负载威灵仙总皂苷丝素蛋白微载体复合软骨细胞促进兔膝关节软骨缺损修复的实验研究  

Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair

作　　者：涂鹏程 马勇 潘娅岚[2,4] 汪志芳 孙杰[1,2] 陈凯 杨光露[1,2] 王礼宁[2,3] 刘孟敏 郭杨 TU Pengcheng;MA Yong;PAN Yalan;WANG Zhifang;SUN Jie;CHEN Kai;YANG Guanglu;WANG Lining;LIU Mengmin;GUO Yang(Department of Orthopedics,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing Jiangsu,210029,P.R.China;Laboratory of New Techniques of Restoration and Reconstruction of Orthopedics and Traumatology,Nanjing University of Chinese Medicine,Nanjing Jiangsu,210023,P.R.China;School of Chinese Medicine,School of Integrated Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing Jiangsu,210023,P.R.China;Nursing Institute of Integrated Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing Jiangsu,210023,P.R.China;Department of Orthopedics,Zhangjiagang Affiliated Hospital of Nanjing University of Chinese Medicine,Suzhou Jiangsu,215699,P.R.China)

机构地区：[1]南京中医药大学附属医院骨伤科,南京210029 [2]南京中医药大学骨伤修复与重建新技术实验室,南京210023 [3]南京中医药大学中医学院·中西医结合学院,南京210023 [4]南京中医药大学中西医结合护理研究所,南京210023 [5]南京中医药大学张家港附属医院中医骨伤科,江苏苏州215699

出　　处：《中国修复重建外科杂志》2022年第3期343-351,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金面上项目(81673995);江苏高校优势学科(中西医结合)建设工程资助项目(苏政办发[2018]87号);江苏省研究生科研创新计划(KYCX17_1308、KYCX21_1683);2018年地方高校国家级大学生创新训练计划(201810315008)。

摘　　要：目的制备负载威灵仙总皂苷(clematis total saponins,CTS)丝素蛋白微载体,探讨其复合软骨细胞后促进兔膝关节软骨缺损修复的效果。方法取5%丝素蛋白溶液与10 mg/mL CTS溶液、甘油混匀后,利用高压静电场结合冷冻干燥方法制备负载CTS丝素蛋白微载体,扫描电镜对样本表征并检测CTS累积释放量;同时制备丝素蛋白微载体。取6只4周龄新西兰大白兔膝关节软骨,分离培养软骨细胞并传代。取第3代细胞分别与两种微载体在微重力条件下共培养7d,期间倒置相差显微镜及扫描电镜观察软骨细胞在微载体上黏附情况,细胞计数试剂盒8(cell counting kit 8,CCK-8)检测细胞增殖活性,并与正常培养细胞比较。取30只3月龄新西兰大白兔制备双侧膝关节软骨缺损模型后随机分为3组(n=20),A组膝关节软骨缺损不作任何处理,B、C组分别采用丝素蛋白微载体-软骨细胞复合物、负载CTS丝素蛋白微载体-软骨细胞复合物填充关节软骨缺损。术后12周取材,ELISA检测关节液基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)、MMP-13、MMP组织抑制因子1(tissue inhibitor of MMP 1,TIMP-1)水平;大体观察及组织学观察(HE、甲苯胺蓝染色)评估软骨缺损修复情况;Western blot检测Ⅱ型胶原及蛋白聚糖表达;组织学及诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)免疫组织化学染色观察关节滑膜炎症程度。结果负载CTS丝素蛋白微载体呈类球形,直径主要在300~500μm之间,表面呈多孔结构,孔隙率达35.63%±3.51%;药物缓释检测示微载体中CTS可长期缓慢释放。微重力条件下随培养时间延长,两种微载体表面黏附的软骨细胞逐渐增多,培养24 h时软骨细胞增殖活性均较正常培养细胞提高(P<0.05),两种微载体间差异无统计学意义(P>0.05)。动物体内实验观察,与A、B组相比,C组关节液中MMP-9、MMP-13含量均降低(P<0.05),TIMP-1含量上调(P<0.05)。与A组相比,B、C组Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins(CTS)(CTSsilk fibroin microcarrier),and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair.Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5%silk fibroin solution,10 mg/mL CTS solution,and glycerin.The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected.Meanwhile,unloaded silk fibroin microcarrier was also prepared.Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured.The 3 rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment.During this period,the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope,and the proliferation activity of cells was detected by cell counting kit 8(CCK-8),and compared with normal cells.Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into3 groups(n=20).Knee cartilage defects in group A were not treated,and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes,respectively.At 12 weeks after operation,the levels of matrix metalloproteinase 9(MMP-9),MMP-13,and tissue inhibitor of MMP 1(TIMP-1)in articular fluid were detected by ELISA.The cartilage defects were collected for gross observation and histological observation(HE staining and toluidine blue staining).Western blot was used to detect the expressions of collagen typeⅡand proteoglycan.The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase(iNOS)immunohistochemical staining.Results The CTS-silk fibroin microcarrier was sph

关 键 词：软骨组织工程 软骨修复 丝素蛋白 威灵仙总皂苷 微重力 细胞培养 兔 

分 类 号：R684[医药卫生—骨科学]