Modulation of angiogenic potential of collagen matrices by covalent incorporation of heparin and loading with vascular endothelial growth factor

One of the prominent shortcomings of matrices for tissue engineering is their poor ability to support angiogenesis. We report here on experiments to enhance the angiogenic properties of collagen matrices. Our aim is to achieve this goal by covalently incorporating heparin into collagen matrices and by physically immobilizing angiogenic vascular endothelial growth factor (VEGF) to the heparin. The immobilization of heparin was performed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Carboxyl groups on the heparin are activated to succinimidyl esters, which react with amino functions on the collagen to zero length cross-links. This modification leads--in addition to the incorporation of heparin--to gross changes in in vitro degradation behavior and water-binding capacity. As a first approach to testing angiogenic capabilities, endothelial cells were exposed to nonmodified and heparinized collagen matrices. This exposure leads to an increase in endothelial cell proliferation. The increase can be further enhanced by loading the (heparinized) collagen matrices with VEGF. Evaluation of the angiogenic potential of heparinized matrices was further investigated by exposing them to the chorioallantoic membrane of chicken embryos and to the subcutaneous tissue of rats. Both approaches show that heparinized matrices have substantially increased angiogenic potential. In particular, the loading of heparinized matrices with VEGF invokes a further increase in angiogenic potential. It is apparent that the physical binding of VEGF to heparin allows for a release that is beneficial to angiogenesis. By varying the heparin and EDC/NHS concentrations during the modification process and by varying the loading with VEGF, the angiogenic potential as well as the degradation behavior can be adapted to obtain matrices that fulfill specific angiogenic requirements in the field of tissue engineering.     

The impact of proteinase-induced matrix degradation on the release of VEGF from heparinized collagen matrices

The in vivo application of engineered matrices in human wound healing processes is often hampered by the slow rate of vascularization. Therefore much research is directed towards enhancing the angiogenic properties of such matrices. One approach for enhancing the vascularization is the incorporation of angiogenic growth factors. Recently, we and others have reported on immobilizing such factors into collagen matrices either by covalent attachment or by physical binding to covalently incorporated heparin. Especially the latter procedure has been shown to lead to substantial increase rates in vascularization in in vivo experiments. The increases have been proposed to depend on the sustained release of the incorporated angiogenic growth factors from the heparinized collagen matrices. In this paper, we report on investigations to study the release of vascular endothelial growth factor (VEGF) from collagen matrices under conditions which mimic potential in vivo situations. Relevant proteinase concentrations were deduced from in vitro experiments in which we evaluated the secretion of selected matrix metalloproteinases from fibroblasts in contact with collagen. The release of VEGF from non-modified, cross-linked and heparinized collagen matrices in the absence and in the presence of varying concentrations of proteinases was then determined by ELISA and liquid scintillation counting. The release behaviour appears to be controlled by both the presence of heparin and the levels of proteinases applied. Experiments with matrices containing radioactively labelled heparin suggest that VEGF release results from the consecutive and simultaneous release of three species of VEGF molecules that differ in their binding affinities to the differently modified collagen matrices. The species binding specifically to heparin most likely accounts for the previously observed increases in angiogenic potential between loading VEGF to non-heparinized and heparinized collagen matrices.     

Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

Effects of modified collagen matrices on human umbilical vein endothelial cells

The most commonly used biomaterials fail to ensure sufficient angiogenesis for fast in vivo incorporation. This results in central necrosis and consequent infection. One way of obtaining a high angiogenic response is the application of vascular endothelial growth factor (VEGF). To obtain a sustained release of these cytokines, heparin was incorporated into collagen matrices using 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS). The functionality of the heparinized collagen matrices was then enhanced by immobilization of VEGF via its heparin-binding domain. This procedure changed in vitro degradation behavior and water-binding capacity. Accelerated endothelial cell proliferation was also achieved. A range of different heparin and EDC/NHS concentrations in combination with VEGF induced variation in endothelial cell growth and tubulogenic formation. Polymerized collagen scaffolds presented biointeractive systems with integrated angiogenic activity. This may become a useful tool in the clinical therapy of disorders connected with wound healing.     

