中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (22): 3505-3513.doi: 10.12307/2022.278

• 复合支架材料 composite scaffold materials • 上一篇    下一篇

聚左旋乳酸己内酯/丝素蛋白小口径人工血管细胞共培养及体内生物相容性

刘  月1,蒋紫仪1,李晶晶2,3,孟  凯1,赵荟菁1   

  1. 1苏州大学纺织与服装工程学院,江苏省苏州市  215123;2苏州大学心血管病研究所,江苏省苏州市  215021;3苏州大学附属第一医院心脏大血管外科,江苏省苏州市  215006
  • 收稿日期:2021-05-14 修回日期:2021-07-22 接受日期:2021-08-05 出版日期:2022-08-08 发布日期:2022-01-12
  • 通讯作者: 赵荟菁,博士,副教授,苏州大学纺织与服装工程学院,江苏省苏州市 215123
  • 作者简介:刘月,女,1995年生,江苏省连云港市人,汉族,硕士,主要从事生物医用纺织材料研究。
  • 基金资助:
    江苏省高等学校自然科学研究重大基金项目(19KJA610004),项目负责人:赵荟菁;中国纺织工业联合会科技指导性项目(2019021),项目负责人:赵荟菁

Cell co-culture and in vivo biocompatibility of poly(L-lactic caprolactone)/silk fibroin small-diameter artificial blood vessels

Liu Yue1, Jiang Ziyi1, Li Jingjing2,3, Meng Kai1, Zhao Huijing1   

  1. 1College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, Jiangsu Province, China; 2Institute for Cardiovascular Science, Soochow University, Suzhou 215021, Jiangsu Province, China; 3Department of Cardiovascular Surgery, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Received:2021-05-14 Revised:2021-07-22 Accepted:2021-08-05 Online:2022-08-08 Published:2022-01-12
  • Contact: Zhao Huijing, MD, Associate professor, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, Jiangsu Province, China
  • About author:Liu Yue, Master, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, Jiangsu Province, China
  • Supported by:
    Natural Science Foundation for Key Program of Jiangsu Higher Education Institutions, No. 19KJA610004 (to ZHJ); Science and Technology Guidance Project of China Textile Industry Federation, No. 2019021 (to ZHJ)

摘要:

文题释义:
小口径人工血管:人工血管是许多严重狭窄或闭塞性血管的替代品,小口径人工血管的直径< 6 mm。
缝合固位强力:也称为手术缝合线固位强度,指确定手术缝合线从人工血管壁中拉出(即管壁被破坏时)所需的力,主要用于表征管状样品是否能够承受手术缝合线的缝合作用。

背景:自体旁路移植是心血管疾病最佳的手术方案,但自体血管来源有限,远远不能满足巨大的临床需求,因此小口径人工血管的研发具有十分重要的意义,然而小口径人工血管由于易产生血栓和内膜增生等问题还没有真正应用于临床。
目的:观察人脐静脉内皮细胞和血管平滑肌细胞在聚左旋乳酸己内酯/丝素蛋白电纺膜上的生长情况,以及聚左旋乳酸己内酯/丝素蛋白小口径人工血管在大鼠体内的通畅性、组织相容性和降解性能。
方法:采用静电纺丝技术制备聚左旋乳酸己内酯/丝素蛋白小口径双层人工血管,加载血管内皮生长因子于人工血管上,最后将肝素加载至人工血管上。将人脐静脉内皮细胞和血管平滑肌细胞分别接种到加载与未加载血管内皮生长因子的人工血管上,通过激光共聚焦显微镜、扫描电镜和MTT法分析细胞在人工血管材料上的生长情况。以加载与未加载血管内皮生长因子的人工血管电纺膜代替细胞小室中的Transwell膜,将人脐静脉内皮细胞和血管平滑肌细胞共同接种至电纺膜上,通过激光共聚焦显微镜、扫描电镜和MTT法分析细胞在人工血管材料上的生长情况。将加载血管内皮生长因子与肝素的人工血管植入大鼠颈动脉,检测人工血管的通畅性与组织相容性、降解性。
结果与结论:①细胞单独接种于人工血管上:激光共聚焦显微镜、扫描电镜与MTT分析显示,接种在加载血管内皮生长因子人工血管上的人脐静脉内皮细胞多于未加载血管内皮生长因子人工血管,加载与未加载血管内皮生长因子的血管平滑肌细胞数量基本相同。②两细胞共同接种于人工血管上:激光共聚焦显微镜、扫描电镜与MTT分析显示,加载与未加载血管内皮生长因子人工血管上的共培养细胞数量均多于对应人工血管上的单独细胞接种数量,但小于两种单种细胞数之和。③动物体内植入实验:激光多普勒血流成像仪显示,人工血管植入1 d后血流速度有所减小,此后逐渐减小,至第3周时已勘测不到。组织学与免疫组化染色显示,人工血管植入第3天时有炎性细胞,1周后炎性细胞消失,在第3周时管内有细微血流通过,管内壁形成均匀的内皮层且有新血管生成。扫描电镜显示,人工血管植入第6周时纤维基本发生断裂,且纤维明显变细。人工血管植入第4周时缝合固位强力降至原来的42.2%。④结果表明,聚左旋乳酸己内酯/丝素蛋白小口径双层人工血管具有良好的细胞相容性与组织相容性,但通畅性与降解性有待提高。

