Functional 3-D cardiac co-culture model using bioactive chitosan nanofiber scaffolds
Functional 3-D cardiac co-culture model using bioactive chitosan nanofiber scaffolds
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DOI：
10.1002/bit.24727
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发表时间：
2013-02-01
影响因子：
3.8
通讯作者：
Cho, Cheul H.
中科院分区：
文献类型：
--
作者：
Hussain, Ali;Collins, George;Cho, Cheul H.
采用细胞、生物材料和生物分子在体外生成三维（3-D）心肌组织样构建物是心脏组织再生、药物测试和组织工程应用中有前途的策略。尽管在这一领域取得了重大进展，目前的心脏组织模型还不能稳定地保持心肌细胞的功能特性，用于长期培养和治疗目的。本研究的目的是利用静电纺丝技术制备具有生物活性的三维壳聚糖支架，并在三维共培养模型中探索其对长期心脏功能的潜力。壳聚糖是一种天然多糖生物材料，具有生物相容性、生物可降解性、无毒和成本效益。电纺壳聚糖被用来提供结构支架，其特征在于规模和建筑相似的细胞外基质（ECM）在体内。壳聚糖纤维通过吸附作用被纤连蛋白包被，以增强细胞与纤维的粘附和向纤维间环境的迁移。从新生大鼠收获心室心肌细胞并在各种培养条件下研究（即，单培养物和共培养物）的活性和功能。使用α-肌节肌动蛋白（SM-肌动蛋白）和间隙连接蛋白连接蛋白-43（Cx43）的免疫荧光染色检查细胞形态和功能。扫描电子显微镜（SEM）和光学显微镜用于研究细胞形态，空间组织和收缩。用钙指示剂监测搏动心肌细胞的钙离子流量。结果表明，壳聚糖纳米纤维在长期细胞培养中保持其圆柱形形态，并在粘附分子纤连蛋白的存在下表现出良好的细胞附着和铺展。心肌细胞单一培养导致心肌细胞极性丧失和非连贯收缩岛。然而，心肌细胞-成纤维细胞共培养导致极化的心肌细胞形态，并保留其形态和功能，用于长期培养。Cx43在成纤维细胞共培养中的表达高于心肌细胞单培养和内皮细胞共培养。此外，成纤维细胞共培养物表现出涉及大型组织样细胞网络的同步收缩。据我们所知，这是第一次尝试测试壳聚糖支架作为3-D心脏共培养模型。我们的研究结果表明，壳聚糖纳米纤维可以作为一个潜在的支架，可以保持心脏的结构和功能。这些研究将提供有用的信息，开发一种策略，使我们能够产生工程化的3-D心脏组织结构，使用生物相容性和生物降解性壳聚糖支架的许多组织工程应用。Biotechnol. Bioeng. 2013; 110：637647. (c)2012 Wiley Periodicals，Inc.
The in vitro generation of a three-dimensional (3-D) myocardial tissue-like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long-term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3-D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long-term cardiac function in the 3-D co-culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non-toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono- and co-cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha-sarcomeric actin (SM-actin) and gap junction protein, Connexin-43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long-term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono-cultures resulted in loss of cardiomyocyte polarity and islands of non-coherent contractions. However, the cardiomyocyte-fibroblast co-cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long-term culture. The Cx43 expression in the fibroblast co-culture was higher than the cardiomyocytes mono-culture and endothelial cells co-culture. In addition, fibroblast co-cultures demonstrated synchronized contractions involving large tissue-like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3-D cardiac co-culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3-D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications. Biotechnol. Bioeng. 2013; 110: 637647. (c) 2012 Wiley Periodicals, Inc.