Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture

Timely vascularization is essential for optimal performance of bone regenerative constructs. Vascularization is efficiently stimulated by vascular endothelial growth factor (VEGF), a substance with a short half-life time. This study investigates the controlled release of VEGF from gelatin microparticles (GMPs) as a means to prolong VEGF activity at the preferred location within 3D bioprinted scaffolds, and the effects on subsequent vascularization. The release of VEGF from GMPs was continuous for 3 weeks during in vitro studies, and bioactivity was confirmed using human endothelial progenitor cells (EPCs) in migration assays. Traditional and real-time migration assays showed immediate and efficient EPC migration in the presence of GMP-released VEGF, indistinguishable from VEGF-solution that was added to the medium. Matrigel scaffolds containing EPCs and VEGF, which was released either in a fast or sustained fashion by application of GMPs, were investigated for their in vivo vasculogenic capacity. Implantation in subcutaneous pockets in nude mice for one week demonstrated that vessel formation was significantly higher in the VEGF sustained-release group compared to the fast release group. In addition, regional differences with respect to VEGF release were introduced in 3D bioprinted EPC-laden scaffolds and their influence on vasculogenesis was investigated in vivo. The different regions were retained and vessel formation occurred analogous with the results seen in the Matrigel plugs. We conclude that GMPs are suitable to generate sustained release profiles of bioactive VEGF, and that they can be used to create defined differentiation regions in 3D bioprinted heterogeneous constructs, allowing a new generation of smart scaffold design. The prolonged presence of VEGF led to a significant increase in scaffold vascularization when applied in vivo.