Low-dose ionizing radiation in vivo unlocks the therapeutic potential of prevascularized dermal spheroids in chronic wounds

Chronic wounds (CWs), particularly in diabetic patients, remain a major clinical challenge because of impaired angiogenesis and fibroblast dysfunction. This study investigated the therapeutic synergy between low-dose ionizing radiation (LDIR; 0.3 Gy) and prevascularized spheroids composed of human dermal fibroblasts (HDFs) and endothelial colony-forming cells (ECFCs) embedded in a xeno-free fibrin hydrogel. <i>In vitro</i>, LDIR enhanced endothelial sprouting and fibroblast outgrowth from HDF-ECFC spheroids without affecting morphology or viability. Gene and protein analyses revealed LDIR-induced upregulation of angiogenic and profibrotic markers, including VEGFR2, CD31, VE-cadherin, COL4A2, α-SMA, TGF-β1 and LH2. Conditioned media from irradiated spheroids exhibited a proangiogenic secretome and significantly increased neovascularization in the chorioallantoic membrane assay. Notably, spheroids composed exclusively of fibroblasts failed to exhibit these LDIR-induced effects, highlighting the importance of endothelial-stromal interactions. Importantly, <i>in vivo</i>, the combination of nonirradiated HDF-ECFC spheroids followed by local LDIR significantly accelerated wound healing and restored skin remodeling, skin architecture and histological integrity by reducing pathological skin remodeling in the <i>db/db</i> diabetic mouse model. In contrast, neither LDIR <i>per se</i> nor preirradiated spheroids on their own promoted wound closure or histological repair. These findings demonstrate that LDIR enhances the regenerative capacity of prevascularized dermal spheroids through molecular activation, paracrine signaling and stromal‒endothelial crosstalk but requires <i>in vivo</i> irradiation of the wound niche to achieve therapeutic benefit. This combinatorial strategy offers a clinically translatable approach to modulate the wound microenvironment and overcome current limitations in CW management.