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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.

The realization of three-dimensional (3D) degradable matrices which slowly release bio-active components represents a major challenge in the field of tissue engineering. In this paper we report on the usage of commercially available bifunctional agents for both the covalent coupling of proteins to and the cross-linking of collagen matrices. Proteins – horse radish peroxidase (HRP) was used as a model protein – were cross-linked with either a homobifunctional (disuccinimidyldisuccinatepolyethylene-glycol) or a heterobifunctional (N-hydroxysuccinimidylvinylsulfonepolyethyleneglycol) agent. In the case of the heterobifunctional cross-linking agent the collagen matrices were previously modified with succinimidylacetylthioacetate in order to introduce sulfhydryl groups. As compared with control experiments a 10-fold and 50-fold increase of immobilized proteins were achieved with the homobifunctional and heterobifunctional cross-linker resp. The HRP–PEG conjugates demonstrated a better long-term stability as compared to the non-treated HRP. The effects of the cross-linking agents and the thiolation reagent succinimidylacetylthio acetate on the in vitro degradation of the collagen matrices by collagenase were also investigated. In particular the reaction with succinimidylacetylthio acetate appears to offer interesting opportunities both for coupling active proteins and modulating the degradation times of collagen matrices.