中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (14): 2203-2208.doi: 10.3969/j.issn.2095-4344.0767

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

胶原/硫酸软骨素/成纤维生长因子复合人工眼角膜的制备

毛宝亮，胡 斌，贾 磊，单恒悦，李 响，王 颖，袁琬娟，张凤超，陈敬华   

  1. 江南大学药学院制药工程系，江苏省无锡市 214122
  • 收稿日期:2017-12-06 出版日期:2018-05-18 发布日期:2018-05-18
  • 通讯作者: 陈敬华，教授，江南大学药学院，药剂学与药剂材料学研究室，江苏省无锡市 214122
  • 作者简介:毛宝亮，男，1995年生，福建省宁德市人，汉族，江南大学药学院本科在读，主要从事药剂学研究。
  • 基金资助:

    江南大学自主科研计划青年项目(JUSRP115A36)；江南大学自主科研计划重点项目(JUSRP51709A)；2017年校大学生创新计划项目(2395210232170990)

Artificial cornea preparation using collagen/chondroitin sulfate/fibroblast growth factor composite film

Mao Bao-liang, Hu Bin, Jia Lei, Shan Heng-yue, Li Xiang, Wang Ying, Yuan Wan-juan, Zhang Feng-chao, Chen Jing-hua   

  1. School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu Province, China
  • Received:2017-12-06 Online:2018-05-18 Published:2018-05-18
  • Contact: Chen Jing-hua, Professor, School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu Province, China
  • About author:Mao Bao-liang, School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu Province, China
  • Supported by:

    Jiangnan University Research Program, No. JUSRP115A36, JUSRP51709A; 2017 Student Innovation Program of Jiangnan University, No. 2395210232170990

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摘要：

文章快速阅读：

 

文题释义：
成纤维细胞生长因子：又称为肝素结合生长因子，是一种能促进成纤维细胞生长的多肽类物质，有酸性(pH 5.6)和碱性(pH 9.6)两种，能促进成纤维细胞有丝分裂、中胚层细胞的生长，还可刺激血管形成，在创伤愈合及肢体再生中发挥作用。
 
 
背景：传统角膜支架材料的强度和生物相容相差，采用天然角膜组织成分的胶原和硫酸软骨素制备人工角膜尚未见报道。
目的：制备具有缓释生长因子、高强度和透光性、良好生物相容性的胶原/硫酸软骨素/成纤维生长因子复合人工眼角膜。
方法：将不同浓度(1%、5%、10%)的胶原溶液采用流延法制备为再生胶原膜，通过生物力学测试筛选出5%胶原溶液制备的再生胶原膜生物力学性能最优，用于制备复合膜。通过EDC-NHS交联法将不同质量浓度(2，20，80 g/L)的硫酸软骨素固定于再生胶原膜上，得到胶原/硫酸软骨素复合膜，通过透光率筛选出20 g/L硫酸软骨素制备的胶原/硫酸软骨素复合膜透光性最好，用于制备载生长因子复合膜。将胶原/硫酸软骨素复合膜与不同质量浓度(5，25，50 mg/L)的成纤维细胞因子10溶液共混于PBS中24 h，制备载生长因子胶原/硫酸软骨素复合膜，将其浸泡于PBS中，ELISA法检测上清液中生长因子水平。将再生胶原膜、胶原/硫酸软骨素复合膜及载生长因子胶原/硫酸软骨素复合膜分别与角膜上皮细胞共培养48 h，MTT法检测细胞增殖，筛选最佳载生长因子胶原/硫酸软骨素复合膜。将载生长因子胶原/硫酸软骨素复合膜与角膜上皮细胞共培养， 48 h后，采用共聚焦显微镜观察细胞形态；72 h后，采用扫描电镜观察细胞形态。

结果与结论：①成纤维生长因子10的释放量随复合膜中初始装载生长因子水平的增加而增加。同时，3种复合膜上的生长因子释放缓慢，并在72 h时分别达到11%、23%和30%，释放行为符合药代动力学过程；②通过MTT实验确定复合膜最佳生长因子负载质量浓度为25 mg/L；③共聚焦显微镜及扫描电镜显示，载生长因子胶原/硫酸软骨素复合膜可促进角膜上皮细胞的黏附、生长及增殖；④结果表明，载生长因子胶原/硫酸软骨素复合膜有望成为一种优良的人工角膜支架材料。

ORCID: 0000-0003-1723-771X(陈敬华)

关键词: 胶原蛋白, 硫酸软骨素, EDC-NHS交联剂, 角膜支架, 仿生, 成纤维生长因子10, 角膜上皮细胞, 缓释, 强度, 生物材料

Abstract:

BACKGROUND: The traditional corneal scaffolds exhibit poor strength and biological compatibility. Little is reported on the artificial cornea prepared by collagen and chondroitin sulfate (CS), which consist of the natural corneal tissue.

OBJECTIVE: To prepare the collagen/CS/fibroblast growth factor (FGF) composite artificial cornea with slow-release growth factor, high strength and light transmittance, as well as good biocompatibility.
METHODS: Regenerated collagen films were prepared by 1%, 5%, 10% collagen solutions using flow casting method, and the regenerated collagen film with the best bioactivity that was prepared by 5% collagen solution was screened through a biomechanical test. Then, the CS/collagen composite film was achieved by cross-linking the CS (2, 20, 80 g/L) with collagen by using N-(3-Dimethylaminopropyl)- N’S-ethylcarbodiimide hydrochloride-N-Hydroxysuccinimide. The composite film made of 20 g/L CS was confirmed to have the best transparency, which was used to be mixed with 5, 25, 50 mg/L FGF in PBS for 24 hours to prepare the collagen/CS/FGF composite films. ELISA method was used to detect the FGF level in the supernatant. Afterwards, corneal epithelial cells were co-cultured with regenerated collagen film, collagen/CS composite film and collagen/CS/FGF composite film, respectively. After 48 hours of co-culture, cell proliferation was detected by MTT method, based on which we could screen the optimal collagen/CS/FGF composite film. After co-culture with the collagen/CS/FGF composite film for 48 and 72 hours, cell morphology was observed by confocal microscope and scanning electron microscope, respectively.

RESULTS AND CONCLUSION: The release amount of FGF from the composite films was dependent on the initial loading amount of FGF. Meanwhile, FGF released slowly from the three kinds of composite films, and the release amount was 11%, 23%, 30% at 72 hours after culture, in accordance with the pharmacokinetic process. MTT findings indicated that the optimal loading concentration of FGF was 25 mg/L. Under the microscope, the collagen/CS/FGF composite film promoted the adhesion, growth and proliferation of corneal epithelial cells. To conclude, the collagen/CS/FGF composite film is expected to be an ideal scaffold material for artificial cornea preparation.

Key words: Collagen, Cornea, Dermatan Sulfate, Tissue Engineering

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