ultimately accelerating the repair of diabetic wounds.

Diabetic wound; Exosomes; Fractional carbon dioxide; Mesenchymal stem cells; Photothermal effect; Sphingosine-1-phosphate.