Influence of modified extracellular matrices on TI6AL4V implants on binding and release of VEGF

Besides osteoconductive and osteoinductive signals, angiogenesis plays a crucial role in bone development and regeneration and consequently in the integration of implants. Therefore we investigated in this study the binding and release behaviour of vascular endothelial growth factor (VEGF) from Ti6Al4V surfaces coated with 3-dimensional collageneous matrices, some additionally modified with heparin. The heparin was incorporated using different methods: a) adsorptive immobilization b) crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) or c) incorporating during self-assembly of fibrils followed by cross-linking. For unmodified samples, maximum VEGF adsorption was reached with 85 ng VEGF/cm(2). On all 3d-collagen coated surfaces studied (with or without heparin), no saturation could be observed in the range of 0-256 ng VEGF/cm(2).Improved release kinetics were observed for the modified coatings. The initial burst of VEGF within the first 24 h was diminished. From the third day of delivery heparinized matrices showed a higher release of VEGF than the pure collagen matrix and the unmodified reference surface, respectively. In vitro, the proliferation of human dermal microvascular endothelial cells was increased with released VEGF from all investigated samples compared to a VEGF-free control. After 7 days highest increases in cell numbers were observed with solutions from heparinized matrices.It is concluded that functionalization of Ti6Al4V surfaces with heparinized collageneous matrices and VEGF leads to advantageous properties concerning the impact on angiogenesis and thus may improve bone regeneration in the microenvironment of implants.     

Sustaining neovascularization of a scaffold through staged release of vascular endothelial growth factor-A and platelet-derived growth factor-BB

Tissue regeneration into a three-dimensional scaffold requires the stimulation of blood vessel ingrowth. We have employed a freely interconnecting porous scaffold developed by us to determine the utility of a covalently bound heparin surface coating for the delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) in vivo. The heparin surface was shown to release VEGF far more rapidly than PDGF-BB in vitro (VEGF: 75?ng/h for 24?h; PDGF-BB: 86?pg/h for >7 days). In rat subcutaneous implants, at 10 days the heparin surface alone increased vessel ingrowth substantially (p<0.05 vs. unmodified scaffold), release of VEGF resulted in a further increase (p<0.05 vs. heparinized scaffold), whereas PDGF-BB had no additional effect. The increase induced by the combination of growth factors was similar to VEGF alone. After 2 months, PDGF-BB, but not VEGF delivery, resulted in a substantial increase in vascularization above that induced by heparin (p<0.05). At the longer time point the combination of growth factors was similar to PDGF-BB. However, only the combination of growth factors significantly elevated the number of ingrowing arterioles (p<0.05 vs. heparinized scaffold). Thus, the covalent modification of a porous scaffold with heparin allows for the differential release of VEGF and PDGF-BB that results in both a rapid and sustained increase in scaffold vascularization.     

Microporous collagen spheres produced via thermally induced phase separation for tissue regeneration

Collagen is an abundant protein found in the extracellular matrix of many tissues. Due to its biocompatibility, it is a potentially ideal biomaterial for many tissue engineering applications. However, harvested collagen often requires restructuring into a three-dimensional matrix to facilitate applications such as implantation into poorly accessible tissue cavities. The aim of the current study was to produce a conformable collagen-based scaffold material capable of supporting tissue regeneration for use in wound repair applications. Microporous collagen spheres were prepared using a thermally induced phase separation (TIPS) technique and their biocompatibility was assessed. The collagen spheres were successfully cross-linked with glutaraldehyde vapour, rendering them mechanically more stable. When cultured with myofibroblasts the collagen spheres stimulated a prolonged significant increase in secretion of the angiogenic growth factor, vascular endothelial growth factor (VEGF), compared with cells alone. Control polycaprolactone (PCL) spheres failed to stimulate a similar prolonged increase in VEGF secretion. An enhanced angiogenic effect was also seen in vivo using the chick embryo chorioallantoic membrane assay, where a significant increase in the number of blood vessels converging towards collagen spheres was observed compared with control PCL spheres. The results from this study indicate that microporous collagen spheres produced using TIPS are biologically active and could offer a novel conformable scaffold for tissue regeneration in poorly accessible wounds.     

