胶原/硫酸软骨素/成纤维生长因子复合人工眼角膜的制备  

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

作　　者：毛宝亮 胡斌 贾磊 单恒悦 李响 王颖 袁琬娟 张凤超 陈敬华 Mao Bao- liang;Hu Bin;Jia Lei;Shan Heng- yue;Li Xiang;Wang Ying;Yuan Wan- juan;Zhang Feng- chao;Chen Jing-hua(School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu Province, China)

机构地区：[1]江南大学药学院制药工程系,江苏省无锡市214122

出　　处：《中国组织工程研究》2018年第14期2203-2208,共6页Chinese Journal of Tissue Engineering Research

基　　金：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)~~

摘　　要：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。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 concentr

关 键 词：胶原 角膜 硫酸皮肤素 组织工程 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 

分 类 号：R318[医药卫生—生物医学工程]