摘要
背景:细胞体外培养情况下无法在远离营养物质200μm以上的区域存活,血管网络构建对组织工程领域厚组织和器官再生至关重要,同轴细胞打印为体外构建类血管通道提供了一种新的方式。目的:优化生物墨水的同轴细胞打印性能,制备具有类血管结构的组织工程支架。方法:通过间歇式巴氏灭菌制备无菌海藻酸钠溶液,冷冻保存;以脱胶蚕丝为原料制备无菌丝素蛋白冻干粉,密封保存;将丝素蛋白冻干粉加入解冻的海藻酸钠溶液中,再加入人脐静脉内皮细胞,作为生物墨水;将生物3D打印机的外轴连接生物墨水,内轴连接交联剂,同轴打印类血管支架材料,进行光学相干层析成像扫描、扫描电镜观察;拉伸测试海藻酸钠与丝素蛋白/海藻酸钠同轴打印环形试件(不含细胞)的弹性模量。采用冷冻保存7 d的海藻酸钠溶液与人脐静脉内皮细胞制作同轴打印支架,冷冻保存7 d的海藻酸钠溶液、人脐静脉内皮细胞与密封保存6个月的丝素蛋白冻干粉制作同轴打印支架,培养24 h后死活染色观察细胞存活率。设计打印串联与并联结构的类血管支架,培养1,3,7,10,14 d后检测细胞增殖情况。结果与结论:①光学相干层析成像扫描显示,该混合生物墨水最高打印高度为9层,整体厚度约为4.4mm;扫描电镜显示,类血管支架的中空纤维丝外壁呈无规则条状卷曲,存在微米级内部连通孔隙结构,中空纤维丝内壁具有更致密的孔隙结构;②丝素蛋白/海藻酸钠同轴打印环形试件的弹性模量大于单纯海藻酸钠同轴打印环形试件(P<0.05);③采用保存7 d海藻酸钠溶液制作的支架细胞存活率为(86.7±3.4)%,加入丝素蛋白冻干粉支架的细胞存活率为(98.1±1.2)%,说明冷冻保存7 d的海藻酸钠溶液未染菌,丝素蛋白的保质期可达6个月;④并联结构类血管支架培养7,10,14 d的细胞增殖活性高于串联结构的类血管支架(P<0.05);⑤结果表明,实验制备的类血管支架材料具有良好的生物相容性与机械性能。
BACKGROUND:Cells cannot survive in the area 200μm away from nutrients in vitro.Vascular network construction is crucial for thick tissue and organ regeneration in tissue engineering.Coaxial cell printing provides a new way to construct vascular-like channels in vitro.OBJECTIVE:To optimize the coaxial cell printing performance of bioink and to build the tissue-engineered scaffolds with vascular-like structure.METHODS:The aseptic sodium alginate solution was prepared by intermittent pasteurization and then frozen.Freeze-dried powder of aseptic silk fibroin was prepared from degummed silk and sealed.The thawed sodium alginate solution was added to the silk fibroin protein freeze-dried powder and human umbilical vein endothelial cells were added to prepare the bioink.The outer axis of the biological three-dimensional printer was connected with the bioink,and the inner axis was connected with the crosslinking agent.The scaffolds were prepared by coaxial printing,and performed by optical coherence tomography,scanning electron microscopy observation and tensile test.Coaxial scaffolds were made by freeze-preserved sodium alginate solution for 7 days with human umbilical vein endothelial cells.Coaxial scaffolds were also made by freeze-dried sodium alginate solution for 7 days with human umbilical vein endothelial cells and silk fibroin protein sealed for 6 months.The cell survival rate was detected by dead and alive staining after 24 hours of culture in vitro.Vascular-like scaffolds with series and parallel structures were designed and printed.The cell proliferation was detected after 1,3,7,10,and 14 days of culture.RESULTS AND CONCLUSIONS:(1)The optical coherence tomography showed that the maximum printing height of the bioink was 9 layers and the overall thickness was about 4.4 mm.Scanning electron microscopy showed that the outer wall of hollow fiber-filament of vascular-like scaffolds presented irregular strip-shaped crimp with micron-scale internal connected pore structure,while the inner wall of hollow fiber-filament had denser pore structure.(2)The elastic modulus of silk protein freeze-dried scaffold was higher than that of sodium alginate solution(P<0.05).(3)The cell survival rate of scaffolds treated with sodium alginate solution for 7 days was(86.7±3.4)%,and that of scaffolds treated with silk protein freeze-dried powder for 7 days was(98.1±1.2)%,indicating that the sodium alginate solution freezepreserved for 7 days was free of bacteria and the shelf-life of silk protein could be up to 6 months.(4)The proliferation activity of cells cultured with parallel structure for 7,10,and 14 days was higher than that with series structure(P<0.05).(5)These results imply that the scaffolds have good biocompatibility and mechanical properties.
作者
张一帆
张佳颖
徐铭恩
王玲
张翮
Zhang Yifan;Zhang Jiaying;Xu Mingen;Wang Ling;Zhang He(School of Life Information and Instrument Engineering,Hangzhou Dianzi University,Hangzhou 310018,Zhejiang Province,China;Zhejiang Provincial Key Lab of Medical Information and Three-Dimensional Bio-Printing,Hangzhou 310018,Zhejiang Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2020年第22期3553-3558,共6页
Chinese Journal of Tissue Engineering Research
基金
国家重点研发计划(2017YFC1103400),项目负责人:徐铭恩
国家自然科学基金(61675059),项目负责人:王玲。
关键词
双网络生物墨水
同轴打印
类血管结构
生物3D打印
组织工程支架
通道网络
串并联
细胞活性
dual network bioink
coaxial printing
vascular-like structure
three-dimensional bioprinting
tissue-engineered scaffold
channel network
series parallel
cell activity
