The spatial presentation of immobilized extracellular matrix (ECM) cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP)-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP). Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10–12 hours in the more crosslinked regions to 4–6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.

Cell behavior is guided by the complex interplay of matrix mechanical properties as well as soluble and immobilized biochemical signals. The development of synthetic scaffolds that incorporate key functionalities of the native extracellular matrix (ECM) for support of cell proliferation and tissue regeneration requires that stiffness and immobilized concentrations of ECM signals within these biomaterials be tuned and optimized prior to in vitro and in vivo studies. A detailed experimental sensitivity analysis was conducted to identify the key polymerization conditions that result in significant changes in both elastic modulus and immobilized YRGDS within visible light photopolymerized poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Among the polymerization conditions investigated, single as well as simultaneous variations in N-vinylpyrrolidinone (NVP) and precursor concentrations of Acryl-PEG3400-YRGDS resulted in a broad range of hydrogel elastic modulus (81 – 1178 kPa) and YRGDS surface concentration (0.04 – 1.72 pmol/cm2). Increasing the YRGDS surface concentration enhanced fibroblast cell adhesion and proliferation for a given stiffness, while increases in hydrogel elastic modulus caused decreases in cell adhesion and increases in proliferation. The identification of key polymerization conditions is critical for the tuning and optimization of biomaterial properties and the controlled study of cell-substrate interactions.

The spatial presentation of soluble growth factors, immobilized extracellular matrix molecules, as well as matrix rigidity, plays an important role in directed and guided cell migration. Synthetic hydrogel scaffolds offer the ability to systematically introduce gradients of these factors contributing to our understanding of how the 3D arrangement of biochemical and mechanical cues influence cell behavior. Using a novel photopolymerization technique, perfusion-based frontal photopolymerization (PBFP), we have engineered poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with gradients of mechanical properties and immobilized biofunctionality. The controlled delivery of a buoyant photoinitiator, eosin Y, through a glass frit filter results in the formation and subsequent propagation of a polymer reaction front that is self-sustained and able to propagate through the monomeric mixture. Propagation of this front results in monomer depletion, leading to variations in cross-linking, as well as spatial gradients of elastic modulus and immobilized concentrations of the YRGDS cell adhesion ligand within PEGDA hydrogels. Furthermore, the magnitudes of the resulting gradients are controlled through alterations in polymerization conditions. Preliminary in vitro cell-culture studies demonstrate that the gradients generated stimulate directed 2D cell growth on the surface of PEGDA hydrogels. By day 14, fibroblast aggregates spread roughly twice as far in the direction parallel to the slope of the gradient as compared to the perpendicular direction. The presented technique has great potential in controlling gradients of mechanical properties and immobilized biofunctionality for directing and guiding 3D cell behavior within tissue-engineered scaffolds.

No abstract

Poly(ethylene glycol) diacrylate (PEGDA) hydrogels are extensively used as scaffolds in tissue engineering. The ability to spatially control hydrogel properties is critical for designing scaffolds that direct cell behavior and tissue regeneration. To this end, we have recently developed a polymerization technique, perfusion‐based frontal photopolymerization, to generate tunable gradients in PEG hydrogels. This study explores the effects of polymerization conditions on the velocity of the propagating front and its influence on gradients in hydrogel swelling. Alterations in photoinitiator perfusion rate result in the largest variations in frontal velocity and in the magnitude of the swelling gradient among all polymerization conditions investigated.