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

Abstract

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.

Engineering bacterial protein polymers to support human pluripotent stem cell growth and differentiation in culture

“…This stands in contrast to other variants where these locations do have activity, e.g. the Caf1-NT-Ln shown here and also the Caf1-L5-Fn and Caf1-L5-YIGSR variants that have been shown to be bioactive with other cell types in previous studies 13,14 . These results are consistent with the view that, although a particular motif may be able to bind to a cell surface receptor, it must be presented in an accessible fashion and that this may differ for different motifs.…”

“…1C) chosen included a fibronectin mimic (Caf1-L5-Fn), laminin mimics (Caf1-L5-YIGSR, Caf1-Ln variants) and vitronectin mimics (Caf1-Vn variants) where L5 indicates that the bioactive motif is inserted into loop 5 of the Caf1 protein. Caf1-L5-Fn and Caf1-L5-YIGSR have been shown to be proadhesive for other cell types in previous studies 13,14 . Caf1-Ln and Caf1-Vn had not been previously tested until this study and motifs were placed at both loop 5 and also the N-terminus (NT) of the protein.…”

“…With these factors in mind, the systems in which Caf1 can be deployed to useful effect is increasing. For example, a recent study shows how Caf1 laminin and VEGF mimicking modules can be linked in a single polymer in order to facilitate the adhesion and migration of human endothelial cells within a 3D hydrogel network 14 . Another study shows the effect of Caf1 osteopontin/BMP2 mimicking mosaic polymers on primary human MSCs, triggering differentiation and the early stages of bone formation without the need for supplementary growth factors 15 .…”

“…1D ). We have previously utilized this system to create functional mimics of ECM proteins such as fibronectin 13 and laminin 14 , as well as growth factors such as BMP2 15 and VEGF 14 .…”

Cell–Material Interactions 2022

“…This lays an important basis for the selection of suitable cell-material combinations for treating articular cartilage lesions. Guiding the behavior of endothelial cells by spatially controlled adhesion (based on the peptidic YISGR sequence) and angiogenic functional motifs (vascular endothelial growth factor, VEGF) was studied by Le Bao et al [ 4 ]. For this purpose, they developed a locally controlled attachment of molecules applied to hydrogels using electrostatic interactions.…”