Spatially Controlled Embedded Bioprinting of Prevascularized Spheroids-Containing Biopatch Enables Functional Skin Regeneration.

Abstract

Functional vascularization remains a critical challenge in the regeneration of full-thickness skin defects. Here, we present a 3D embedded bioprinting strategy for engineering vascularized biopatches through the precise integration of prevascularized multicellular spheroids within a skin-derived decellularized extracellular matrix (SdECM) bioink. Rheological results reveal that a 1.0% (w/v) SdECM formulation exhibits yield-stress behavior, pronounced shear-thinning, and rapid shear recovery, enabling high-fidelity of spheroids while preserving cell viability and structural integrity. Using this platform, endothelial spheroids with tunable diameters are fabricated to investigate size-dependent vascular self-organization. Intermediate-sized spheroids (∼700 µm) demonstrate robust endothelial network formation, with enhanced vascular coverage, branching complexity, and angiogenic gene expression while avoiding excessive hypoxia. Spatial organization of prevascularized spheroids critically regulates paracrine-mediated wound repair, with enhanced fibroblast migration observed when inter-spheroid distances were within 1.8 mm. Furthermore, incorporation of mesenchymal stem cells (MSCs) provides perivascular-like support, significantly stabilizing microvascular networks, with an optimized endothelial cell-to-MSC ratio of 1:0.5. In a full-thickness murine wound model, multicellular spheroids-laden SdECM biopatches significantly accelerate wound closure, enhance re-epithelialization, promoted angiogenesis, and improve blood perfusion. Collectively, this study establishes a scalable embedded bioprinting for engineering prevascularized biopatches and provides design principles for spheroid-based soft tissue regeneration.