Enhancing angiogenesis in collagen matrices by covalent incorporation of VEGF

Abstract

Since the survival of ingrowing cells in biomaterials for regenerative processes largely depends on the supply of nutrients and oxygen, angiogenesis plays an important role in the development of new materials for tissue engineering. In this study we investigated the possibility of enhancing the angiogenic properties of collagen matrices by covalent incorporation of the vascular endothelial growth factor (VEGF). In a previous paper we already reported the use of homo- and heterobifunctional cross-linking agents for modifying collagen matrices [1]. In the present work the angiogenic growth factor was linked to the collagen with the homobifunctional cross-linker disuccinimidyldisuccinatepolyethyleneglycol (SS-PEG-SS) in a two step procedure. The efficiency of the first reaction step-the reaction of SS-PEG-SS with VEGF--was evaluated by western blot analysis. After 10 minutes virtually all of the dimeric molecules VEGF were on average modified by conjugation with 1 cross-linking molecule. The biological activity of the conjugate was investigated by exposing endothelial cells to non-modified VEGF and to VEGF conjugated to the cross-linker. The conjugation only had a limited effect on the mitogenic activity of VEGF. We therefore applied the cross-linking reaction to the VEGF-collagen system. In a first approach the changes were evaluated by the in vitro exposure of HUVECs to non-modified matrices, to matrices in which the VEGF was simply admixed and to matrices in which the VEGF was covalently incorporated. The angiogenic properties were evaluated in vivo with the chorioallantois membrane model. In this assay the chorioallantois membrane of the chicken embryo was exposed to the same set of matrices. The covalent incorporation of VEGF has a small but significant effect both on the formation of microvessels in the chorioallantois membrane and the tissue ingrowth into the implant. The covalent incorporation of angiogenic growth factors may thus be considered as a promising approach for enhancing the angiogenic capabilities of collagen matrices. Also the cross-linking with the homobifunctional cross-linking agent has a positive effect on the angiogenic potential of the collagen matrices.

Vascular endothelial growth factor immobilized in collagen scaffold promotes penetration and proliferation of endothelial cells

“…These studies showed increased proliferation of ECs on VEGF-immobilized substrates. Koch and colleagues immobilized VEGF in 3D collagen matrices using a homobifunctional crosslinker, with the goal of enhancing angiogenesis in wound healing applications [41]. They demonstrated that ECs cultured in a 2D tissue culture plate showed increased proliferation upon exposure to VEGF-immobilized collagen matrices in vitro; furthermore, VEGF-immobilized collagen matrices enhanced angiogenesis of the surrounding environment upon implantation into the chorioallantoic membrane of the chicken embryo.…”

Vascularization of Engineered Tissues: Approaches to Promote Angiogenesis in Biomaterials

“…In another study, a potent angiogenic factor, VEGF, was covalently incorporated into collagen gels using homobifunctional crosslinking reagent directed to thiol groups in collagen and VEGF. When implanted on chicken chorioallantoic membrane, the collagen gels modified with VEGF enhanced capillary formation and tissue ingrowth [47]. VEGF also has been genetically modified to express N-terminal cysteine, and the recombinant protein was conjugated to fibronectin via thiol-directed bifunctional crosslinking reagent without loss in bioactivity [48].…”

Spatio-Temporal Modification of Collagen Scaffolds Mediated by Triple Helical Propensity

“…11,16 Synthetic collagen model systems, also known as collagen mimetic peptides (CMPs), have been useful in elucidating collagen structure and factors responsible for the stabilization of the triple helix. 9,[17][18][19] CMPs form a triple helical structure almost identical to that of natural collagens; however, unlike collagen molecules, CMPs are small (<3 KDa) and, therefore, exhibit reversible melting behaviors. 11,13,17 CMPs based on ProProGly or ProHypGly trimers have been widely studied and their melting temperatures (T m ) vary from 21 to 75 °C, depending on the molecular weight and amino acid composition.…”