A comparative study on the effects of tumor necrosis factor-α (TNF-α), human angiogenic factor (h-AF) and basic fibroblast growth factor (bFGF) on the chorioallantoic membrane of the chick embryo

The chorioallantoic membrane (CAM) assay is a widely used bioassay for testing angiogenic activities. In the present study we compared the gross and micromorphological effects of three angiogenic factors applied in Elvax? carriers on the CAM: Tumor necrosis factor-α (TNF-α), human angiogenic factor (h-AF), and basic fibroblast growth factor (bFGF). Our question was whether the CAM responds to these factors which have very different actions with a stereotype or with a factor specific reaction. By microangiography and light microscopy, all positive reactions appeared as a spoke-wheel vascular pattern with a bundle of small capillary blood vessels in the center. These vessels were predominatly of a distended type in h-AF and TNF experiments, while narrower capillary vessels followed bFGF application. Chorioallantoic ectoderm and endoderm were thickened by cell accumulation and the mesenchymal stroma of the CAM was edematous and infiltrated with leucocytes in all three reactions. Additionally, bFGF experiments showed areas of densely arranged fibroblasts. Observations in vivo showed chorioallantoic tissue movements as a possible mechanism for the spokewheel vascular pattern. As compared with our results from studies of cytokinetics with bromodeoxyuridine, these current findings indicate that chemotaxis is responsible for the chorioallantoic angiogenic reaction rather than cellular proliferation. © 1992 Wiley-Liss, Inc.     

Nerve growth factor (NGF) promotes angiogenesis in the quail chorioallantoic membrane.

Angiogenesis, the formation of new blood vessels, is tightly regulated by growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). The authors hypothesize that nerve growth factor (NGF), a well known neurotrophin, may play a direct angiogenic role. To test this hypothesis, the authors measured the effects of NGF on the natural vascularization of the quail chorioallantoic membrane (CAM). The angiogenic effect of NGF was compared to that of human recombinant VEGF165 (rhVEGF) and basic FGF (rhbFGF). In comparison to phosphate-buffered saline-treated controls, NGFs from different biological sources (mouse, viper, and cobra) increased the rate of angiogenesis in a dose-dependent fashion from 0.5 to 5 microg. For quantitative morphometry, grayscale images of the blood vessels end points of the CAM arteries were binarized for visualization and skeletonized for quantization by fractal analysis. In mouse NGF-treated embryos the fractal dimension (Df), indicative of arterial vessel length and density, increased to 1.266 +/- 0.021 compared to 1.131 +/- 0.018 (p < .001) for control embryos. This effect was similar to that of 0.5 microg rhVEGF (1.290 +/- 0.021, p < .001) and 1.5 microg rhbFGF (1.264 +/- 0.028, p < .001). The mouse NGF-induced angiogenic effect was blocked by 1 microM K252a (1.149 +/- 0.018, p < .001), an antagonist of the NGF/trkA receptor, but not by 1 microM SU-5416 (1.263 +/- 0.029, p < .001), the VEGF/Flk1 receptor antagonist, indicating a direct, selective angiogenic effect of NGF via quail embryo trkA receptor activation. These results confirm previous observations that NGF has angiogenic activity and suggest that this neurotrophin may also play an important role in the cardiovascular system, besides its well-known effects in the nervous system. The angiogenic properties of NGF may be beneficial in engineering new blood vessels and for developing novel antiangiogenesis therapies for cancer.     

Angiogenesis in lipoma: An experimental study in the chick embryo chorioallantoic membrane

Lipoma is one of the most common benign mesenchymal tumors. Its ability to trigger an angiogenic response is a critical step for its growth. Because adipose tissue serves as an important conduit for the vasculature, it is conceivable that the angiogenic properties of this tissue may modulate the growth of the vasculature in a paracrine manner. We investigated in vivo the angiogenic potential of bioptic fragments of human lipoma by using the chick embryo chorioallantoic membrane (CAM), a useful model for such an investigation. The angiogenic response in pathological and control implants was assessed on histologic sections by a morphometric method, 96 h after grafting. Results showed that pathological samples were surrounded by numerous allantoic vessels with a radially arranged pattern around the implant. The vascular counts in the CAMs treated with lipoma implants were comparable to that of FGF-2. The role played in vasoproliferative response by angiogenic cytokines (FGF-2, VEGF) released by adipocytes, by endogenous cytokines, such as FGF-2, stored in the CAM extracellular matrix and by angiogenic growth factors released by perivascular mononuclear cells around the newly-formed blood vessels, were supported by this study.     