黄芪-脉络宁胶原促进体外胸主动脉内皮细胞血管新生的实验研究

目的:探讨黄芪-脉络宁胶原对血管新生的细胞学影响及其可能机制,为其应用于临床提供理论依据。方法:应用增殖、迁移、管腔形成实验观察黄芪-脉络宁胶原对大鼠胸主动脉内皮细胞在血管新生各阶段的作用,用Western Bloting实验检测大鼠胸主动脉血管内皮细胞生长因子(VEGF)的表达率。结果:黄芪-脉络宁胶原能明显促进大鼠胸主动脉内皮细胞的增殖率(与对照组比较,增加了90.22%,P<0.01)、迁移率(与对照组比较增加了211.43%,P<0.01)、管腔形成数(是对照组的2.46倍,P<0.01),同时刺激VEGF蛋白表达比对照组增加63.8%(P<0.01)。结论:黄芪-脉络宁胶原能明显促进内皮细胞增殖、迁移、管腔形成,具有显著促体外血管新生作用,其机制可能与增加VEGF蛋白的表达有关。     

In vivo biocompatibility of carbodiimide-crosslinked collagen matrices: Effects of crosslink density, heparin immobilization, and bFGF loading

Collagen matrices, crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (E) and N-hydroxysuccinimide (N), were previously developed as a substrate for endothelial cell seeding of small-diameter vascular grafts. In the present study, the biocompatibility of various EN-crosslinked collagen matrices was evaluated following subcutaneous implantation in rats for periods up to 10 weeks. The effects of the crosslink density, referred to as the number of free primary amino groups per 1,000 amino acid residues (EN10, EN14, EN18, or EN22), the amount of heparin immobilized to EN14, and the effect of preloading heparinized EN14 with basic fibroblast growth factor (bFGF) on the induced tissue reaction were studied. EN-crosslinked collagen was biocompatible at both early and late time intervals, and matrices with high crosslink densities (i.e., EN14, EN10) especially demonstrated a significantly decreased antigenic response when compared to noncrosslinked collagen. Furthermore, increased crosslinking resulted in a decreased degradation rate. Immobilization of heparin onto EN14 resulted in a similar to EN14 (thus without heparin) or somewhat reduced tissue reaction, but fibrin formation and vascularization were increased with increasing quantities of immobilized heparin. Matrices preloaded with bFGF also demonstrated good biocompatibility, especially in combination with higher amounts of immobilized heparin. The latter matrices [EN14 with high heparin and bFGF, thus EN14-H (0.4)F and EN14-H(1.0)F] demonstrated significantly increased vascularization for periods up to 3 weeks. Neither heparin immobilization nor bFGF preloading induced an increased antigenic response. It is concluded that the results of this study justify further evaluation of bFGF preloaded, heparin immobilized EN14 collagen, as a matrix for endothelial cell seeding in experimental animals.     

Delivery of VEGF using collagen-coated polycaprolactone scaffolds stimulates angiogenesis

Establishing sufficient vascularization in scaffold remains a challenge for tissue-engineering. To improve angiogenesis, we incorporated vascular endothelial growth factor (VEGF) in collagen-coating over the porous polycaprolactone (PCL) scaffolds. The release kinetics of loaded VEGF from collagen-coated PCL (col-PCL) scaffolds was same as from scaffolds without the collagen. The bioactivity of VEGF delivered by the col-PCL scaffolds was confirmed by human umbilical vein endothelial cell (HUVEC) proliferation and chorioallantoic membrane (CAM) assay. The col-PCL scaffolds were implanted subcutaneously in mice for 7 and 14 days. At day 7, vascularization within scaffolds loaded with VEGF was superior to that in the scaffolds without VEGF. However, the vessel connectivity to host circulatory system was incomplete and restricted to the scaffold edges. At day 14, blood vessels in scaffolds reached density similar to the subcutaneous tissue and were perfusable throughout the implant thickness. Prewashing the scaffolds with saline to remove the unbound growth factor decreased the initial burst release and sustained the VEGF-mediated angiogenesis in vivo. In conclusion, our study demonstrates that physically adsorbed VEGF stimulated angiogenesis in collagen-coated PCL scaffolds.     

