Abstract

BACKGROUND:A lot of researches have proved that polyglicolide acid (PGA), as a stent material, has been successfully used to construct engineered tissues, such as cartilage, bone and tendons, in nude mice or even big mammal. Whether the incubation of vascular endothelial cells and smooth muscle cells in the polyglicolide acid may subcutaneously construct vessel-like structure in nude mice needs a further study.
OBJECTIVE: To verify the feasibility of forming vessel-like structure in the nude mice by subcutaneous implantation of polyglicolide acid cocultured with vascular endothelial cells and smooth muscle cells derived from newborn umbilical vein.
DESIGN: Contrast study.
SETTING: Tissue Engineering Key Laboratory, Medical College, Shanghai Jiao Tong University.
MATERIALS: This study was performed at Tissue Engineering Key Laboratory, Medical College, Shanghai Jiao Tong University from January to June 2002. Belly band was derived from newborn babies in our department of obstetrics & gynecology. The parturien provided the informed consent, and this study was approved by the local research ethics committee. Twenty-six nude mice (3-4 weeks old, clean grade, irrespective of gender) were selected in this study. The animal experiment received confirmed consent from the local ethic committee. Polyglicolide acid was provided by Albany International Research Co.
METHODS: Vascular endothelial cells and smooth muscle cells derived from newborn umbilical vein were incubated on a piece of polyglicolide acid to produce cell-material compound. In addition, the compound covered around the silicone tube to form a tube-like structural substance. Subsequently, the tube-like structural substance was subcutaneously implanted in 20 nude mice, which were regarded as an experimental group. And then, polyglicolide acid alone was subcutaneously transplanted in the rest 6 nude mice, which was regarded as a control group.
MAIN OUTCOME MEASURES: Gross observations of cell-material compound by 2 and 6 weeks after transplantation; HE staining and immunohistochemical staining detection; expression of factor Ⅷ and α-smooth muscle actin.
RESULTS: Twenty-six nude mice were included in the final analysis. ① Gross observation: At 2 weeks after implantation, both the experimental and control groups formed tubular structures, however, at 6 weeks after implantation, the tubular structure still remained in experimental group but not in the controls. ② Histological observation and immunohistochemical detection: The histological examination of the engineered vessel showed that at 2 weeks, the vessels in both group contained mainly undegraded PGA fibers, while at 6 weeks, the PGA fibers were almost completely degraded in both groups and in the control group only fibrous-like tissue formed. Contrastly, in experimental group a typical vascular structure formed, Masson's trichrome stain, which stains collagen green, smooth muscle fibers red and cells purple, showed significant amounts of stainable collagen and smooth muscle fibers in the wall of the engineered vessel, furthermore, immunohistochemistry examination revealed that there were an endothelial cell layer formed in the inner surface of the engineered vessel which was confirmed by positive staining of von Willebrand factor, meanwhile, the smooth muscle cells in the wall of the engineered vessel were confirmed by the positive staining of smooth muscle α-actin.
CONCLUSION: The subcutaneous implantation of polyglicolide acid cocultured with vascular endothelial cells and smooth muscle cells may form vessels, which are similar to normal vascular histological structure.

1Department of Plastic and Reconstructive Surgery, Shanghai 9th People's Hospital, Medical College, Shanghai Jiao Tong University, Shanghai 200011, China; 2Shanghai Tissue Engineering Center, Medical College, Shanghai Jiao Tong University, Shanghai 200011, China

Liu Yang☆, Doctor, Associate chief physi-cian, Department of Plastic and Reconstruc-tive Surgery, Shanghai 9th People's Hospital, Medical College, Shanghai Jiao Tong University, Shanghai 200011, China
twinspingan@gmail.
com

Supported by: Shanghai Postdoctoral Science Foundation, No. 200116*

Received: 2007-08-31 Accepted: 2007-12-19 (07-50-8-4754/YWY)