The effects of growth factors on the day 13 chorioallantoic membrane (CAM): a study of VEGF165 and PDGF-BB.

Summary The in vivo effects of two growth factors, VEGF₁₆₅ and PDGF-BB, were studied in the chick chorioallantoic membrane (CAM). The factors were airdried on Thermanox discs and the inverted discs were placed on the day-13 CAM for a period of 3 days. The specimens were then fixed, examined under a stereomicroscope and processed for semi- and ultrathin sectioning. VEGF₁₆₅ induces marked vascular growth. Many new blood vessels emerge from the precapillary arterioles, and a brush-like formation of vessels can be seen in this region. In the venous part of the vascular system, the formation of sinusoidal or lacunar vessels can be seen. Edema does not develop. PDGF-BB induces thickening of the CAM due to extracellular-matrix production and the proliferation or immigration of fibrocytes. These lie just beneath the ectodermal epithelium and are oriented parallel to it. Out of the four factors we have already studied (PDGF-BB, VEGF₁₆₅, Angiogenin, bFGF), only VEGF₁₆₅ specifically induces the growth of blood vessels.     

Histology and ultrastructure of the chorioallantoic membrane of the mallard duck (Anas platyrhynchos)

The histology and fine structure of the chorioallantoic membrane of the mallard duck (Anas platyrhynchos), and the density of vessels per millimeter of membrane were assessed between days 12 and 24 of incubation. Light and transmission electron microscopy of the chorioallantoic membrane of the mallard duck after various days of incubation was carried out. Blood vessels within the mesoderm were counted per millimeter of membrane by light microscopy (40x). The chorioallantoic membrane had three distinct layers from day 12 to 24 of incubation, the chorionic epithelium, the mesoderm, and the allantoic epithelium. After day 12, chorionic epithelium consisted of two layers of flattened, elongated epithelial cells interfaced by numerous desmosomes, and separated from the underlying mesoderm by a basement membrane. At this stage, the allantoic epithelium consisted of a single layer of flattened, overlapping cells. Blood capillaries were observed in the mesoderm close to the chorionic epithelium on days 12 and 13; by day 14, these capillaries were located within the chorionic epithelium, forming a capillary sinus. Between days 14 and 16, the chorion underwent cellular and cytological differentiation into three cell types: capillary covering cells, villus cavity cells, and less differentiated basal cells. The mesoderm was composed of a loose matrix of mesenchymal cells and collagen fibrils through which coursed blood and lymphatic vessels. The vascular density in the mesoderm increased rapidly from 4.2+/-0.6 vessels per mm (n = 12) on day 12 to a maximum of 9.4+/-0.3 vessels per mm (n = 15) by day 16. From day 16, the allantoic epithelium had two to three layers of elongated and overlapping cells. The luminal layer of allantoic epithelial cells had microvillus projections and varying numbers of membrane-bound dense vesicles at all stages from day 12 onward. The histologic and ultrastructural features of mallard duck chorioallantoic membrane from day 12 to 24 of incubation were very similar to those described in the chorioallantoic membrane of the chicken (Gallus gallus) from day 8 to 20 of incubation. Much of the information available concerning the CAM of the chicken also may apply to the CAM of the mallard, with timing adjusted to match the developmental time-frame recorded here.     

The chick embryo choriallantoic membrane as a bioassay for angiogenesis factors: reactions induced by carrier materials.

A variety of filter materials, sponges, and gels were placed on the chick chorioallantoic membrane (CAM), the reactions of it investigated and compared with those induced by natural egg materials (white eggshell membrane, coagulated albumen and yolk). Independently of the kind and nature of the naterials the CAM reacted nearly regularly underneath these diverse materials with a proliferation of ectodermal cells, fibroblasts, and blood vessels forming a highly capillarized granulation tissue. The area of the CAM surrounding the foreign materials frequently displayed an increase of small blood vessels macroscopically discernible and showing a radial arrangement (spoke-wheel-appearance according to FOLKMAN 1974). It is concluded that this type of vascular reaction cannot be considered as a characteristic feature for the action of a special tumour angiogenesis factor, since it can be induced by a variety of stimuli leading to an inflammatory reaction in the CAM. For the detection of special angiogenetic activities an objective quantification of the vessel reactions is necessary under consideration of the reactions induced by the mere presence of such foreign materials.     

