Bioprinting of pre-vascularized constructs for enhancedin vivoneo-vascularization

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 onin vivoneo-vessel formation has not been studied. In this study, we developed a functional pre-vascularized construct that significantly promotes graft vascularization and conductedin vivoevaluations of the micro-vascular patterns (μVPs) in various printed designs.μVP formation, composed of high-density capillaries, was induced by the co-printing of endothelial cells and adipose-derived stem cells (ADSC). We implanted the printed constructs with variousμVP designs into a murine femoral arteriovenous bundle model and evaluated graft vascularization via 3D visualization and immune-histological analysis of the neo-vessels. TheμVP-distal group (μVP located away from the host vessel) showed approximately two-fold improved neo-vascularization compared to theμVP-proximal group (μVP located near the host vessel). Additionally, we confirmed that theμVP-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 thanμVP, was added to theμVP + AMP group design. TheμVP + AMP group showed approximately 1.5- and 1.9-fold higher total sprouted neo-vessel volume than theμVP only and AMP only groups, respectively. In immunohistochemical staining analysis, theμVP + AMP group showed two-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.