Choi Daheui, Komeda Misaki, Heo Jiwoong, Hong Jinkee, Matsusaki Michiya, Akashi Mitsuru
Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea.
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
ACS Biomater Sci Eng. 2018 May 14;4(5):1833-1842. doi: 10.1021/acsbiomaterials.8b00100. Epub 2018 Apr 2.
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 long-term 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.
为了增强细胞功能并维持生长因子的生物活性，人们已经开发出了具有化学固定生长因子的支架。然而，在生长因子结合过程中可能会出现细胞毒性和生长因子结构变形等问题，这会对细胞产生负面影响。因此，我们设计了一种方法，通过将主要的促血管生成生长因子血管内皮生长因子（VEGF）纳入多层膜中来改善生长因子的长期储存以及靶细胞进行血管生成的能力。我们使用纤连蛋白、肝素和单宁酸的层层组装技术，制备了一种含有VEGF的多层膜（VEGF膜），该膜光滑且稳定，能使细胞增殖增加至对照组细胞的2.5倍。此外，我们将VEGF膜直接制备在内皮细胞上，以最大限度地提高VEGF的功效，并且我们观察到由于多层膜的硬度，细胞漂浮在生长培养基中。尽管由于膜的硬度细胞难以附着在培养板表面，但细胞的存活和增殖得以维持。为了评估VEGF膜包被后内皮细胞进行血管生成前期的程度，我们检测了血管生成标志物CD31的表达。应用VEGF膜后CD31表达增加，细胞呈现出细长的形态，形成紧密连接以形成簇。因此，我们得出结论，含有VEGF的多层膜诱导内皮细胞进行血管生成前期，表明其在组织工程应用中的潜在用途。
