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.




INTRODUCTION

The lack of ideal arterial grafts has been a great challenge to cardiovascular and plastic surgeons, though artificial vessels have been widely used in the clinic of vascular surgery, its application in substituting small-caliber arterial defects is still limited due to the early thrombosis formation. The development of tissue engineering has highlighted this problem[1].
The early engineered tissues such as cartilage, bone and tendons are all produced in nude mice. The successful creation of the above tissues in nude mice has not only proved the scientific significance and promised the wide practical application of tissue engineering, but also supplied the necessary bases and data for engineering tissues in immune-potent animals and its clinical applications. Contrast to tissues such as bone, cartilage and tendons which are composed of one kind of cells, blood vessel consists of several kinds of cells, therefore vascular engineering is more complicated and difficult. This study was conducted to explore the method of engineering small-caliber artery in nude mice as well as to supply the necessary bases and data for engineering vessels and its application in immune-potent animals and humans.

MATERIALS AND METHODS

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 obstet-rics & gynecology. The parturien provided the informed consent, and this study was approved by the local research ethics com-mittee. Twenty-six nude mice (3-4 weeks old, clean grade, irre-spective of gender) were provided by Shanghai Animal Institute of Chinese Academy of Science. The animal experiment re-ceived confirmed consent from the local ethic committee. Poly-glicolide acid (PGA) was provided by Albany International Re-search Co.; CO2 incubator by Forma Scientific Company, USA; super clean bench by Suzhou Antai Air Technology Co., Ltd.; Biofuge primo low speed centrifuge by Heraeus Company, Germany; TS100 inverted phase contrast microscope by Nikon Company, Japan.

Methods
Cell isolation and culture: Procedures used for cell culture was described in detail in reference[1]. Briefly, a 20-25 cm neo-nate umbilical cord was immediately harvested after the de-livery. Endothelial cells were obtained from the umbilical vein by using a collagenase instillation technique for 15-20 minutes at 37 ℃ at 95% oxygen and 5% carbon dioxide (0.1% collagenase type I in Medium 199) and cultured in tissue culture flasks by using Medium 199 supplemented with 20% fetal bovine serum, 20 mg/mL VEGF and 50 IU/mL heparin. The media was changed either every 2-3 days or during passage. The cells were passaged by 0.1% trypsin and 0.02% EDTA in phosphate buffered solution. Cell samples of each pas-sage were collected for transmission electromicroscopic exami-nation and immunocytochemical examinations.
Vascular smooth muscle cells were obtained by tissue explant method from the umbilical artery. The umbilical artery was dis-sected from the umbilical cord and was minced into 1 mm2 pieces and cultured in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal bovine serum. Smooth muscle cells began to migrate from the explant from day 3-6 after harvest and would become confluent within 2 weeks, cells were passaged by 0.1% typsin and 0.02% EDTA in phosphate buffered solution and were also examined by transmission elec-tromicroscopy and immunocytochemistry.
Biodegradable polymer: The unwoven PGA were purchased from Smith and Nephew Inc. 25 mg PGA were weighed for each piece and made into a sheet sized 2 cm width, 3 cm length and 1 mm thickness, the PGA sheet were sterilized by immersion in 75% alcohol for 30 minutes and then washed by PBS for 3 times. After the sterilized PGA sheet became dry cell seeding were conducted.
Cell seeding: After (21±4) days in cell culture, 1×107 smooth muscle cells suspended in 100 μL DMEM were dripped on PGA sheet and the cell-PGA construct were placed stationary at 37 ℃ at 95% oxygen and 5% carbon dioxide. Af-ter 4 hours, adequate DMEM supplemented with 10% fetal bo-vine serum were added to the culture flask and the cell-PGA construct were cultured at 37 ℃ at 95% oxygen and 5% carbon dioxide. The culture medium was changed every 2-3 days dur-ing the culture period. Five to seven days later, 5×106 endothe-lial cells were seeded on top of the smooth muscle cell-PGA construct with the same procedures as the above and cultured in DMEM/M199 (1∶1) for another 2-3 days. The cell-PGA mix-ture were observed under phase contrast microscope everyday and before implantation to nude mice, a small piece of the mix-ture were cut off and prepared for scanning microscopic exami-nation.
Implantation: Before the cell-PGA construct was implanted subcutaneously in nude mice, the cell-PGA sheet was wrapped around a silicone tube (2 mm in diameter) to form a tubular structure. Nude mice were anesthetized by inhalation of isofluorane and the tubular structure was placed subcutaneously in them as experimental group (n=20). In control group (n =6), PGA tube alone without cells were implanted. The mice were feed routinely for 4-6 weeks before sacrifice.
Evaluation of the tissue-engineered small-caliber artery: Ex-cept for the gross observation, the engineered artery was fixed in 4% polyethyrene and examined by HE staining, Masson's trichrome staining and immunohistochemistry.

