To achieve the task of fabricating functional tissues, scaffold materials that can be sufficiently vascularized to mimic functionality and complexity of native tissues are yet to be developed. Here, we report development of synthetic, biomimetic hydrogels that allow the rapid formation of a stable and mature vascular network both in vitro and in vivo. Hydrogels were fabricated with integrin binding sites and protease-sensitive substrates to mimic the natural provisional extracellular matrices, and endothelial cells cultured in these hydrogels organized into stable, intricate network of capillarylike structures. The resulting structures were further stabilized by recruitment of mesenchymal progenitor cells that differentiated into smooth muscle cell lineage and deposited collagen IV and laminin in vitro. In addition, hydrogels transplanted into mouse cornea were infiltrated with host vasculature, resulting in extensive vascularization with functional blood vessels. These results indicate that these hydrogels may be useful for applications in basic biological research, tissue engineering, and regenerative medicine.

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The development and use of functional tissue-engineered products is currently limited by the challenge of incorporating microvasculature. To this end, we have investigated strategies to facilitate vascularization in scaffold materials, in this case poly(ethylene glycol) (PEG) hydrogels. These hydrogels are hydrophilic and resist protein adsorption and subsequent non-specific cell adhesion, but can be modified to contain cell-adhesive ligands and growth factors to support cell and tissue function. Additionally, the hydrogel matrix can include proteolytically degradable peptide sequences in the backbone of the structure to allow cells to control scaffold biodegradation, allowing three-dimensional migration. Vascular endothelial growth factor (VEGF), a potent angiogenic signal, and the cell-adhesive peptide RGDS were each covalently attached to PEG monoacrylate linkers. PEGylated RGDS and VEGF were then covalently immobilized in PEG-diacrylate (PEGDA) hydrogels in 2D and 3D. Immobilized VEGF increased endothelial cell tubulogenesis on the surface of non-degradable PEGDA hydrogels 4-fold compared to controls without the growth factor. Endothelial cell behavior in 3D collagenase-degradable hydrogels modified with RGDS and VEGF was observed using time-lapse confocal microscopy. Bulk immobilization of VEGF in 3D collagenase-degradable RGDS-modified hydrogels increased endothelial cell motility 14-fold and cell-cell connections 3-fold. Covalent incorporation of PEGylated VEGF in PEG hydrogels can be a useful tool to promote endothelial cell migration, cell-cell contact formation and tubulogenesis in an effort to produce vascularized tissue-engineered constructs.

Spontaneous formation of endothelial tubules was restricted to patterned micron-scale regions presenting cell adhesion ligands and angiogenic signaling protein on poly(ethylene glycol) hydrogels. Arginine-glycine-aspartic acid-serine (RGDS), an integrin ligand, and vascular endothelial growth factor (VEGF), a rate-limiting signaling protein involved in angiogenesis, were covalently bound through photopolymerization via laser scanning lithography to the surface of poly(ethylene glycol) hydrogels in patterned micron-scale regions. Endothelial cells cultured in this restricted environment underwent accelerated tubulogenesis, forming endothelial tubes within 2 days, whereas cells cultured on larger patterned areas remained spread and did not form tubules by day 2. Tubules formed in 2 days on RGDS and VEGF patterns were observed to possess lumens; however, tubule-like structures formed in 2 days on RGDS-only control patterns did not have observable lumens. Additionally, tubules that formed on restricted areas of RGDS and VEGF expressed more VEGF receptor 1, VEGF receptor 2, and ephA7 surface markers, in addition to higher expression of laminin, than cells remaining spread on wide patterned lines. This work reports spatial control and acceleration of endothelial tubule formation using biocompatible hydrogel materials to allow the formation of highly organized vascularized tissues.