人关节软骨脱细胞基质制备实验(英文)

Preparation of human articular cartilage acellular matrix

背景:对天然的细胞外基质进行处理,剔除其抗原成分,保留了组织结构的完整性,这种材料具有良好的生物相容性,能为细胞创造尽可能接近体内的培养环境,因此应是组织工程中细胞培养支架的首选。目的:制备人关节软骨脱细胞基质,为进一步研究关节软骨基质作为细胞外支架材料提供方法学资料。设计:以骨组织标本为实验对象,单一样本研究。单位:解放军总医院骨科研究所。材料:实验于2004-01/05在解放军总医院骨科研究所完成。材料来自因股骨颈头下型骨折行关节置换而切除的股骨头。方法:切取人关节软骨,剪裁成3.5mm×4.5mm×2.0mm大小共10块,冻干处理12h,采取化学去污剂TritonX-100及DNA酶和RNA酶等试剂制备脱细胞的人关节软骨。用苏木精-伊红、番红O及软骨蛋白聚糖免疫组化染色等方法进行关节软骨脱细胞定性检测。主要观察指标:脱细胞关节软骨的组织学观察以及软骨蛋白聚糖免疫组织化学染色结果。结果:①苏木精-伊红染色、番红O染色均显示细胞陷窝内已无细胞结构。②软骨蛋白聚糖免疫组织化学染色阳性,提示脱细胞关节软骨基质内仍存在软骨蛋白聚糖。结论:人关节软骨冻干后,经去污剂-酶等处理方法可脱去软骨的细胞成分,成功制备人关节软骨脱细胞基质。其中保留的软骨蛋白聚糖可能仍存在原有的耐压特性。

BACKGROUND:Elimination of antigenic substances from natural extracellular matr ix with the integrity of the tissue structure retained renders the matrix to pos sess better biocompatibility and provides a cell culture environment close to co nditions of the internal environment.Such materials are the primary choice for c ell culture scaffold in tissue engineering. OBJECTIVE:To prepare human articular cartilage acellular matrix so as to provi de a methodological basis for further study of articular cartilage acellular mat rix as cell scaffold materials. DESIGN:A single sample study of bone tissues. SETTING:The experiment was performed in Institute of Orthopedics,General Hospi tal of PLA,between January and May in 2004.The specimens were obtained from pati ents requiring joint replacement for femoral neck fracture. MATERIAIS:The experiment was conducted in the Department of Orthopedics, Gener al Hospital of PLA from January to May in 2004.Human articular cartilage specime ns were obtained from the femoral head of patients with total hip arthroplasty f or femoral neck fracture. METHODS:Totally 10 specimens of fresh articular cartilage(3.5 mm ×4.5 mm×2.0 mm) were obtained and freeze dried for 12 hours. Cartilage acellular matrix wa s prepared using Triton X 100,DNase and RNase and identified by means of hemato xylin eosin(HE) and safranine O staining and immunohistochemical staining for c artilage proteoglycan. MAIN OUTCOME MEASURES:Histological observation of the articular cartilage acel lular matrix and immunohistochemical staining of cartilage proteoglycan. RESULTS:HE and safranine O staining both showed no cellular structure in the m atrix with only recesses left by the removed cells. Immunohistochemical staining for cartilage proteoglycan yielded positive results,suggesting the presence of cartilage proteoglycan in the acellular matrix. CONCLUSION:Human articular cartilage acellular matrix can be prepared using th e modified four step procedures with detergent and enzymatic extraction with ly ophilization,and the preserved cartilage proteoglycan in the material may retain good pressure resistance.