VEGF decoration of fibrin scaffolds for in vitro and in vivo engineering of vascularized tissues

Introduction. Generation of functional vascular networks is an unresolved challenge for 3D engineered tissues, both in vivo to promote progenitor survival and differentiation and in vitro to produce vascularized organoids. Physiological growth of vascular networks depends on controlled signaling by the morphogenic factor VEGF, which is physiologically regulated through interaction with the extracellular matrix. Here we decorated fibrin hydrogels with an engineered VEGF protein to generate an optimal matrix-associated angiogenic microenvironment and promote the rapid self-assembly of micro-vascular networks in vitro that can 1) be efficiently perfused by the host vasculature in vivo and 2) be exploited to generate a hierarchical vascular tree in vitro and to vascularize bone-like and breast cancer organoids.
Methods. Fibrin matrices were prepared by mixing 10 mg/ml of human fibrinogen, 3 U/ml of thrombin and 3 U/ml of factor XIIIa, endothelial cells (HUVEC) and adipose stromal cells (ASC) as vascular support. VEGF164 protein was fused to the transglutaminase substrate peptide NQEQVSPL (TG-VEGF) to enable its covalent cross-linking to fibrin to a final concentration of 100 ng/ml. Functional perfusion and survival of the self-assembled microvascular networks were tested by subcutaneous implantation in SCID mice.
Results. Co-culture of HUVEC with ASC self-assembled into physiologically differentiated vascular networks, displaying apico-basal polarization, marked by basal laminin and luminal podocalyxin, and open lumens. Vessel formation was influenced by fibrinogen concentration, cell density and TG-VEGF: gels of 10 mg/ml of fibrinogen containing 5x106 cells/ml with 100 ng/ml of TG-VEGF resulted in a 5.4-fold improvement in vascular density, compared to no-VEGF controls, after both 7 and 14 days. TG-VEGF significantly accelerated endothelial proliferation speed, as shown by pospho-histoneH3 staining. Vessel diameters remained compatible with micro-circulation, with a median of 17 μm. Human-derived vascular structures formed after 7 days of in vitro culture and could rapidly connect to the host vasculature upon subcutaneous implantation and be perfused by the systemic circulation. TG-VEGF enabled vascular connection and perfusion of hybrid vessels by promoting human micro-vascular networks growth and survival, as well as improving graft invasion by the host blood vessels. Notably, matrix-bound TG-VEGF was necessary for the in vivo survival of pre-assembled microvascular networks, as these regressed almost completely with the supplementation of soluble VEGF.
In vitro, the self-assembled micro-vascular networks allowed the formation and the growth of breast cancer clusters. Further, within 7 days micro-vascular structures connected to chondrogenic pellets and to an endothelialized artificial macro channel of 325 µm diameter, to form a perfusable hierarchical vascular tree.
Conclusions. Decoration of fibrin matrix with 100 ng/ml of TG-VEGF promotes the efficient self-assembly of 3D, perfusable, lumenized and physiologically differentiated micro-vascular networks in vitro within 7 days. This engineered angiogenic microenvironment can be exploited for the rapid in vivo vascularization of 3D tissue engineered grafts and the generation of in vitro vascularized organoids.