MMP-Sensitive PEG Diacrylate Hydrogels with Spatial Variations in Matrix Properties Stimulate Directional Vascular Sprout Formation

Abstract

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.

Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks

“…This results in morphological differences of HUVECs seeded on the hydrogel, with cells maintaining a round morphology in the softer region, and spreading out in the stiffer region [68]. In a different study, Turturro et al report a gradient RGD-functionalized PEG hydrogel prepared using perfusion-based frontal photopolymerization, with the elastic modulus decreasing from 3.2 to 0.62 kPa over a span of 10 mm [69]. The gradient results in a decrease in the overall amount of vascular invasion from co-culture aggregates of endothelial cells and smooth muscle cells, but in a clear increase in the anisotropy of the vascular structures, with an alignment parallel to the direction of the gradient ( Figure 3B).…”

“…This corresponds with the institution of a long-range VEGF gradient in the interstitial cells, indicating that local tension can shape the angiogenic microenvironment in tissues. [66], [69], [73], and [74], respectively. Abbreviations: RGD, Arg-GlyAsp; PEG, polyethylene glycol.…”

Harnessing developmental processes for vascular engineering and regeneration

“…These include cell-cell interactions (Stratman and Davis, 2012), cell interactions with the surrounding ECM through specific cell-adhesion molecules (Estrach et al, 2011) and cell signaling via secreted or ECM-sequestered GFs or cytokines (Stratman et al, 2011). Other physicochemical parameters include proteolytic matrix remodeling and matrix stiffness, which affect vascular morphogenesis and network formation Busnadiego et al, 2013;Turturro et al, 2013). Oxygen availability has also been shown to play a crucial role in early vascular development (Imanirad et al, 2014).…”

“…Various kinds of engineered materials can mimic the ECM in order to support vascular differentiation, morphogenesis and tube formation, with the aim of recapitulating the in vivo microenvironment of vascular formation. The engineered ECM-like scaffold provides a structural framework and is capable of presenting various physiologically relevant signals, including cell-adhesion sites and proteolytically degradable sites (Cuchiara et al, 2012;Hanjaya-Putra et al, 2012;Kusuma et al, 2013;Turturro et al, 2013). In this section, we discuss the application of fundamental theories of vascular development either to create engineered vasculatures in vitro, or to promote neovascularization in situ.…”

“…They then applied the hydrogel to a microfluidic system in order to mimic native vascular organization and function (Moon et al, 2010). PEG-based hydrogels tailored with RGD-and MMP-sensitive peptides have been widely used for supporting vascular network formation (Moon et al, 2009(Moon et al, , 2010Cuchiara et al, 2012;Turturro et al, 2013). The authors first encapsulated HUVECs and mesenchymal progenitors (10T1/2 cells) that exhibited a perivascular cell morphology within PEG hydrogels containing both RGD peptides and MMP-sensitive sites (Cuchiara et al, 2012).…”