中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (5): 697-705.doi: 10.12307/2023.885

• 水凝胶材料Hydrogel materials • 上一篇    下一篇

负载外泌体水凝胶修饰3D打印支架构建血管化的气道替代物

沈子青1，夏  天1，单一波2，朱睿君1，万昊鑫1，丁  浩1，潘  枢1，赵  军1   

  1. 1苏州大学附属第一医院胸外科，江苏省苏州市  215006；2扬州大学医学院转化学研究院，江苏省扬州市  225000
  • 收稿日期:2022-11-24 接受日期:2023-01-05 出版日期:2024-02-18 发布日期:2023-08-16
  • 通讯作者: 赵军，博士，教授，主任医师，苏州大学附属第一医院胸外科，江苏省苏州市 215006
  • 作者简介:沈子青，男，1996年生，江苏省苏州市人，汉族，苏州大学第一临床医学院在读硕士，主要从事组织工程气管替代治疗的研究。
  • 基金资助:
    江苏省自然科学基金青年项目(BK20200196)；项目负责人：潘枢；苏州大学企业委托项目(H221026)，项目负责人：赵军

Vascularized tracheal substitutes constructed by exosome-load hydrogel-modified 3D printed scaffolds

Shen Ziqing1, Xia Tian1, Shan Yibo2, Zhu Ruijun1, Wan Haoxin1, Ding Hao1, Pan Shu1, Zhao Jun1   

  1. 1Department of Thoracic Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China; 2Institute of Translational Research, College of Medicine, Yangzhou University, Yangzhou 225000, Jiangsu Province, China
  • Received:2022-11-24 Accepted:2023-01-05 Online:2024-02-18 Published:2023-08-16
  • Contact: Zhao Jun, PhD, Professor, Chief physician, Department of Thoracic Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • About author:Shen Ziqing, Master candidate, Department of Thoracic Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Supported by:
    Natural Science Foundation for the Youth of Jiangsu Province, No. BK20200196 (to PS); Enterprise Commissioned Project of Soochow University, No. H221026 (to ZJ)

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摘要：


文题释义：

外泌体：是一种由细胞分泌的包含有核酸、脂质和蛋白质的纳米囊泡状结构，在细胞间的交流中发挥着不可或缺的调节作用。
甲基丙烯酰化透明质酸水凝胶：透明质酸是由D-葡萄糖醛酸和N-乙酰-D-氨基葡萄糖作为双糖结构单元的天然糖胺多糖聚合物，在细胞增殖、分化、形态发生和伤口愈合等生物学过程中发挥重要作用。在其分子链上接枝丙烯酸基团获得甲基丙烯酰化透明质酸，使其进一步具有光固化特性。


背景：对于长段气管缺损的替代治疗，尽管近年来组织工程研究取得了一定的进展，但仍然不尽完善，其中最大的难点就是不能快速实现气管替代物的血运重建。

目的：初步探索经负载外泌体水凝胶修饰的聚己内酯支架构建可快速血管化气管替代物的潜力。
方法：提取SD大鼠骨髓间充质干细胞来源外泌体，制备甲基丙烯酰化透明质酸溶液，将外泌体溶液与甲基丙烯酰化透明质酸溶液按照体积比1∶1混合，紫外光照射5 min制备负载外泌体的甲基丙烯酰化透明质酸水凝胶。检测未负载外泌体水凝胶的降解情况，负载外泌体水凝胶外泌体的控制释放情况。采用3D打印法制备聚己内酯支架，将单纯的甲基丙烯酰化透明质酸溶液与负载外泌体的甲基丙烯酰化透明质酸水溶液分别滴加至支架表面，紫外光照射后获得水凝胶修饰的支架与外泌体修饰的支架。将30只SD大鼠采用随机数字表法分为3组，每组10只，分别于皮下包埋单纯的支架、水凝胶修饰的支架与外泌体修饰的支架，术后30 d取出各组支架及周围组织，通过苏木精-伊红及Masson染色观察新生血管生成情况，免疫荧光检测CD31的表达。

