Vascular Endothelial Growth Factor Incorporated Multilayer Film Induces Preangiogenesis in Endothelial Cells

Scaffolds featuring chemically immobilized growth factors have been developed to enhance cellular functions and maintain growth factor bioactivity. However, problems including cytotoxicity and growth factor structural deformation may occur during growth factor conjugation, which can negatively affect the cells. Therefore, we designed a method to improve the longterm storage of growth factors and the target cells' ability to undergo angiogenesis by incorporating the primary proangiogenic growth factor vascular endothelial growth factor (VEGF) into a multilayer film. Using the layer-by-layer (LbL) assembly technique with fibronectin, heparin, and tannic acid, we prepared a VEGF-incorporated multilayer film (VEGF film) that is smooth and stable and increases cell proliferation by up to 2.5 times that of the control group cells. In addition, we prepared the VEGF film directly onto the endothelial cells to maximize the efficacy of VEGF, and we observed cells floating in the growth medium owing to the stiffness of the multilayer film. Although the cells were hard to attach to the culture plate surface due to film stiffness, cell survival and proliferation were maintained. To evaluate the extent of the preangiogenesis undertaken by the endothelial cells after VEGF film coating, we examined the expression of the angiogenic marker CD31. CD31 expression was increased after applying the VEGF film, and the cells adopted an elongated morphology, forming tight connections to make clusters. Thus, we conclude that the VEGF-incorporated multilayer film induced endothelial cells to undergo preangiogenesis, suggesting its potential use in tissue engineering applications.