https://orcid.org/0000-0002-4021-5706 (刘月) ;https://orcid.org/0000-0003-0250-3579 (赵荟菁)

中国组织工程研究杂志出版内容重点:生物材料;骨生物材料口腔生物材料纳米材料缓释材料材料相容性;组织工程

关键词: 小口径人工血管, 聚左旋乳酸己内酯, 丝素蛋白, 细胞共培养, 体内实验

Abstract: BACKGROUND: The autologous bypass grafting is the best surgical scheme. However, the source of autologous blood vessels is limited, which is far from meeting the huge clinical needs. Therefore, the research and development of small-diameter artificial blood vessels are of great significance. However, small-diameter artificial blood vessels have not been truly applied in clinical practice due to the problems of easy thrombosis and intimal hyperplasia. 
OBJECTIVE: To explore the growth of human umbilical vein endothelial cells and vascular smooth muscle cells on poly(L-lactic caprolactone)/silk fibroin electrospun membrane, the patency, histocompatibility and degradability of poly(L-lactic caprolactone)/silk fibroin small-diameter artificial blood vessels in rats.
METHODS: Electrospinning technology was used to prepare poly(L-lactic caprolactone)/silk fibroin small-caliber artificial blood vessels. Vascular endothelial growth factors were loaded on the artificial blood vessel, and heparin was finally loaded on the artificial blood vessel. Human umbilical vein endothelial cells and vascular smooth muscle cells were respectively seeded on artificial blood vessels loaded and unloaded with vascular endothelial growth factor. The growth of cells on artificial blood vessels was analyzed by confocal laser scanning microscope, scanning electron microscope, and MTT assay. Transwell membrane in cell chamber was replaced with poly(L-lactic caprolactone)/silk fibroin electrospun membrane. Human umbilical vein endothelial cells and vascular smooth muscle cells were inoculated on poly(L-lactic caprolactone)/silk fibroin material for cell co-culture. The growth of cells on poly(L-lactic caprolactone)/silk fibroin materials was analyzed by confocal laser scanning microscope, scanning electron microscope, and MTT assay. The artificial blood vessel loaded with vascular endothelial growth factor and heparin was implanted into the rat carotid artery. The patency, histocompatibility, and degradability of the artificial blood vessel were detected.
RESULTS AND CONCLUSION: (1) Cells separately seeded on artificial blood vessels: Confocal laser scanning microscope, scanning electron microscope, and MTT assay showed that the number of human umbilical vein endothelial cells seeded on the artificial blood vessel loaded with vascular endothelial growth factor was more than that of the artificial blood vessel without vascular endothelial growth factor. The number of vascular smooth muscle cells loaded with and without vascular endothelial growth factor was basically the same. (2) Two kinds of cells seeded together on the artificial blood vessel: Confocal laser scanning microscope, scanning electron microscope, and MTT assay showed the number of cells in co-culture was larger than that of single cells in non-co-culture environment, but less than the sum of the number of two single cells. (3) Implantation experiments in animals: The laser Doppler perfusion imaging showed that the blood flow velocity decreased at 1 day after implantation of the artificial blood vessel, and then gradually decreased, until the third week it had not been surveyed. Histological and immunohistochemical staining exhibited inflammatory cells appeared on the third day after implantation of artificial blood vessel, and disappeared one week later. At the third week, blood flow was observed in the sample, and a uniform endodermis and new angiogenesis were formed on the inner wall of the sample. Scanning electron microscopy showed that the fibers were basically broken at the 6th week of implantation of the artificial blood vessel, and the fibers became thinner. At the 4th week, the retention strength of artificial blood vessel suture decreased to 42.2% of the original data. (4) These findings suggest that poly(L-lactic caprolactone)/silk fibroin small-diameter artificial blood vessels have good cytocompatibility and histocompatibility, but the patency and degradability need to be improved. 

Key words: small-diameter artificial blood vessel, poly(l-lactide-co-ε-caprolactone), silk fibroin, cell co-culture, in vivo experiment

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