丝素蛋白/壳聚糖复合支架有良好的细胞相容性与渗透性  

Good cell compatibility and permeability of silk fibroin/chitosan composite scaffolds

作　　者：章晓云[1] 陈跃平[1] 宋世雷 张驰 卓映宏 杨楠 占华松 许灿宏 Zhang Xiaoyun;Chen Yueping;Song Shilei;Zhang Chi;Zhuo Yinghong;Yang Nan;Zhan Huasong;Xu Canhong(Department of Orthopedics,Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine,Nanning 530011,Guangxi Zhuang Autonomous Region,China;Graduate School of Guangxi University of Traditional Chinese Medicine,Nanning 530001,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]广西中医药大学附属瑞康医院骨科,广西壮族自治区南宁市530011 [2]广西中医药大学研究生学院,广西壮族自治区南宁市530001

出　　处：《中国组织工程研究》2020年第16期2544-2550,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81760796,81960803)，项目负责人：陈跃平;广西高校青年教师基础能力提升项目(2019KY0352,KY2016YB204)，项目负责人：章晓云;广西中医药大学2019年校级科研课题(2019QN027)，项目负责人：章晓云;广西中医药大学一流学科课题(2019XK029)，项目负责人：章晓云;广西自然科学基金课题(2015GXNSFAA139136)，项目负责人：陈跃平;广西卫生厅重点课题(S201419-05)，项目负责人：陈跃平;2016年全国名老中医传承工作室建设项目，项目参与者：章晓云~~

摘　　要：背景:丝素蛋白与壳聚糖为组织工程常用的支架材料,但二者单独应用均存在一定的不足,将两者混合使用可以互为改性,充分发挥优点,获取理想的复合支架材料。目的:制备丝素蛋白/壳聚糖复合支架并对其进行性能测定。方法:通过冷冻干燥方法制备丝素蛋白/壳聚糖复合支架,采用电镜扫描检测复合支架的形态结构,并进行热重分析、力学性能及细胞毒性检测。制备季铵化壳聚糖,利用核磁共振仪表征其核磁氢谱,Zeta电位仪检测其电位和粒径分布,凝胶电泳实验检测其保护DNA的情况,透射电镜观察其与DNA结合情况。结果与结论:①扫描电镜显示丝素蛋白/壳聚糖复合支架具体良好的三维孔洞结构,孔径为50-100μm;②热重分析显示当温度小于200℃时,丝素蛋白/壳聚糖复合支架的质量损失下降速度较低;当温度上升至200-500℃时,支架质量损失速度开始加快,损失量增多;在800℃时,复合支架的残余质量为38%;③丝素蛋白/壳聚糖复合支架的最大应变可以达到94.94%,最大承受应力为7.01 MPa;④CCK-8实验显示,丝素蛋白/壳聚糖复合支架对兔骨髓间充质干细胞没有细胞毒性,具有良好的细胞相容性;⑤核磁氢谱检测显示,季铵化壳聚糖的季铵化程度约为20%;⑥季铵化壳聚糖的粒径分布为(588.56±52.39)nm,季铵化壳聚糖颗粒的表面带正电荷,电位为(16.3±3.92)mV,有利于与DNA结合;⑦凝胶电泳实验显示,季铵化壳聚糖材料的比例越高,对DNA的包裹越好,当其与DNA的比例为1∶3时,对DNA达到包裹作用;⑧透射电镜显示,季铵化壳聚糖/DNA大部分的微粒呈实心圆形,微粒粒径差别较小,平均粒径约为200 nm;⑨结果表明,丝素蛋白/壳聚糖复合支架有良好的细胞相容性与细胞渗透性,利于细胞在支架间的生长。BACKGROUND: Silk fibroin and chitosan are commonly used as scaffolds in tissue engineering, but there are some shortcomings in th eir separate application. When they are mixed, they can be modified each other. They give full play to each other’s advantages and become ideal composite scaffolds. OBJECTIVE: To prepare Silk fibroin/chitosan composite scaffold and determineits properties. METHODS: The silk fibroin/chitosan composite scaffolds were prepared by freeze-drying method. The morphology and structure of the composite scaffolds were examined by scanning electron microscopy. The properties of the composite scaffolds were tested by thermogravimetric analysis, mechanical properties test, and cytotoxicity test. The quaternion chitosan was prepared. The nuclear magnetic resonance spectrum was detected by nuclear magnetic resonance instrument. The potential and particle size distribution were detected by Zeta potentiometer. The protection of DNA was detected by gel electrophoresis. The binding with DNA was observed by transmission electron microscope. RESULTS AND CONCLUSION:(1) The results of scanning electron microscopy showed that the silk fibroin/chitosan composite scaffolds had a good three-dimensional pore structure, with a pore size of 50-100 μm.(2) The results of thermogravimetric analysis showed that when the temperature was less than 200 °C, the mass loss rate of silk fibroin/chitosan composite scaffold was lower. When the temperature increased to 200-500 °C, the mass loss rate of the scaffold began to accelerate, and the loss amount increased. At 800 °C, the residual mass of the composite scaffold was 38%.(3) The maximum strain of silk fibroin/chitosan composite scaffold reached 94.94%, and the maximum stress was 7.01 MPa.(4) The results of CCK-8 experiment showed that silk fibroin/chitosan composite scaffolds had no cytotoxicity to rabbit bone marrow mesenchymal stem cells and had good cell compatibility.(5) The results of nuclear magnetic resonance spectra showed that the quaternion degree of quate

关 键 词：丝素蛋白/壳聚糖复合支架 生物降解速率 力学性能 细胞毒性 季铵化壳聚糖 蚕丝蛋白 骨髓间充质干细胞 生物相容性 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]