RESULTS

Quantitative analysis of the experimental animals
Twenty-six nude mice were included in the final analysis.

Cell culture
Under transmission electromicroscope, typical W-P bodies were observed in the cytoplasma of the cultured endothelial cells, together with the positive staining of von Willebrand factor ex-amined by immunocytochemistry revealed the endothelial phe-notype of the cells. Meanwhile, the smooth muscle cell pheno-type was proved by the present of dense body and smooth mus-cle α-actin in the cytoplasma of the cultured cells.

Observation of cell-PGA mixture
Under phase contrast microscope, smooth muscle cells began to attach to the PGA fibers and migrate along them at 4 hours and the attachment became tight at 24 hours after seeding, during the following days the attached cells continued to grow and secret a lot of matrix filling up the spaces of the PGA sheet. Scanning electromicroscope showed that the endothelial cells were ar-ranged regularly and formed a complete endothelium layer on one side of the cell-PGA construct, on the other side, a lot of matrix spotted with some smooth muscle cells were observed.

Gross observation
At 2 weeks after implantation, both the experimental and control groups formed tubular structures, however, at 6 weeks after im-plantation, the tubular structure still remained in experimental group but not in the controls. Only a thin fibrous tissue was formed around the silicone tube (Figure 1).

Histological observation of the engineered vessels
The histological examination of the engineered vessel showed that at 2 weeks, the vessels in both group contained mainly un-degraded 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 experi-mental 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 ex-amination 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 engi-neered vessel were confirmed by the positive staining of smooth muscle α-actin (Figures 2-4).

