Inspired by Nature: Hydrogels as Versatile Tools for Vascular Engineering

Abstract

Significance: Diseases related to vascular malfunction, hyper-vascularization, or lack of vascularization are among the leading causes of morbidity and mortality. Engineered, vascularized tissues as well as angiogenic growth factor-releasing hydrogels could replace defective tissues. Further, treatments and testing of novel vascular therapeutics will benefit significantly from models that allow for the study of vascularized tissues under physiological relevant in vitro conditions. Recent Advances: Inspired by fibrin, the provisional matrix during wound healing, naturally derived and synthetic hydrogel scaffolds have been developed for vascular engineering. Today, engineers and biologists use commercially available hydrogels to pre-vascularize tissues, to control the delivery of angiogenic growth factors, and to establish vascular diseases models. Critical Issue: For clinical translation, pre-vascularized tissue constructs must be sufficiently large and stable to substitute function-relevant tissue defects and integrate with host vascular perfusion. Moreover, the continuous integration of knowhow from basic vascular biology with innovative, tailorable materials and advanced manufacturing technologies is key to achieving near-physiological tissue models and new treatments to control vascularization. Future Directions: For transplantation, engineered tissues must comprise hierarchically organized vascular trees of different caliber and function. The development of novel vascularization-promoting or -inhibiting therapeutics will benefit from physiologically relevant vessel models. In addition, tissue models representing treatment-relevant vascular tissue functions will increase the capacity to screen for therapeutic compounds and will significantly reduce the need for animals for their validation.

Keywords: angiogenesis; blood vessel; engineering; growth factor; hydrogel; tissue model.

Martin Ehrbar, PhD

Figure 1.

Engineering neovascularization in hydrogels inspired by the template function of the ECM during blood vessel development and maturation. (A) The establishment of new blood capillaries occurs through vasculogenesis and angiogenesis in an angio-competent milieu generated by growth factors and the ECM. (B) Tools for vascular engineering derived from the natural processes of blood vessel development include vascular cells, growth factors, and ECM-inspired hydrogel scaffolds. Hydrogels can be engineered from natural ECM components or synthetic ECM analogues. (C) Hydrogel-based strategies to generate new functional vascular networks in vivo. Pre-vascularization follows the concept of engineering vascular networks within hydrogels in vitro by the application of endothelial cells that self-assemble into micro-capillary networks. On transplantation, pre-vascularized hydrogel constructs can anastomose to the host vasculature and become perfused. In an alternative strategy, the delivery of soluble, matrix-bound, or on-demand releasable angiogenic growth factors can promote the formation of new vessels in situ. 3D, three-dimensional; ANG-1/ANG-2, angiopoietin 1/2; ECM, extracellular matrix; FGF-2, fibroblast growth factor 2; HA, hyaluronic acid; PDGF-BB, platelet-derived growth factor BB; PEG, poly(ethylene glycol); SDF-1α, stromal cell-derived factor 1α; TGF-β, transforming growth factor-β To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 2.

Pre-vascularization of fibrin hydrogels leads to the rapid anastomosis of engineered micro-capillaries with the host vasculature. Micro-capillaries were engineered in vitro by the co-culture of human fibroblasts with HUVECs in fibrin hydrogels. Pre-vascularized tissue (A), 7 days in vitro pre-culture, was perfused by host blood cells more rapidly than non-pre-vascularized tissue (B), 1 day in vitro pre-culture, on implantation. (C) HUVEC-cultures in the absence of supporting fibroblasts do not form perfusable structures. (D-O) Histological tissue sections from 3 to 14 days post-implantation, with red blood cells being evident in the pre-vascularized tissue starting at day 5 post-implantation. Black arrowheads: lumen, unperfused, red arrowheads: perfused lumen, filled with red blood cells. Scale bars: 100 μm. Adapted figure from Chen et al. with re-print permission. HUVECs, human umbilical vein endothelial cells; IT, implant tissue; MT, mouse tissue To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 3.

In situ neovascularization by engineered, hydrogel-immobilized VEGF delivery. (A) Skin vascularization response to fibrin hydrogels containing no VEGF (control), soluble VEGF, or engineered, fibrin-immobilized VEGF. Although the immobilized VEGF outperforms soluble VEGF, the vascularization response decreases as the fibrin hydrogel becomes degraded over time. Figure re-print from Largo et al. (B) Improvement of fibrin-immobilized VEGF treatment by aprotinin engineered, long-lasting fibrin hydrogels. No VEGF (control) or engineered, fibrin-immobilized VEGF (0.5 and 5 μg/mL) were compared in a hindlimb ischemia mouse model. Fibrin hydrogels used for the delivery were stabilized by fibrin-immobilization of the fibrinolysis inhibitor aprotinin. Tissues analyzed 4 weeks after hydrogel delivery for endothelial cells (CD31, in red), pericytes (NG2, in green), and smooth-muscle cells (α-smooth muscle actin, in cyan, scale bar: 20 μm) or for microcirculation by Laser-Doppler Imaging of non-ischemic and ischemic limbs (left and right legs, respectively). Figure re-print from Sacchi et al. VEGF, vascular endothelial growth factor. *p < 0.05 vs. negative control, **p < 0.01 vs. negative control, ***p < 0.001 vs. negative control, δ p < 0.05 between 0.5 and 5 μg/mL conditions. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 4.

Perfused vasculature on-a-chip model as potential cancer and drug screening platform. (A) Vascular networks formed by co-culture of human ECFC-EC and human fibroblasts in fibrin hydrogels. Vascular networks inside the three tissue chambers connect to microfluidic channels and can be perfused (70 kDa fluorescein isothiocyanate-dextran). (B) VMTs. HCT116 cancer cells were embedded together with endothelial/fibroblast co-cultures into fibrin hydrogels and grown into microtumors surrounded by perfused vascular networks. Scale bars: (A, B) 100 μm; (C) 50 μm. (C) VMTs used for anti-cancer drug screening. The collapse of the tumor (vasculature) can be monitored on treatment with Food and Drug Administration-approved anti-cancer drugs. Adapted figure from Phan et al. with re-print permission. ECFC, endothelial colony forming cells; VMT, vascularized microtumor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 5.

Engineering of vascularized tissue models using hydrogels. Vascularized tissue models largely depend on engineered hydrogel-based, cell-instructive microenvironments. The culture of adult (stem) cells in these cell-instructive microenvironments enables the study of basic vascular biology under defined in vitro conditions. Engineering physiologically relevant human tissue and cancer models requires the integration of knowhow from vascular biology and biofabrication techniques. In the future, the use of defined three-dimensional culture systems together with patient derived cells will allow for the screening of therapeutics and the testing for treatment toward personalized medicine. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound