Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells
Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells
复制标题
DOI：
10.1097/00029330-200704020-00016
复制
发表时间：
2007-04-20
影响因子：
6.1
通讯作者：
Pan Luan-feng
中科院分区：
文献类型：
--
作者：
Fang Ning-tao;Xie Shang-zhe;Pan Luan-feng
背景 组织工程心脏瓣膜有可能克服现有心脏瓣膜置换术的局限性。本研究旨在利用人脐带血来源的内皮祖细胞(EPCs)和脱细胞瓣膜支架开发组织工程心脏瓣膜。方法以新鲜猪心脏瓣膜为原料制备脱细胞瓣膜支架。通过密度梯度离心从新鲜人脐带血中分离 EPC，在 EGM-2-MV 培养基中培养 3 周，通过免疫荧光染色评估，此时所得细胞群本质上变成内皮细胞。将EPC衍生的内皮细胞以3 X 10(6)细胞/cm(2)接种到脱细胞支架上，并在静态条件下培养7天。使用MTT测定检测支架上接种细胞的增殖。通过 HE 染色、免疫荧光染色和扫描电子显微镜对组织工程心脏瓣膜进行分析。通过血小板粘附实验和逆转录聚合酶链反应（RT-PCR）分析内皮一氧化氮合酶（eNOS）和组织型纤溶酶原激活剂（t-PA）的表达来评估工程心脏瓣膜上内皮的抗血栓形成功能。结果EPC来源的内皮细胞呈现组织溶解鹅卵石形态，表达内皮细胞谱系的特异性标记物，包括冯·维勒布兰德因子(vWF) 和 CD31，结合人内皮细胞特异性凝集素 Ulex Europaeus 凝集素-1 (UEA-1)，并吸收 Dil 标记的低密度脂蛋白 (Dil-Ac-LDL)。接种到脱细胞支架上后，细胞表现出优异的代谢活性和增殖能力。通过 HE 染色以及 vWF 和 CD31 免疫染色评估，细胞在脱细胞基质顶部形成汇合的内皮单层。扫描电子显微镜证明形成汇合单层的细胞之间存在紧密连接。血小板粘附实验表明新内皮细胞是非血栓形成的。新生内皮细胞中eNOS和t-PA基因的表达水平与人脐静脉内皮细胞中的表达水平非常相似。结论从人脐带血中分离的EPCs可以在体外分化为内皮细胞，并在脱细胞心脏瓣膜支架上形成功能性内皮细胞。因此，EPCs可能是构建组织工程心脏瓣膜的有前途的细胞来源。
Background Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds.Methods Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 X 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).Results EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells.Conclusions EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.