Spatial-Controlled Coating of Pro-Angiogenic Proteins on 3D Porous Hydrogels Guides Endothelial Cell Behavior

In tissue engineering, the composition and the structural arrangement of molecular components within the extracellular matrix (ECM) determine the physical and biochemical features of a scaffold, which consequently modulate cell behavior and function. The microenvironment of the ECM plays a fundamental role in regulating angiogenesis. Numerous strategies in tissue engineering have attempted to control the spatial cues mimicking in vivo angiogenesis by using simplified systems. The aim of this study was to develop 3D porous crosslinked hydrogels with different spatial presentation of pro-angiogenic molecules to guide endothelial cell (EC) behavior. Hydrogels with pores and preformed microchannels were made with pharmaceutical-grade pullulan and dextran and functionalized with novel pro-angiogenic protein polymers (Caf1-YIGSR and Caf1-VEGF). Hydrogel functionalization was achieved by electrostatic interactions via incorporation of diethylaminoethyl (DEAE)–dextran. Spatial-controlled coating of hydrogels was realized through a combination of freeze-drying and physical absorption with Caf1 molecules. Cells in functionalized scaffolds survived, adhered, and proliferated over seven days. When incorporated alone, Caf1-YIGSR mainly induced cell adhesion and proliferation, whereas Caf1-VEGF promoted cell migration and sprouting. Most importantly, directed cell migration required the presence of both proteins in the microchannel and in the pores, highlighting the need for an adhesive substrate provided by Caf1-YIGSR for Caf1-VEGF to be effective. This study demonstrates the ability to guide EC behavior through spatial control of pro-angiogenic cues for the study of pro-angiogenic signals in 3D and to develop pro-angiogenic implantable materials.