Pre-vascularization has been receiving significant attention for developing implantable engineered 3D tissues. While various pre-vascularization techniques have been developed to improve graft vascularization, the effect of pre-vascularized patterns on in vivo neo-vessel formation has not been studied. In this study, we developed a functional pre-vascularized construct that significantly promotes graft vascularization and conducted in vivo evaluations of the micro-vascular patterns (µVP) in various printed designs. µVP formation, composed of high-density capillaries, was induced by the co-printing of endothelial cells (EC) and adipose-derived stem cells (ADSC). We implanted the printed constructs with various µVP designs into a murine femoral arteriovenous bundle model and evaluated graft vascularization via 3D visualization and immune-histological analysis of the neo-vessels. The µVP-distal group (µVP located away from the host vessel) showed approximately 2-fold improved neo-vascularization compared to the µVP-proximal group (µVP located near the host vessel). Additionally, we confirmed that the µVP-distal group can generate the angiogenic factor gradient spatial environment for graft vascularization via computational simulations. Based on these results, the ADSC mono pattern (AMP), which secretes four times higher angiogenic factors than µVP, was added to the µVP + AMP group design. The µVP + AMP group showed approximately 1.5- and 1.9-fold higher total sprouted neo-vessel volume than the µVP only and AMP only groups, respectively. In immunohistochemical staining analysis, the µVP + AMP group showed 2-fold improved density and diameter of the matured neo-vessels. To summarize, these findings demonstrate graft vascularization accelerated due to design optimization of our pre-vascularized constructs. We believe that the developed pre-vascularization printing technique will facilitate new possibilities for the upscaling of implantable engineered tissues/organs.

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