Fibrin gel-immobilized VEGF and bFGF efficiently stimulate angiogenesis in the AV loop model

Abstract

The modulation of angiogenic processes in matrices is of great interest in tissue engineering. We assessed the angiogenic effects of fibrin-immobilized VEGF and bFGF in an arteriovenous loop (AVL) model in 22 AVLs created between the femoral artery and vein in rats. The loops were placed in isolation chambers and were embedded in 500 microL fibrin gel (FG) (group A) or in 500 microL FG loaded with 0.1 ng/microL VEGF and 0.1 ng/microL bFGF (group B). After two and four weeks specimens were explanted and investigated using histological, morphometrical, and ultramorphological [scanning electron microscope (SEM) of vascular corrosion replicas] techniques. In both groups, the AVL induced formation of densely vascularized connective tissue with differentiated and functional vessels inside the fibrin matrix. VEGF and bFGF induced significantly higher absolute and relative vascular density and a faster resorption of the fibrin matrix. SEM analysis in both groups revealed characteristics of an immature vascular bed, with a higher vascular density in group B. VEGF and bFGF efficiently stimulated sprouting of blood vessels in the AVL model. The implantation of vascular carriers into given growth factor-loaded matrix volumes may eventually allow efficient generation of axially vascularized, tissue-engineered composites.

Figure 1

The cylindrical chamber with rounded lid is made of heat-resistant medical-grade Teflon (inner diameter 10 mm, height 6 mm) (A). In the center of the chamber, four tubes consisting of medical plastic are placed to avoid a dislocation of the loop (B).

Figure 2

A. An arteriovenous loop is constructed between the left femoral artery and vein using a contralateral vein graft. B: The loop is placed in a custom-made Teflon isolation chamber and is embedded in 500 μL fibrin gel.

Figure 3

Hematoxylin-eosin staining of matrices explanted after two (A, B, E, F) and four (C, D, G, H) weeks. After two weeks vascular density was low in group A (without growth factors) (A, E) and considerably increased in group B (treated with growth factors) (B, F). After four weeks construct size was decreased in both groups (compare A, B with C, D). Compared to group A (C, G), however, the number of India ink-positive vessels was higher in the growth factor group B (D, H). Arrows indicate India Ink-perfused, patent parts of the AVL. Scale bars A-D = 1 mm, scale bars E-H = 50 μm.

Figure 4

CD31 Immunohistology of the specimens two weeks after implantation (group A). Functional vessels are stained red after two weeks. Specimens were injected with India ink injected before explantation. Scale bar = 50 μm.

Figure 5

Scanning electron microscopy analysis of vascular corrosion casts of the specimens two weeks after implantation. In all specimens the neocapillary network displayed characteristics of an immature vascular bed, such as absence of a distinct vascular hierarchy and vivid angiogenesis in terms of sprout formation. In comparison to group A (A) the density of the neocapillaries was higher in the treatment group B (B). (magnification x 500).

Figure 6

Morphometric analysis of construct size. There is a significantly decreased construct size in the growth factor (GF) group (group B) compared to group A (without growth factors) (p<0.05) after two weeks. Moreover, the construct size is considerably decreased after four weeks in group B compared to group A. In both groups, cross section area of the implants decreased between week two and four (P > 0.05).

Figure 7

Morphometric analysis of vascular density. In the growth factor (GF) group (group B), a significantly higher vascular density compared to group A (without growth factors) (p<0.05) is observed at two weeks. Similar results, without reaching statistical significance are seen in the four week groups (P > 0.05).

Figure 8

Morphometric analysis of absolute number of blood vessels. The absolute number of vessels per construct was significantly higher in group B compared to group A after two weeks (P > 0.05). The same trend was observed at four weeks without reaching statistical significance (P > 0.05).