Mechanisms of vascular endothelial growth factor-induced pathfinding by endothelial sprouts in biomaterials

A critical property of biomaterials for use in regenerative medicine applications is the ability to promote angiogenesis, the formation of new vascular networks, to support regenerating tissues. Recent studies have demonstrated that a complex interplay exists between biomechanical and biochemical regulators of endothelial cell sprouting, an early step in angiogenesis. Here, we use a microfluidic platform to study the pathfinding behaviors induced by various stable vascular endothelial growth factor (VEGF) gradients during sprouting morphogenesis within biomaterials. Quantitative, time-lapse analysis of endothelial sprouting demonstrated that the ability of VEGF to regulate sprout orientation during several stages of sprouting morphogenesis (initiation, elongation, and turning navigation) was biomaterial dependent. Identical VEGF gradients induced different types of coordinated cell movements depending on the density of the surrounding collagen/fibronectin matrix. In denser matrices, sprouts were more likely to have an initial orientation aligned parallel to the VEGF gradient. In contrast, in less dense matrices, sprouts were more likely to initially misalign with the VEGF gradient; however, these sprouts underwent significant turning and navigation to eventually reorient to be parallel to the VEGF gradient. These less dense matrices required shallower VEGF gradients and demonstrated lower activating VEGF thresholds to induce proper sprout alignment and pathfinding. These results encourage the future use of microfluidic platforms to probe fundamental aspects of matrix effects on angiogenesis, to screen biomaterials for angiogenic potential, and to design ex vivo tissues with aligned vascular networks.     

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

A key challenge in engineering functional tissues in vitro is the limited transport capacity of oxygen and nutrients into the tissue. Inducing vascularization within engineered tissues is a key strategy to improving their survival in vitro and in vivo. The presence of vascular endothelial growth factor (VEGF) in a three-dimensional porous collagen scaffold may provide a useful strategy to promote vascularization of the engineered tissue in a controlled manner. To this end, we investigated whether immobilized VEGF could promote the invasion and assembly of endothelial cells (ECs) into the collagen scaffolds. We conjugated VEGF onto collagen scaffolds using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride chemistry, and measured the concentrations of immobilized VEGF in collagen scaffolds by direct VEGF enzyme-linked immunosorbent assay. We demonstrated that immobilized VEGF (relative to soluble VEGF) promoted the penetration and proliferation of ECs in the collagen scaffold, based on results of cell density analysis in histological sections, immunohistochemistry, XTT proliferation assay, glucose consumption and lactate production. Furthermore, we observed increased viability of ECs cultured in scaffolds with immobilized VEGF relative to soluble VEGF. This research demonstrates that immobilization of VEGF is a useful strategy to promote the invasion and proliferation of ECs into a scaffold, which may in turn lead to a vascularized scaffold.     

Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2)

Tissue integration between a tissue-engineered corneal equivalent and the host eye is of critical importance in ensuring long-term implant success. A novel dendrimer cross-linked collagen scaffold has previously shown good corneal epithelial cell compatibility in vitro particularly when the highly functional dendrimer cross-linkers were functionalized to introduce additional biological groups. Herein we investigated heparinization of these materials and their potential to facilitate the delivery of basic fibroblast growth factor (FGF-2) in an active form, ultimately for use as a corneal tissue scaffold. Collagen gels cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry, and varying amounts of polypropyleneimine octaamine generation 2 (G2) dendimer and heparin were synthesized. Swelling studies and differential scanning calorimetry characterization indicated higher gel stability with the introduction of dendrimer cross-linking, which was not compromised by heparin integration. Dendrimer cross-linked gels with or without heparin gave multiple denaturation peaks, as did the heparinized EDC gels. This is thought to be the result of the heterogeneous cross-linking possible between collagen, the dendrimer and heparin. Release of FGF-2 from collagen gels showed typical first-order kinetics, with an initial burst followed by a prolonged gradual release. Heparinized dendrimer cross-linked gels released approx. 40% of the growth factor over a 2-week period, with significance maintenance of growth factor activity. Incorporation of heparin resulted in somewhat prolonged release from these systems.     

Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2)

Tissue integration between a tissue-engineered corneal equivalent and the host eye is of critical importance in ensuring long-term implant success. A novel dendrimer cross-linked collagen scaffold has previously shown good corneal epithelial cell compatibility in vitro particularly when the highly functional dendrimer cross-linkers were functionalized to introduce additional biological groups. Herein we investigated heparinization of these materials and their potential to facilitate the delivery of basic fibroblast growth factor (FGF-2) in an active form, ultimately for use as a corneal tissue scaffold. Collagen gels cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry, and varying amounts of polypropyleneimine octaamine generation 2 (G2) dendimer and heparin were synthesized. Swelling studies and differential scanning calorimetry characterization indicated higher gel stability with the introduction of dendrimer cross-linking, which was not compromised by heparin integration. Dendrimer cross-linked gels with or without heparin gave multiple denaturation peaks, as did the heparinized EDC gels. This is thought to be the result of the heterogeneous cross-linking possible between collagen, the dendrimer and heparin. Release of FGF-2 from collagen gels showed typical first-order kinetics, with an initial burst followed by a prolonged gradual release. Heparinized dendrimer cross-linked gels released approx. 40% of the growth factor over a 2-week period, with significance maintenance of growth factor activity. Incorporation of heparin resulted in somewhat prolonged release from these systems.     

Preparation and characterization of a collagen/chitosan/heparin matrix for an implantable bioartificial liver

A new type of collagen/chitosan/heparin matrix, fabricated by gelation of collagen/ chitosan with heparin sodium containing ammonia, was produced to construct livers by tissue engineering and regenerative engineering. The obtained collagen/chitosan/heparin matrix was found to be highly porous, swelled rapidly in PBS solution and was stable in vitro for at least 60 days in collagenase/lysozyme containing buffered aqueous solution (PBS, pH 7.4) at 37 degrees C. The collagen/chitosan/heparin matrix resulted in a superior blood compatibility compared to the ammonia-treated collagen and collagen/chitosan matrices. The morphology and behavior of the cells on the collagen/chitosan/heparin membrane were found to be similar to those on the collagen membrane but different from those on the collagen/chitosan membrane. Hepatocytes cultured on the collagen/chitosan/heparin matrices exhibited highest urea and triglyceride secretion functions 25 days post seeding. These results suggest that this collagen/chitosan/heparin matrix is a potential candidate for liver tissue engineering.     

The enhancement of VEGF-mediated angiogenesis by polycaprolactone scaffolds with surface cross-linked heparin

This study investigates the effect of surface cross-linked heparin on vascular endothelial growth factor (VEGF)-mediated angiogenesis in porous polycaprolactone (PCL) scaffolds in vivo. We tested the hypothesis that VEGF delivered by scaffolds coated with a sub-micron thick layer of immobilized heparin would accelerate angiogenesis. The bioactivity of retained VEGF was confirmed by its phosphorylation of VEGF receptor-2. After 7 and 14 days of subcutaneous implantation in mice, the heparin-PCL scaffolds loaded with VEGF displayed significantly higher infiltration of blood vessels which traversed the entire scaffold thickness (2 mm). The stability and function of the newly formed vessels were confirmed by smooth muscle cell coverage and vessel perfusability, respectively. The contribution of individual components was assessed by varying the VEGF dose and heparin thickness. Prolonging the cross-linking reaction on PCL scaffolds resulted in higher heparin content, thicker heparin layer, and higher VEGF retention. While a dose dependent angiogenic response was observed with VEGF, higher amount of cross-linked heparin did not translate into additional improvement in angiogenesis for a given dose of VEGF. The synergism of immobilized heparin and VEGF in stimulating angiogenesis was observed in vivo.     

