Harnessing inter-spheroid spacing and structural connectivity to direct collective cell migration and host vessel integration in 3D engineered tissue

Abstract

Engineering functional vascular networks within three-dimensional (3D) tissues remains a major challenge in regenerative medicine. Here, we introduce a hybrid platform that integrates spheroids with gelatin methacryloyl (GelMA) hydrogels and 3D-printed poly(ε-caprolactone) (PCL) scaffolds to generate pre-vascularized 3D constructs. The scaffold architecture was designed with spatially defined chambers and interconnected pores incorporating catching strands that anchor spheroid positioning and direct cell migration from embedded spheroids. Additionally, the GelMA matrix establishes a permissive microenvironment conducive to cell adhesion and matrix remodeling, and the PCL framework preserves macroscopic structural integrity. Co-culture spheroids composed of human umbilical vein endothelial cells (HUVEC) and human adipose-derived stem cells (hADSC) enhanced sprouting and collective migration compared with suspended single cells, and also provided significant upregulation of extracellular matrix (ECM) remodeling genes and pro-angiogenic markers. Spatially distributed spheroids with 3D structure strongly modulated vascular network morphogenesis, whereby the inter-spheroid distance finely tuned cell migration dynamics and ECM remodeling. Scaffold microarchitecture further governed host tissue integration in vivo, with higher pore interconnectivity facilitating superior vascular infiltration and increased co-localization between host and engineered vessels. Notably, host vessels aligned preferentially along the inferior catching strands, suggesting geometry-associated vascular integration. Collectively, these findings demonstrate that precise spatial organization of spheroids combined with tailored 3D-printed scaffold architecture supports robust pre-vascularization of engineered tissues, offering a promising strategy for a variety of biomedical applications