Importance of vascular phenotype by basic fibroblast growth factor, and influence of the angiogenic factors basic fibroblast growth factor/fibroblast growth factor receptor-1 and ephrin-A1/EphA2 on melanoma progression

The expression of several angiogenic factors and receptors was examined in a series of vertical growth phase cutaneous melanomas using high-throughput tissue microarray technology and immunohistochemistry. The results were correlated with microvessel density, clinicopathological features, and patient survival. Expression of basic fibroblast growth factor (bFGF) was significantly associated with increased microvessel density. Also, we found an independent prognostic importance of vascular phenotype by endothelial cell expression of bFGF; cases with positive vessels had the best prognosis and these tumors revealed a low frequency of vascular invasion (14%) when compared with bFGF-negative vessels (47%). This bFGF-negative phenotype was significantly increased in metastatic lesions. Strong tumor cell expression of FLT-4, ephrin-A1, and EphA2 was associated with increased melanoma thickness, and ephrin-A1 staining was related to decreased survival (P = 0.039). Expression of EphA2 in tumor cells was associated with increased tumor cell proliferation (Ki-67 positivity), indicating possible autocrine growth stimulation. Thus, our findings indicate the presence of phenotypic diversity among tumor-associated vessels, and subgroups defined by bFGF expression may be of clinical importance. bFGF was associated with microvessel density, whereas the ephrin-A1/EphA2 pathway might also be important for tumor cell proliferation and patient survival.     

Engineered adipose tissue supplied by functional microvessels

A volume-persistent culture of adipose tissue under in vivo conditions can be achieved only by early vascularization after cell transplantation. Cotransplantation of autologous preadipocytes with endothelial cells may enable the early formation of a capillary network. Investigations were performed in vivo in a specially adapted chorioallantoic membrane (CAM) model. Fertilized White Leghorn eggs were incubated and opened on day 3 of incubation and human dermal microvascular endothelial cell (HDMVEC) spheroids and preadipocytes were transferred in a fibrin matrix to the CAM. On day 7 after incubation the composites were explanted and immunohistologically investigated. Numerous vessels consisting of HDMVECs could be detected and the lumena of these vessels were perfused by chick erythrocytes. These results show the formation of a capillary network consisting of transplanted HDMVECs. The microcirculation of chick erythrocytes in vessels consisting of human endothelial cells proves the continuity of a newly formed capillary system to the host vessel system. The experiments demonstrate the first patent connection of tissue-engineered microvessels in adipose tissue to a host vessel system without applying exogenous angiogenic growth factors or transient transfection. The cotransplantation of endothelial cell spheroids with angiogenic mesenchymal cells may lead to the engineering of complex three-dimensional implants.     

Basic fibroblast growth factor (bFGF, FGF-2) potentiates leukocyte recruitment to inflammation by enhancing endothelial adhesion molecule expression

Basic fibroblast growth factor (bFGF, FGF-2) is a potent angiogenic factor and endothelial cell mitogen. Although bFGF levels are increased in chronically inflamed tissue, its role in inflammation is unclear. We investigated the effect of bFGF on acute dermal inflammation and the recruitment of monocytes, T cells, and neutrophils. Leukocyte recruitment to inflamed sites was quantified with radiolabeled leukocytes. Intradermal injection of bFGF in rats did not induce leukocyte recruitment or inflammation. However, the recruitment of leukocytes to inflammation induced by tumor necrosis factor-alpha, interferon-gamma, C5a, or a delayed hypersensitivity reaction was enhanced by bFGF by 55 to 132% (P < 0.05). Either acute or prolonged bFGF treatment of dermal sites had this effect. The potentiating effect of bFGF on leukocyte recruitment was also seen in joints. There was no associated modulation of vascular permeability, blood flow, or angiogenesis in the sites by bFGF. However, the expression of the endothelial cell adhesion molecules (CAMs) for leukocytes, P-selectin, E-selectin, and ICAM-1, was significantly up-regulated in the inflamed tissue by bFGF, as quantified by radiolabeled anti-CAM antibody binding in vivo. Thus, although not directly proinflammatory, bFGF synergistically potentiates inflammatory mediator-induced leukocyte recruitment, at least in part, by enhancing CAM up-regulation on endothelium.     

Osteogenic protein-1, a bone morphogenetic protein, induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor-beta1

Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-betas themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF-beta superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein-1 (OP-1; BMP-7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP-1 at both ortho- and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF-beta1. The single application of relatively high doses of TGF-beta1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP-1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP-1) dose-dependent angiogenic response. The binary application of a relatively low dose of OP-1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF-beta1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis.     

