Hyaluronic Acid Hydrogels Support Cord-Like Structures from Endothelial Colony-Forming Cells

Abstract

The generation of functional vascular networks has the potential to improve treatment for vascular diseases and to facilitate successful organ transplantation. Endothelial colony-forming cells (ECFCs) have robust proliferative potential and can form vascular networks in vivo. ECFCs are recruited from a bone marrow niche to the site of vascularization, where cues from the extracellular matrix instigate vascular morphogenesis. Although this process has been elucidated using natural matrix, little is known about vascular morphogenesis by ECFCs in synthetic matrix, a xeno-free scaffold that can provide a more controllable and clinically relevant alternative for regenerative medicine. We sought to study hyaluronic acid (HA) hydrogels as three-dimensional scaffolds for capillary-like structure formation from ECFCs, and to determine the crucial parameters needed to design such synthetic scaffolds. We found that ECFCs express HA-specific receptors and that vascular endothelial growth factor stimulates hyaluronidase expression in ECFCs. Using a well-defined and controllable three-dimensional HA culture system, we were able to decouple the effect of matrix viscoelasticity from changes in adhesion peptide density. We determined that decreasing matrix viscoelasticity, which corresponds to a loose ultrastructure, significantly increases ECFC vascular tube length and area, and that the effect of local delivery of vascular endothelial growth factor within the hydrogel depends on the makeup of the synthetic environment. Collectively, these results set forth initial design criteria that need to be considered in developing vascularized tissue constructs.

Controlled activation of morphogenesis to generate a functional human microvasculature in a synthetic matrix

“…11,25,26 Synthesis of AHA hydrogels AHA hydrogels were prepared as was previously reported. 27 Briefly, we synthesized AHA using a 2-step protocol: (1) we synthesized the tetrabutylammonium salt of HA (HA-TBA) by reacting sodium hyaluronate (64 kDa; Lifecore Biomedical) with the highly acidic ion exchange resin Dowex-100 and neutralizing with 0.2M TBA-OH; (2) we coupled acrylic acid (2.5/equivalent [Eq]) and HA-TBA (1 Eq, repeat unit) in the presence of dimethylaminopyridine (DMAP; 0.075 Eq) and di-tert-butyl dicarbonate (1.5 Eq) in DMSO, followed by dialysis and lyophilization; we used the 1 H NMR spectrum to confirm the final percent modification of the AHA.…”

“…The Young modulus (substrate viscoelasticity) was calculated by E ϭ 2GЈ(1 ϩ ). Acrylated HA hydrogels are assumed to be incompressible 23,25,26 such that their Poisson ratios () approach 0.5 and the relationship becomes E ϭ 3GЈ. 28…”

“…ITGA2, ITGA5, ITGAV, ITGB1, ITGB3, Fn, HPRT1, and ␤-ACTIN, and samples were examined in triplicate, analyzed, and graphed (n ϭ 3) as we previously described. 25,26 …”

Harnessing developmental processes for vascular engineering and regeneration

“…Of these, VEGFs and their receptors (Flk and Flt) are considered to be master determinants of vasculogenesis and angiogenesis, as together they regulate the survival, differentiation, proliferation, morphogenesis and migration of ECs (Gentile et al, 2013;Hang et al, 2013;Nakayama et al, 2013). Given their crucial role in vascular development, genes encoding VEGFs have been widely used as therapeutic pro-angiogenic factors in clinical trials or to regulate vascular differentiation and morphogenesis to create vascular network structures in vitro (Hanjaya-Putra et al, 2010;Yee et al, 2011). PDGFs are also essential for vascular development, as they recruit mural cell precursors that promote vessel tightness and stabilization (Zhou et al, 2014).…”

“…HA can be used in hydrogel scaffolds in conjunction with RGD and MMP-sensitive peptides to support vascular morphogenesis and network formation Yee et al, 2011;Kusuma et al, 2013). ECFCs, which are progenitors of ECs, have been shown to undergo a wide range of coordinated morphogenic processes, including vacuole and lumen formation, as well as branching and sprouting, when placed within a synthetic HA hydrogel tailored with integrin α 5 β 1 -and α v β 3 -mediated cell adhesion domains, and MMP-sensitive sites .…”