Liu Y, Zhang YZ, Chen JJ, Yin DM, Cao Y, Xu ZC, Liu W, Cui L, Cao YL.Artificial vessel construction in nude mice by subcutaneous implantation of poly-glicolide acid cocul-tured with vascular endothelial cells and smooth muscle cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(10): 1958-1961(China)
[www.zglckf.com/zglckf/ejournal/upfiles/ 12-10/10k-1958
(ps).pdf]

摘要
背景：有实验表明聚羟基乙酸支架材料已成功在裸鼠及大型哺乳动物体内构建形成了组织工程化软骨、骨、肌腱等组织，将血管内皮细胞与平滑肌细胞接种于聚羟基乙酸能否在裸鼠皮下形成血管样结构？
目的：采用新生婴儿脐静脉血管内皮细胞与平滑肌细胞接种于片状聚羟基乙酸形成的复合物移植于裸鼠皮下，验证其形成血管样结构的可行性。
设计：对比观察。
单位：原上海第二医科大学组织工程重点实验室。
材料：实验于2002-01/06在上海第二医科大学组织工程重点实验室完成。新生婴儿脐带来源于本院妇产科健康新生儿，经产妇知情同意，实验经过医院伦理委员会的批准。选用26只3~4周龄清洁级裸鼠，雌雄不拘，实验过程中对动物的处置符合动物伦理学标准。聚羟基乙酸购自Albany International Research Co.。
方法：将体外培养、扩增的新生婴儿脐静脉血管内皮细胞与平滑肌细胞接种于片状聚羟基乙酸材料上，形成细胞-材料复合物，将该复合物包绕于硅胶管使成管状结构后移植于20只裸鼠皮下为实验组，将未接种细胞的单纯聚羟基乙酸置入其余6只裸鼠皮下。
主要观察指标：分别于细胞-生物材料复合物置入后第2，6周后取出两组移植物进行大体观察，并进行苏木精-伊红染色及免疫组织化学染色，检测VIII因子及α-平滑肌肌动蛋白的表达。
结果：裸鼠26只均进入结果分析。①大体观察结果：移植物置入2周后，两组移植物均有管状结构形成；置入6周后，对照组管形结构消失，围绕硅胶管有极薄的纤维组织包裹形成，抽去硅胶管后，纤维组织失去支撑，无法维持管型结构。实验组抽去硅胶管后,新生组织仍维持管型结构。②组织学观察及免疫组化检测结果：移植物置入2周后，两组均可在镜下观察到大量未完全降解的聚羟基乙酸，对照组内少有细胞成分，实验组内可见多量细胞存在；置入6周后，两组聚羟基乙酸成分基本消失，对照组只有菲薄的纤维结缔组织形成，实验组显示有新生血管组织形成，其最内层有VIII因子阳性细胞覆盖，管壁内有大量细胞外基质及散在的α-平滑肌肌动蛋白阳性细胞存在。
结论：血管内皮细胞、平滑肌细胞-聚羟基乙酸复合物置入裸鼠皮下可形成具有与正常血管组织学结构相似的血管。
关键词：人工血管； 血管内皮细胞； 平滑肌细胞； 生物材料；组织工程；组织构建

刘 阳1，张燕中2，陈瑾君2，殷德民2，曹 颖2，许志成2，刘 伟2，崔 磊2，曹谊林2
1上海交通大学医学院附属第九人民医院整复外科，上海市 200011；2上海交通大学医学院组织工程研究中心，上海市 200011
刘 阳☆，女，1970年生，辽宁省沈阳市人，汉族，2000年解放军第二军医大学毕业，博士，副主任医师，主要从事整形外科临床及血管组织工程研究工作。
上海市博士后科研资助计划（200116）*

中图分类号: R318 文献标识码: A 文章编号: 1673-8225(2008)10-01958-04
刘阳，张燕中，陈瑾君，殷德民，曹颖，许志成，刘伟，崔磊，曹谊林.血管内皮细胞和平滑肌细胞-聚羟基乙酸复合物置入裸鼠皮下构建人工血管[J].中国组织工程研究与临床康复，2008，12(10):1958-1961
[www.zglckf.com/zglckf/ejournal/upfiles/12-10/10k-1958(ps).pdf]
(Edited by Lu L/Ji H/Wang L)