Mechanism of the induction of angiogenesis by human neoplastic lymphoid tissue: studies on the chorioallantoic membrane (CAM) of the chick embryo

The angiogenic activity of various neoplastic and control tissues, cells and extracts has been tested on the chorioallantoic membrane of the chick (CAM). The vascular response was assessed macroscopically and also by histological examination. Angiogenesis was induced by a number of neoplastic implants the most potent being derived from Hodgkin's disease, histiocytic lymphoma or glioma tissue. Boiled tumour tissue was ineffective. Lymphocytes extracted from human lymphomas, activated normal peripheral blood lymphocytes and established lymphoid cell lines of neoplastic origin were generally effective in inducing neovascularisation through millipore membranes as were 90,000-100,000 MW fractions of human tumour tissue. In all cases examined histologically a mononuclear cell infiltrate in the CAM mesoderm accompanied a positive vascular response. These results implicate host monocytes in the generation of neovascularisation by neoplastic tissue.     

Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis

BACKGROUND: Angiogenesis is a prerequisite for airway remodeling in bronchial asthma. Several growth factors may play important roles in inflammation and angiogenesis through effects on inflammatory cell infiltration or neovascularization. OBJECTIVE: We sought to compare bronchial vascularity and expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin in bronchial biopsy specimens from asthmatic and healthy control subjects. METHODS: Bronchial biopsy specimens were obtained from 16 asthmatic subjects and 9 normal control subjects. The number of vessel profiles and the vascular area per unit area on a histologic section were estimated by using computerized image analysis after staining for type IV collagen in vessel walls. Numbers of VEGF+, bFGF+, and angiogenin+ cells were determined by means of immunoreactivity. RESULTS: The airways of asthmatic subjects had significantly more vessels (P < .05) and greater vascular area (P < .001) than that observed in control subjects. Asthmatic subjects exhibited higher VEGF and bFGF and angiogenin immunoreactivity in the submucosa than did control subjects (P < .001, respectively). Significant correlations were detected between the vascular area and the numbers of angiogenic factor-positive cells (VEGF: rs = 0.93, P < .001; bFGF: rs = 0.83, P < .001; angiogenin: rs = 0.88, P < .001) within the asthmatic airways. Furthermore, the degree of vascularity was inversely correlated with airway caliber and airway responsiveness. Colocalization analysis revealed that the angiogenic factor-positive cells were CD34+ cells, eosinophils, and macrophages. CONCLUSION: Our results suggest that increased vascularity of the bronchial mucosa in asthmatic subjects is closely related to the expression of angiogenic factors, which may then contribute to the pathogenesis of asthma.     

Quantitation and physiological characterization of angiogenic vessels in mice: effect of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and host microenvironment.

A prerequisite for the development of novel angiogenic and anti-angiogenic agents is the availability of routine in vivo assays that permit 1) repeated, long-term quantitation of angiogenesis and 2) physiological characterization of angiogenic vessels. We report here the development of such an assay in mice. Using this assay, we tested the hypothesis that the physiological properties of angiogenic vessels governed by the microenvironment and vessel origin rather than the initial angiogenic stimulus. Gels containing basic fibroblast growth factor (bFGF) or vascular endothelial growth (VEGF) were implanted in transparent windows in the dorsal skin or cranium of mice. Vessels could be continuously and non-invasively monitored and easily quantified for more than 5 weeks after gel implantation. Newly formed vessels were first visible on day 4 in the cranial window and day 10 in the dorsal skinfold chamber, respectively. The number of vessels was dependent on the dose of bFGF and VEGF. At 3000 ng/ml, bFGF- and VEGF-induced blood vessels had similar diameters, red blood cell velocities, and microvascular permeability to albumin. However, red blood cell velocities and microvascular permeability to albumin were higher in the cranial window than in the dorsal skinfold chamber. Leukocyte-endothelial interaction was nearly zero in both sites. Thus, newly grown microvessels resembled vessels of granulation and neoplastic tissue in many aspects. Their physiological properties were mainly determined by the microenvironment, whereas the initial angiogenic response was stimulated by growth factors.     

Osteogenic protein‐1, a bone morphogenetic protein,induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor‐β1

Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor‐β (TGF‐β) superfamily, including TGF‐βs themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF‐β superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein‐1 (OP‐1; BMP‐7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP‐1 at both ortho‐ and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF‐β1. The single application of relatively high doses of TGF‐β1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP‐1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP‐1) dose‐dependent angiogenic response. The binary application of a relatively low dose of OP‐1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF‐β1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis. Anat Rec 259:97‐107, 2000. © 2000 Wiley‐Liss, Inc.     

