Aim
To develop an in vitro three-dimensional (3-D) angiogenesis system to analyse the capillary sprouts induced in response to the concentration ranges of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and to quantify their synergistic activity.
Conclusion
A microcarrier-based in vitro three-dimensional angiogenesis model can be developed in fibrin. It offers a unique system for quantitative analysis of angiogenesis. Both bFGF and VEGF exert their angiogenic effects on HMVECs synergistically and in a dose-dependent manner.
Methods
Microcarriers (MCs) coated with human microvascular endothelial cells (HMVECs) were embedded in fibrin gel and cultured in 24-well plates with assay media. The growth factors bFGF, or VEGF, or both were added to the system. The wells (n = 8/group) were digitally photographed and the average length of capillary-like sprouts (ALS) from each microcarrier was quantitated.
Results
In aprotinin-stabilized fibrin matrix, human microvascular endothelial cells on the MCs invaded fibrin, forming sprouts and capillary networks with lumina. The angiogenic effects of bFGF or VEGF were dose-dependent in the range from 10 to 40 ng/mL. At d 1, 10 ng/mL of bFGF and VEGF induced angiogenesis with an ALS of 32.13 +/- 16.6 microm and 43.75 +/- 27.92 microm, respectively, which were significantly higher than that of the control (5.88 +/- 4.45 microm, P<0.01), and the differences became more significant as the time increased. In addition, the combination of 10 ng/mL of bFGF and VEGF each induced a more significant effect than the summed effects of bFGF (10 ng/mL) alone and VEGF (10 ng/mL) alone when analyzed using SPSS system for general linear model (GLM) (P = 0.011), and that also exceeded the effects by 20 ng/mL of either bFGF or VEGF.