Engineering Fibrous Hydrogels to Support 3D Endothelial Cell Networks

Microvascular networks (MVN) are essential for tissue survival and function, making them critical for physiologically relevant tissue models and engineered tissues. Current in vitro MVN that rely on natural matrices face limitations including batch variability, undefined composition, poor mechanical stability, and inadequate control over key matrix properties. Recent research shows matrix degradability and stress-relaxation are crucial for cellular self-organization. Building on these insights, we developed fiber-based hydrogels (FHs) with inherent porosity and cell-permissive properties that mimic native extracellular matrix architecture. Our central hypothesis is that the unique attributes of the FH system will facilitate cellular self-organization and sprouting of microvasculature, addressing limitations of both natural and synthetic matrices. This platform will support engineered vascularized systems for diverse biomedical applications, from microphysiological models to functional engineered tissues.

To create FHs, electrospun hydrogel fibers were first created from a solution of norbornene-modified polyethylene glycol and thiol-modified polyethylene glycol (PEG-SH). This solution also contained a thiolated cell adhesive ligand, RGD. The ratio of norbornenes to thiols in solution were stoichiometrically controlled to ensure excess thiols were present after fiber-crosslinking and RGD ligation for later FH stabilization. The solution was electrospun to fabricate dry fibers that are collected and UV-crosslinked, then rehydrated to form hydrogel fibers. To form the FH, hydrogel fibers were segmented by homogenization, suspended in PBS, and centrifuged at 10,000 rcf for 5 minutes. The resulting pellet was considered the fully packed, base material for the FH. To generate lower fiber densities within FHs, the fully packed material could be diluted volumetrically with medium. FHs were formed by including PEG-SH and photoinitiator within the fluid in the interstitial space of the packed hydrogel fibers and photocrosslinking the fibers to one another, with the PEG-SH serving as crosslinker between fiber surfaces. To incorporate endothelial cells within the 3D FH gels, HUVECs were included at 110⁷ cells/mL within FH prior to crosslinked to stabilize the scaffold. After 4 days cells were fixed and stained using immunofluorescence for visualization of human umbilical vein endothelial cells (HUVECs) within the scaffolds. Additionally, confocal imaging and rheology measurements confirmed the high porosity and permissive properties of FH.

We measured porosities in systems that were composed of 100% fully packed fibers and, 50% fully packed fibers/50% medium, and 20% fibers/80% medium. Observed porosities were ~20%, ~50% and ~80%, correspondingly. In situ photo-rheology demonstrated the formation of a stable FH scaffold after UV crosslinking. The high aspect ratio (fiber length: diameter ~22:1) of the fibers allowed for fiber entanglement leading to the system’s unique ability to have long range material interactions, which could be observed through yielding at increased strains compared to other granular systems. We observed cellular morphological response and expression of vascular protein markers in 3D cultures as a function of fiber density. Our results indicated that increasing porosity, or reduced fiber density, affected the formation of microvascular network formation in these materials. Immunofluorescent images demonstrated that HUVECs organized into microvascular networks over 4 days within FH. The 20% FH allowed for increased cellular interactions compared to those with the highest, fully packed, fiber concentrations. We ascribe the enhanced cellular interactivity to the high porosity allowing the cells to more easily move, proliferate, and organize within the scaffold.

Based on these results, we expect that by altering the fiber concentration of granular hydrogels, we can create an optimal system to study cells in vitro and understand what factors effect cellular behaviors within FH. Ongoing work is considering additional degrees of porosity and the effect of fiber modification with additional cell-instructive ligands. We expect that this novel biomaterial system can be engineered to present unique properties and support MVN formation critical to biomedical and tissue engineering applications.