Endometrial angiogenesis throughout the human menstrual cycle

BACKGROUND: The timing and mechanisms of new blood vessel formation in the endometrium during the menstrual cycle are still largely unknown. In the present study we used the chick embryo chorioallantoic membrane (CAM) as an in-vivo assay for angiogenesis to assess the angiogenic potential of endometrium obtained at different stages of the menstrual cycle. METHODS: Endometrial fragments were explanted onto the CAM and, after 4 days of incubation, slides of the treated area were taken in ovo through a microscope for computerized image analysis. The vascular density index (VDI), a stereological estimate of vessel number and length, was obtained by counting the intersections of vessels with five concentric circles of a circular grid superimposed on the computerized image. RESULTS: We demonstrated that human endometrium has angiogenic potential throughout the menstrual cycle. Furthermore, there was a significant difference in angiogenic response between the stages of the menstrual cycle (P = 0.01). The VDIs of the early proliferative, early and late secretory stage were significantly higher than the VDI of the late proliferative phase. CONCLUSIONS: Elongation of existing vessels during the early proliferative phase as well as growth and coiling of the spiral vessels during the secretory phase may demand far higher angiogenic activity than outgrowth and maintenance of vessels during the late proliferative phase.     

Angiogenesis in the distal femoral chondroepiphysis of the rabbit during development of the secondary centre of ossification

In the developing chondroepiphyses of long bones, the avascular cartilaginous anlage is invaded by numerous blood vessels, through the process of angiogenesis. The objective of this study was to investigate the chronology of this vascular invasion with the spontaneous calcification of the cartilaginous epiphysis during development of the secondary ossification centre in the rabbit distal femur. The time-course of chondroepiphyseal vascular invasion was determined histologically and standardized for eight gestational and four postnatal intervals by plotting kit body mass against crown-rump length. Similarly, microcomputed tomography (micro-CT) helped to visualize calcification at those same gestational and postnatal intervals. To confirm the angiogenic nature of the avascular chondroepiphysis, such samples were assayed on the chick chorio-allantoic membrane (CAM). Neovascular outgrowths from the CAM were apparent 48 h following introduction of an 18-day (gestational) chondroepiphyseal sample. Chondroepiphyseal samples were assayed for the potent developmental angiogenic factors bFGF and VEGF, with the mRNA expression for both these mediators being confirmed using RT-PCR. As angiogenesis and calcification during chondroepiphyseal development occur in a defined tissue environment initially devoid of blood vessels and mineral, those processes provided a unique opportunity to study their progression without complication of injury-related inflammation or extant vasculature and mineral. Furthermore, the discovery of angiogenic, angiostatic or mineral-regulating mediators specific to developing connective tissue may prove useful for analysing the regulation of vascular and mineral pathogenesis in articular tissues.     

Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres

Site-specific delivery of angiogenic growth factors from tissue-engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would ensure cell survival and function. In the present article, we describe the construction of a novel porous alginate scaffold that incorporates tiny poly (lactic-co-glycolic acid) microspheres capable of controlling the release of angiogenic factors, such as basic fibroblast growth factor (bFGF). The microspheres are an integral part of the solid alginate matrix, and their incorporation does not affect the scaffold porosity or pore size. In vitro, bFGF was released from the porous composite scaffolds in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of cardiac fibroblasts. The controlled delivery of bFGF from the three-dimensional scaffolds accelerated the matrix vascularization after implantation on the mesenteric membrane in rat peritoneum. The number of penetrating capillaries into the bFGF-releasing scaffolds was nearly fourfold higher than into the control scaffolds (those incorporating microspheric BSA and heparin but not bFGF). At day 10 posttransplantation, capillary density in the composite scaffolds was 45 +/- 3/mm(2) and it increased to 70 +/- 7/mm(2) by day 21. The released bFGF induced the formation of large and matured blood vessels, as judged by the massive layer of mural cells surrounding the endothelial cells. The control over bFGF delivery and localizing its effects to areas of need, may aid in the wider application of bFGF in therapeutic angiogenesis as well as in tissue engineering.     

Sequential delivery of basic fibroblast growth factor and platelet-derived growth factor for angiogenesis

An externally regulated delivery model that permits temporal separation of multiple angiogenic factors was used for the delivery of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF). While bFGF plays a significant role in the sprouting of new capillaries, PDGF plays a role in the recruitment of mural cells, which stabilize neovessels. However, these two factors have been shown to inhibit each other, when presented together. Using the externally regulated model, sequential delivery of bFGF and PDGF led to not only increased endothelial cell migration, but also endothelial cell and vascular pericyte colocalization. More importantly, this delivery strategy was able to induce red blood cell-filled neovessels, suggesting integration of angiogenesis with the existing vasculature.