脱细胞软骨细胞外基质取向支架复合软骨细胞构建组织工程软骨的实验研究  

Construction of tissue engineered cartilage based on acellular cartilage extracellular matrix oriented scaffold and chondrocytes

作　　者：鹿亮[1] 刘彬[1] 尚希福[1] 张雨 陈伟健[1] 刘舒云[3] 黄靖香[3] 汪爱媛[3] 郭全义[3] 卢世璧[3] LU Liang;LIU Bin;SHANG Xifu;ZHANG Yu;CHEN Weijian;LIU Shuyun;HUANG Jingxiang;WANG Aiyuan;GUO Quanyi;LU Shibi(Department of Orthopedics, Anhui Provincial Hospital, Hefei Anhui, 230001, P.R.China;Department of Orthopedics, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing Jiangsu, 210008, P.R.China;Orthopedics Research Institute, General Hospital of Chinese PLA, Beijing, 100853, P.R.China)

机构地区：[1]安徽省立医院骨科,合肥230001 [2]南京大学医学院附属南京鼓楼医院运动医学与关节外科,南京210008 [3]中国人民解放军总医院骨科研究所,北京100853

出　　处：《中国修复重建外科杂志》2018年第3期291-297,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金面上项目(81473092)~~

摘　　要：目的探讨脱细胞软骨细胞外基质(acellular cartilage extracellular matrix,ACECM)取向支架复合软骨细胞构建组织工程软骨的可行性。方法取市售猪关节软骨组织,分离培养关节软骨细胞并传代。取第3代软骨细胞行PKH26荧光标记,MTT检测标记对细胞增殖无影响后,分别取标记及未标记的软骨细胞复合ACECM取向支架并体外培养后,大体观察支架形态,倒置显微镜、荧光显微镜观察软骨细胞在支架中的黏附、生长和分布情况,扫描电镜观察支架中细胞形态,Ⅱ型胶原免疫荧光染色观察软骨细胞外基质分泌情况。将PKH26标记的软骨细胞-支架复合物植入裸鼠背部皮下腔隙,术后观察裸鼠一般情况,4周后分子荧光活体成像系统无创伤性评估细胞-支架复合物生长情况,取材行大体观察以及番红O、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色观察,评价形成软骨组织的能力。结果细胞-支架复合物体外培养7 d,大体观察呈半透明并具有一定硬度;倒置显微镜和荧光显微镜观察软骨细胞在支架上能良好黏附生长,并沿支架管道方向生长,分泌Ⅱ型胶原。细胞-支架复合物植入裸鼠皮下后,分子荧光活体成像系统观察示细胞均存活;术后4周,大体观察见复合物呈类软骨样组织,组织学染色及Ⅱ型胶原免疫组织化学染色示细胞周围软骨细胞外基质分泌,可见"陷窝"样结构形成。结论 ACECM取向支架有利于软骨细胞的黏附、增殖及取向性分布类似于正常软骨结构,并在裸鼠皮下成功异位构建组织工程软骨。Objective To observe the feasibility of acellular cartilage extracellular matrix（ACECM） oriented scaffold combined with chondrocytes to construct tissue engineered cartilage. Methods Chondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3 rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold（cell-scaffold complex） was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining（Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining）. Results After chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix（typeⅡcollagen） was observed by histological staining and typeⅡcollagen immunohistochemical staining.Conclusion ACECM oriented scaffold is benefit to the cell adhesion,

关 键 词：软骨组织工程 脱细胞软骨细胞外基质取向支架 软骨细胞 支架材料 

分 类 号：R318.08[医药卫生—生物医学工程] R68[医药卫生—基础医学]