Preparation and characterization of a collagen/chitosan/heparin matrix for an implantable bioartificial liver

A new type of collagen/chitosan/heparin matrix, fabricated by gelation of collagen/chitosan with heparin sodium containing ammonia, was produced to construct livers by tissue engineering and regenerative engineering. The obtained collagen/chitosan/heparin matrix was found to be highly porous, swelled rapidly in PBS solution and was stable in vitro for at least 60 days in collagenase/lysozyme containing buffered aqueous solution (PBS, pH 7.4) at 37°C. The collagen/chitosan/heparin matrix resulted in a superior blood compatibility compared to the ammonia-treated collagen and collagen/chitosan matrices. The morphology and behavior of the cells on the collagen/chitosan/heparin membrane were found to be similar to those on the collagen membrane but different from those on the collagen/chitosan membrane. Hepatocytes cultured on the collagen/chitosan/heparin matrices exhibited highest urea and triglyceride secretion functions 25 days post seeding. These results suggest that this collagen/chitosan/heparin matrix is a potential candidate for liver tissue engineering.     

Heparin modification of calcium phosphate bone cements for VEGF functionalization

A promising strategy to promote angiogenesis within an engineered tissue is the local and sustained delivery of an angiogenic factor by the substitute itself. Recently, we reported on functionalization of Biocement D (BioD) and several modifications of this calcium phosphate bone cement with vascular endothelial growth factor (VEGF). Maintenance of biological activity of VEGF after release from the cement was improved by modification of BioD with mineralized collagen type I (BioD/coll). However, BioD/coll composites showed a higher initial burst of VEGF release than do the unmodified BioD. In the present study, VEGF release from BioD/coll composites modified with different amounts of heparin was investigated. We found a distinct reduction of the initial burst of release by adding heparin in a concentration-dependent manner. Moreover, the heparin modification had a positive impact on the biological activity of released VEGF. An advancement of biological properties of BioD/coll by addition of heparin was further shown by improved adhesion of endothelial cells on the cement surface. Characterization of material properties of the heparin-modified BioD/coll composites revealed a finer microstructure with smaller HA-particles and a higher specific surface area than heparin-free BioD/coll. However, higher amounts of heparin resulted in a reduced compressive strength. The rheological properties of these cement pastes have been found to be favorable for good handling particularly with regard to their clinical application.     

The effect of vascular endothelial growth factor (VEGF) presentation within fibrin matrices on endothelial cell branching

Vascular endothelial growth factor (VEGF) has been extensively investigated to promote vascularization at damaged or diseased sites and in tissue implants. Here we are interested in determining if the manner in which VEGF is presented from a scaffold to endothelial cells influences the architecture of the blood vessels formed. We bound VEGF to nanoparticles and placed these nanoparticles inside fibrin hydrogels, which contained human umbilical vein endothelial cells (HUVECs) bound to cytodex beads. Fibroblast cells are plated on top of the fibrin gel to further mimic a physiologic environment. In addition, we used a chorioallantoic membrane (CAM) assay to determine the role of VEGF presentation on angiogenesis in?vivo. We tested VEGF bound in high density and low density to study differences between growth factor presentation in heterogeneous nanodomains and homogenous distribution. VEGF covalently bound to nanoparticles at high density led to an increase in HUVEC tube branching, thickness, and total vessel network length compared to soluble VEGF. While VEGF bound electrostatically exhibited no significant difference with covalently bound VEGF in the tube formation assay, this method failed to promote host vessel infiltration into the fibrin implant on the CAM. Together our data suggest that the mode of VEGF presentation to endothelial cells influences the vessel architecture and vascularization of implants in?vivo.