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Title

Gradient Hydrogels for Neovascularization of Engineered Tissues

Creator

He, Yusheng Jason

Date

2020

Description

The inability to induce extensive and perfusable microvasculature within complex engineered tissues that possess spatial variations in...
Show moreThe inability to induce extensive and perfusable microvasculature within complex engineered tissues that possess spatial variations in mechanical properties, physical architecture and biochemical composition remains as a major hurdle to their clinical translation. Biomaterial strategies focused on designing scaffolds with physiologically relevant gradients provide a promising means for elucidating 3D vascular cell responses to spatial and temporal variations in matrix properties. This work developed a cell-laden hydrogel platform with tunable decoupled and combined gradients of multiple matrix properties critical for maintenance of long term-vascular cell viability, adhesion, migration and invasion outgrowth to elucidate the impact of gradient matrix cues on 3D neovascularization in culture. This was achieved through the completion of three specific aims. First, a novel ascending frontal polymerization (AFP) technique was developed to generate gradient-based PEG hydrogel scaffolds with tunable individual and combined matrix gradients. Using programmable syringe pumps to control the delivery of precursors with distinct composition during crosslinking, we were able to generate gradient scaffolds with decoupled spatial variations in the immobilized concentration of the RGD cell adhesion peptide ligand and elastic modulus. Using this approach, the slope and magnitude of the imposed RGD gradients were readily manipulated without inducing variations in elastic modulus. Vascular spheroids inserted into gradient hydrogel scaffolds supported 3D vascular sprout formation, while the immobilized RGD gradient promoted an increase in sprout length towards the imposed gradient. Next, to create cell-laden scaffolds photopolymerization conditions were optimized to enable viable cell encapsulation during scaffold fabrication. To achieve this, an experimental sensitivity analysis combined with the design of experiments (DOE) was implemented to design isotropic hydrogel scaffolds with a broad range of matrix properties (elastic modulus, immobilized RGD and proteolytic degradation) that supported vascular sprouting in 3D culture. We examined the individual and interaction effects of each matrix property and demonstrated that an optimal combination associated with increases in immobilized RGD and proteolytic degradation of mediate synergistic enhancements in 3D vascular sprouting. Based on the findings from this in vitro study with isotropic hydrogel scaffolds, we designed scaffolds with 5 types of gradient combinations in immobilized RGD, stiffness and protease-sensitivity and explored the impact of spatial variations these matrix cues on vascular sprouting within the constructs in 3D culture. Specifically, we created hydrogel scaffolds with gradients in immobilized RGD with (1) steep and (2) shallow slopes, (3) gradients in elastic modulus, (4) gradients in protease-sensitivityand and (5) opposing gradients of RGD and modulus and concurrent gradients of protease sensitivity and RGD. By encapsulating vascular spheroids in different regions of each gradient scaffold, we observed spatial variations in total sprout length within all gradient scaffolds. We also found that RGD gradient and combined gradient scaffolds induced biased vascular sprouting toward increased RGD concentration and that biased sprouting was enhanced by gradient magnitude and slopes of immobilized RGD concentration. Conversely, directional sprouting responses diminished in scaffolds possessing opposing gradients in RGD (with concurrent gradients of degradation) and modulus. The presented work is the first to demonstrate the use of a cell-laden biomaterial platform to explore the impact of gradients in RGD, proteolytic degradation, and stiffness on vascular sprouting responses in 3D culture. The presented platform and findings of this thesis work hold great potential in the fields of tissue engineering specifically for prevascularization of complex tissues that possess spatial variations in mechanical properties, degradation rate and adhesion ligand composition to facilitate their regeneration.
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