结果与结论：①随着时间的推移，水凝胶逐渐降解，并且包封于水凝胶中的外泌体逐渐释放，可持续释放至30 d以上，并且外泌体的释放速率快于水凝胶自身的降解速率，浸泡30 d时仍有近20%的外泌体未被释放；②扫描电镜下可见，单纯的聚己内酯支架表面粗糙，经水凝胶修饰后，支架孔隙之间覆盖一层凝胶物质，支架表面变得光滑、致密；③大鼠皮下包埋30 d后，苏木精-伊红及Masson染色显示，与水凝胶修饰支架组相比，外泌体修饰支架组支架内部可见更多的新生血管形成，两组支架表面的水凝胶未完全降解；免疫荧光染色显示，外泌体修饰支架组CD31表达高于水凝胶修饰支架组(P < 0.000 1)；④结果表明，甲基丙烯酰化透明质酸水凝胶可以作为外泌体的控释载体，经负载外泌体甲基丙烯酰化透明质酸水凝胶修饰后的3D打印聚己内酯支架，具有良好的生物相容性，且具有促进新生血管形成的潜力。

https://orcid.org/0000-0003-3916-6983(沈子青)

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: 气管重建, 外泌体, 聚己内酯, 组织工程, 3D打印, 血管化, 水凝胶

Abstract: BACKGROUND: For the replacement treatment of long-segment tracheal defects, although tissue engineering research has made some progress in recent years, it is still not perfect, and one of the biggest difficulties is that the hemodynamic reconstruction of the tracheal replacement cannot be achieved rapidly. 
OBJECTIVE: To preliminarily explore the potential of polycaprolactone scaffolds modified with exosome-loaded hydrogels to construct a rapidly vascularized tracheal substitute. 
METHODS: Exosomes were extracted from bone marrow mesenchymal stem cells of SD rats. After preparation of hyaluronic acid methacrylate solution, the exosome solution was mixed with hyaluronic acid methacrylate solution at a volume ratio of 1:1. Hyaluronic acid methacrylate hydrogels loaded with exosomes were prepared under ultraviolet irradiation for 5 minutes. The degradation of exosome-unloaded hydrogels and the controlled release of exosome-loaded hydrogels were detected. Polycaprolactone scaffolds were prepared by 3D printing. The pure hyaluronic acid methacrylate solution and the exosome-loaded hyaluronic acid methacrylate solution were respectively added to the surface of the scaffold. Hydrogel-modified scaffolds and exosome-modified scaffolds were obtained after ultraviolet irradiation. Thirty SD rats were randomly divided into three groups with 10 rats in each group and subcutaneously implanted with simple scaffolds, hydrogel-modified scaffolds and exosome-modified scaffolds, respectively. At 30 days after surgery, the scaffolds and surrounding tissues of each group were removed. Neovascularization was observed by hematoxylin-eosin staining and Masson staining and the expression of CD31 was detected by immunofluorescence. 
RESULTS AND CONCLUSION: (1) As time went by, the hydrogel degraded gradually, and the exosomes enclosed in the hydrogel were gradually released, which could be sustained for more than 30 days. The exosome release rate was faster than the degradation rate of the hydrogel itself, and nearly 20% of the exosomes were still not released after 30 days of soaking. (2) Under a scanning electron microscope, the surface of the simple polycaprolactone scaffold was rough. After hydrogel modification, a layer of gel was covered between the pores of the scaffold, and the scaffold surface became smooth and dense. (3) After 30 days of subcutaneous embedding, hematoxylin-eosin staining and Masson staining showed that more neovascularization was observed inside the scaffolds of the exosome-modified scaffold group compared with the hydrogel-modified scaffold group. The hydrogels on the scaffolds of the two groups were not completely degraded. Immunofluorescence staining showed that CD31 expression in the exosome-modified scaffold group was higher than that in the hydrogel-modified scaffold group (P < 0.000 1). (4) These results indicate that hyaluronic acid methacrylate hydrogels can be used as controlled-release carriers for exosomes. The 3D-printed polycaprolactone scaffold modified by hyaluronic acid methacrylate hydrogel loaded with exosomes has good biocompatibility and has the potential to promote the formation of neovascularization. 

Key words: tracheal reconstruction, exosome, polycaprolactone, tissue engineering, 3D printing, vascularization, hydrogel

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