体外动态环境培育组织工程心血管补片的特点  

In vitro dynamic culture condition for a tissue-engineered cardiovascular patch

作　　者：杨超 杜静 周钧 罗滨 孟春营 李芸 

机构地区：[1]暨南大学医学院第二附属医院,深圳市人民医院心胸外科,广东省深圳市518020

出　　处：《中国组织工程研究与临床康复》2008年第23期4414-4417,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省自然科学基金项目资助(06300946)~~

摘　　要：背景:搏动性生物反应器可以提供体外动态培育环境,给血管组织补片以血流剪切力和培养液的波动灌流,模拟体内血流动力学环境。目的:观察体外动态培育环境下肌成纤维细胞在组织工程心血管补片材料中的生长特点。设计、时间及地点:对比观察实验,于2007-01/12在暨南大学第二临床医学院中心实验室完成。材料:新生儿脐带组织为产妇自愿捐献,并对实验内容知情同意。聚-4-羟基丁酸酯为Tepha公司产品。方法:应用液氮冷冻保存的人脐带血管壁肌成纤维细胞种植在聚-4-羟基丁酸酯材料构建的补片支架材料上,体外动态培育制备组织工程心血管补片。动态组织培育组:细胞-组织材料复合物先体外静态培育14d,再置入生物反应器中继续动态组织培育7d。静态组织培育组:细胞-组织材料复合物体外静态培育21d。主要观察指标:①行苏木精-伊红染色对组织补片进行形态学分析。②补片组织扫描电镜观察其超微结构。③应用免疫组织化学对比分析样本中Ⅰ型胶原蛋白、alpha平滑肌肌动蛋白的合成情况。结果:苏木精-伊红染色显示动态培育的聚合材料表面完全被有序的层状致密组织覆盖。静态培育的材料表面组织形成较少。扫描电镜检测显示,动态培育的补片材料表面组织致密平整,细胞、组织呈轴向性排列。而静态培育的材料表面组织均一性较差,细胞、组织轴向性排列差。免疫组织化学染色显示动态培育环境下补片材料中有更多的胶原蛋白、alpha平滑肌肌动蛋白合成。结论:动态培育环境可显著促进肌成纤维细胞在补片材料中的增殖及合成细胞外基质。BACKGROUND： Pulsating bioreactor can provide dynamic culture environment in vitro, blood shearing force for vascular tissue patch and fluctuating perfusion for culture solution, and can simulate hemodynamic environment in vivo. OBJECTIVE： To investigate the growth characteristics of myofibroblast in tissue-engineered cardiovascular patch under dynamic culture condition in vitro. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Central Laboratory of Second Medical School of Jinan University from January to December 2007. MATERIALS： Neonate umbilical core was voluntarily provided by the puerperant with the written informed consent; poly-4-hydroxy-butyrate polymer was product of Tepha. METHODS： Cryopreserved human umbilical artery myofibroblasts （HUAMs） were seeded with patch scaffolds fabricated from P4HB polymers to construct tissue-engineered cardiovascular patch under dynamic cell culture condition in vitro. In experiment group, the cell-polymer patch constructs were incubated in vitro for 14 days under static culture conditions. On the 15^th day, the constructs were transferred into a bioreactor for another culture of 7 days. As a control, the cell-seeded constructs were incubated in static condition for 21 days. Morphological characterization of tissue-engineered patches included histology and scanning electron microscopy. Extracellular matrix （collagen） and alpha smooth muscle actin （a-SMA） formation was analyzed by immunohistochemistry. MAIN OUTCOME MEASURES： After Hematoxylin and eosin （HE） staining, histological examinations of tissue patches were conducted; ultrastructure of the patches were observed under scanning electron microscope; production of collagen Ⅰ and α -SMA was analysed using immunohistochemical staining. RESULTS： HE staining demonstrated that the dynamic conditioned patch constructs were covered completely with ordered lamellar tissues. There was minimal tissue formation in the control samples. The scanning electr

关 键 词：组织工程 心血管补片 动态培育 脐带血管细胞 细胞冷冻 生物材料 

分 类 号：R318.08[医药卫生—生物医学工程]