中国修复重建外科杂志

中国修复重建外科杂志

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

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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。

ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd 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).ResultsAfter 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.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.

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

Key words: Cartilage tissue engineering; acellular cartilage extracellular matrix oriented scaffold; chondrocytes; scaffold material

引用本文: 鹿亮, 刘彬, 尚希福, 张雨, 陈伟健, 刘舒云, 黄靖香, 汪爱媛, 郭全义, 卢世璧. 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. 中国修复重建外科杂志, 2018, 32(3): 291-297. doi: 10.7507/1002-1892.201710095 复制

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