DISCUSSION

Seed cells, biodegradable polymers and tissue construction consist of the three main parts of tissue engineering research. How to obtain enough healthy and proliferating cells is the first key problem to be solved for tissue engineering. As en-dothelial cells and smooth muscle cells are the main compo-nent cells of blood vessel, we therefore manage to culture the above cells in vitro.
For endothelial cells, we used collagen digestion method and could obtain (5-10)×105 endothelial cells in the primary culture from each umbilical cord, the cells could proliferate and keep its endothelial cell phenotype during the following culture period, which met both the quantitative and qualitative demands of cells for vascular engineering and could be the suitable method for obtaining endothelial cells. However, for sake of practical use, the collagen digestion method was not the appropriate one. Nowadays more and more papers are reporting the presence of endothelial progenitor cells in the peripheral blood and bone marrow[2-4], which could be a new and practical source of obtaining endothelial cells for vascular engineering and our lab have begun the study.
It has been well known that there are two phenotypes of vascu-lar smooth muscle cells in physiologic and pathologic conditions: synthetic phenotype and contractile phenotype[1]. It was reported that smooth muscle cells could be obtained by both the collagen digestion protocols and tissue explant protocols. However, cultures of the two protocols differed significantly in their properties such as cell yield, morphology, contractile proteins and growth charac-teristics[5-7]. Theoretically, more cells are obtained by the explant technique compared to enzyme dispersal, and the collagen diges-tion cultures have a longer population doubling time of (68±2) hours (n=5) compared to tissue explant cultures for which dou-bling time is (35±2) hours (n=5), moreover, the collagen diges-tion cultures contain much high levels of both smooth muscle α-actin and smooth-muscle-specific myosin heavy chain compared to that of tissue explant cultures. We therefore chose tissue explant method to obtain smooth muscle cells in this study. The positive staining of smooth muscle a-actin examined by immunocyto-chemistry revealed the smooth muscle phenotype of the cultured cells. In this study, after the above cells were seeded on the PGA fibers, their proliferation and extracellular matrix synthesis were observed under microscopy and electromicroscopy, furthermore, 6 weeks after the implantaion of cell-PGA construct subcutaneously in nude mice, a tubular structure formed, all these results revealed that the smooth muscle cells cultured by tissue explant method were capable of proliferation and differentiation both in vitro and in vivo and thus could synthesis new tissue. Therefore, we re-garded the tissue explant method was the appropriate method for obtaining the smooth muscle cells for tissue engineering.
The tissue construction method and culturing environment was another important case of tissue engineering. For vascular engineering, it was more complicated and difficult, for vessels were composed of not only one kind of cells and contained sev-eral layers in structure. Shinoka[8] reported that by seeding si-multaneously the endothelial cells and smooth muscle cells in mixture on PGA, the cell-PGA construct formed a new artery containing endothelium layer and middle layer after its implanta-tion in substituting the pulmonary defect in a sheep model, however, in our study, seeding simultaneously the endothelial cells and smooth muscle cells in mixture on PGA could not form vessels containing two layers in nude mice model (data not shown), contrastly, by seeding smooth muscle cells first on PGA and cultured them for a short period in vitro followed by seeding the endothelial cells on top of them resulted in a typical vessel formation after its subcutaneous implantation in nude mice. The difference between our results and the shinoka's might be due to the different environments in which the cell-PGA constructs were placed, in shinoka's model, the cell-PGA construct were placed in blood circulation environment, the mechanical stimula-tions might cause the endothelial cell migrating to the luminal surface of the vessels, whereas, in our model, the cell-PGA con-struct were placed staitionary in nude mice, without any me-chanical stimulation, the endothelial cells just grew and prolifer-ated at the position where they were placed and could not mi-grate to the inner surface to form the typical vascular structures. Moreover, we found that though the engineered vessels in our study contained the vascular endothelial layer and middle layer, comparing to the normal artery, the middle layer of the engi-neered vessel was mainly composed of collagens and a small amount of smooth muscle fibers, we regarded these results were also caused by the lack of appropriate mechanical stimulations to the engineered vessels.
It has been widely accepted that the shear stress and dilatation force to the vessel wall are important factors affecting the mor-phology, proliferation, phenotype, arrangement, extracellular matrix synthesis and distribution of the vascular wall cells[9-10], thus in the future study of the vascular engineering, more efforts should be put on this issue, our lab have begun to develop a bio-reactor mimicking the in vivo environment of the blood vessels in which the engineered vessels would be cultured. We prospect by using the bioreactor and obtaining the seed cells from the peripheral blood or bone marrow, more matured vessels could be made by tissue engineering approach and be put into practical use in the near future.

REFERENCES

1 Langer R, Vacanti JP. Tissue Engineering. Science 1993; 260(5110): 920-926
2 Jeffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973;52(11):2745-2756
3 Bian JY, Zhou D. The influence factors on the in vitro culture of human endothelial cells derived from umbilical veins. Xibao Shengwuxue Zazhi 1997;19(2):66-68
4 Boyer M, Townsend LE, Vogel LM, et al. Isolation of endothelial cells and their progenitor cells from human peripheral blood. J Vasc Surg 2000;31(1 Pt 1):181-189
5 Shi Q, Rafii S, Wu MH, et al. Evidence for circulation bone mar-row-derived endothelial cells.Blood 1998;92(2):362-367
6 Kim BS, Baez CE, Atala A. Biomaterials for tissue engineering. World J Urol 2000;18(1):2-9
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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*☆

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）*
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
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)