脱细胞支架复合兔骨髓间充质干细胞构建组织工程血管

背景:目前临床使用的小口径(<6 mm)人工血管因生物相容性差、远期通畅率低,效果并不理想.因此,学术界一直致力于寻找具有正常血管生物学功能的血管代用品,组织工程血管的构建与功能研究已成为目前热门研究课题.目的:将兔骨髓间充质干细胞与脱细-胞血管支架动态复合培养体外构建组织工程血管,通过体内移植实验,探讨该组织工程血管的组织相容性及通畅率.设计、时间及地点:随机对照实验,细胞学、组织病理学观察,于2006-01/2008-06在南京大学医学院附属鼓楼医院实验室完成.材料:通过去污剂-酶消化法制备兔腹主动脉脱细胞支架;采用密度梯度离心法结合贴壁分离培养法,分离扩增兔骨髓间充质干细胞,将扩增后的干细胞静态种植于脱细胞支架后置于生物反应器中动态培养构建组织工程血管.方法:60只兔随机均分为3组,剪取一段腹主动脉长约1.0 cm,再将移植血管以8/0聚丙烯线间断外翻吻合到腹主动脉上.组织工程血管组:受体为对应抽取骨髓干细胞的实验兔,以组织工程血管为移植血管;脱细胞血管支架组:以脱细胞处理的同种异体腹主动脉为移植血管;同种异体血管组:以同种异体新鲜腹主动脉作为移植血管.主要观察指标:对培养的骨髓间充质干细胞进行免疫组化鉴定;血管移植后3个月行数字减影血管造影、病理切片、扫描电镜等观察移植效果.结果:兔骨髓间充质干细胞在体外培养8 d后形成漩涡状排列,免疫组化结果符合间充质干细胞表型特征:将间充质干细胞与脱细胞支架置于生物反应器培养12 d后,种子细胞在血管腔内黏附生长;血管移植3个月后,组织工程血管组、脱细胞血管支架组通畅率分别为90%,80%,均优于同种异体血管组(25%).移植3个月后苏木精一伊红染色及扫描电镜结果显示,组织工程血管组形成清晰的内、中、外膜3层结构,形态接近正常动脉,内皮细胞覆盖完整;脱细胞血管支架组血管内表面内皮细胞覆盖不完整,伴有附擘血栓形成,内膜轻度增生,伴炎性细胞浸润:同种异体血管组内膜极度增厚、坏死,管腔明显狭窄,伴不同程度的血栓机化.结论:将兔骨髓间充质干细胞复合脱细胞血管支架上,可获得一种具有良好生物相容性和通畅率的生物人工血管.

Tissue-engineered graft constructed by bone marrow mesenchymal stem cells and vascular acellular matrix

BACKGROUND: At present, the commercial artificial small vascular grafts (diameter < 6 mm) are still unsatisfactory, due to poor blocompatibility and low long-term patency rate. Therefore, finding a vascular substitute with normal biological function and studying construction and function of tissue-engineered blood vessel have become hot topics recently.OBJECTIVE: To construct a novel tissue-engineered blood vessel by rabbit bone marrow mesenchymal stem cells (MSCs) and vascular acellular matrix, and to investigate the biocompatibility and patency rate of tissue-engineered blood vessels.DESIGN, TIME AND SETTING: An in vitro randomized controlled study at level of cytology and histopathology was performed at the Laboratory of Affiliated Drum Tower Hospital of Nanjing University Medical School from January 2006 to June 2008.MATERIALS: The decellularized vascular acellular matrix was obtained by a detergent-enzymatic procedure. MSCs from rabbits were isolated using density gradient centrifugation method and cultured in culture flasks coated with fibronectin. Subsequently, the expanded MSCs were seeded on the decellularized scaffolds, and then co-cultured in the self-made bioreactor to construct the tissue-engineered blood vessels.METHODS: Sixty rabbits were randomly divided into three groups. A1.0-cm abdominal aorta was sheared, and a tissue-engineered blood vessel was transplanted on the abdominal aorta using 8/0 polypropylene thread. Tissue-engineered blood vessel group: Tissue-engineered blood vessel was considered as the transplanted vessel; vascular acellular matrix group:Xenoma artery treated by vascular acellular matrix was considered as the transplanted vessel; xenoma artery group: Fresh xenoma artery was considered as the transplanted vessel.MAIN OUTCOME MEASURES: Immunocytochemical staining was used to identify the cultured MSCs. After 3 months of transplantation, the grafts were retrieved for digital subtraction angiography, pathological test and scanning electron microscope examination.RESULTS: Rabbits MSCs presented a whirlpool-like appearance at 8 days after culture. The immunocytochemistry results were consistent with the phenotype of MSCs. After high proliferation, MSCs were seeded onto the vascular acellular matrix for 12 days,and seed cells attached to well in the lumen of blood vessels. Three months after implantation, the patency rate was 90% of tissue-engineered blood vessel group and 80% of vascular acellular matrix group, which was superior to xenoma artery group (25%). At three months after transplantation, HE staining and scanning electron microscope demonstrated that internal, middle,and external membrane were clearly observed in the tissue-engineered blood vessel group, and the membrane morphology was similar to normal artery. The endothelial cells were covered completely. However, the endothelial cells were not covered completely in the vascular acellular matrix group, while mural thrombosis, mild proliferation of intima, and inflammatory cell infiltration were observed. The intima was thick and necrotic in the xenoma artery group, while lumens were stenotic and accompanied with a certain degree of thrombus organization.CONCLUSION: This study provides a new strategy to develop a tissue-engineered blood vessel with excellent biocompatibility and high patency rate constructed by rabbit MSCs and vascular acellular matrix.