SFL-3D-cultured adipose mesenchymal cell-derived extracellular vesicles promote diabetic wound healing by alleviating microvascular endothelial senescence via PI3K/AKT/mTOR/4EBP1-mediated cap-dependent translation.

Background: Diabetic foot ulcers (DFUs) are severe complications of diabetes, and treatment options for DFUs are limited. Current research on impaired angiogenesis in DFUs predominantly relies on generic endothelial cells, which inadequately reflect the pathophysiological microenvironment of the diabetic microvasculature. In contrast, this study focused specifically on human dermal microvascular endothelial cell (HDMEC) senescence as a central mechanism in DFU progression. We used a self-feeder layer 3D (SFL-3D) culture system to reprogram adipose-derived mesenchymal stem cells (ADSCs) to prepare functionally enhanced three-dimensional adipose stem cells (tdASCs) and their extracellular vesicles (tdASC-EVs) to mitigate HDMEC senescence and improve diabetic healing. Methods: , a fat graft model was used to assess the stem cell function of tdASCs, whereas a diabetic wound model was used to evaluate the therapeutic efficacy of tdASC-EVs. Results: tdASC-EVs significantly attenuated high-glucose-induced HDMEC senescence, oxidative stress, and mitochondrial dysfunction while increasing tube formation. Mechanistically, tdASC-EVs activated PI3K/AKT/mTOR signaling, increased 4EBP1 phosphorylation, and promoted eIF4E-eIF4G assembly. In diabetic mice, tdASC-EVs accelerated wound closure and increased microvascular density, which correlated with reduced p16 expression and increased CD31 and p-4EBP1 expression. In the Bama miniature pig diabetic large animal model, tdASC-EVs exhibited markedly superior therapeutic effects compared with those induced by conventional ADSC-EVs, as evidenced by significantly faster wound closure, greater neovascularization density, narrower scar width, and more orderly collagen deposition. Conclusions: tdASC-EVs ameliorated diabetic wound healing by targeting HDMEC senescence through PI3K/AKT/mTOR/4EBP1-mediated cap-dependent translational activation. The preclinical large animal data further confirmed the superior efficacy of tdASC-EVs over ADSC-EVs, highlighting the therapeutic potential of SFL-3D-modified EVs for DFU treatment.