【摘要】 目的:探讨脱细胞关节软骨支架材料的制备方法,制备软骨理想的组织工程支架材料。方法:实验于2005-12/2006-08在兰州大学第二医院骨科研究所实验室完成。实验方法:利用冷冻干燥、化学去污剂等方法制备脱细胞的兔关节软骨。在无菌状态下取青紫蓝兔的新鲜兔关节软骨,剪成3.5mm×3.5mm,厚度0.2～2.0mm,在冷冻干燥器中冻干12h。在10g/L Triton X-100、Tris-HCl液内加入蛋白酶抑制剂-苯甲基黄酰氟,持续振荡48h后标本以双蒸馏水连续冲洗后置于DNase I酶和RNase A酶混合液中消化。置于10g/L Triton X-100、Tris-HCl液中洗脱。实验评估:①大体观察:肉眼观察脱细胞后关节软骨的外观形态。②组织学观察:将制备的脱细胞关节软骨行石蜡包埋切片,行苏木精-伊红染色、Massion三色染色并在光镜下观察。③扫描电镜观察:将制备的脱细胞关节软骨以戊二醛-锇酸双固定后,行扫描电镜观察。结果:①脱细胞后关节软骨外观形态:肉眼下可见正常关节软骨呈白色或淡黄色,脱细胞后关节软骨色呈灰白,半透明状,无光泽,外形与软骨相似并且仍维持了关节软骨的结构。②脱细胞后关节软骨的组织学变化:苏木精-伊红染色显示,软骨细胞消失,软骨巢分辨不清,在巢内没有蓝染的核物质,核细胞碎屑,只有红染为残留的细胞外基质。Massion三色染色显示,脱细胞后关节软骨内主要含有胶原纤维。③脱细胞后关节软骨的超微结构:扫描电镜下显示,脱细胞后关节软骨陷窝呈蜂窝状,未见到残余的细胞核、细胞器,残余的空穴高低不平。结论:经冷冻干燥、化学去污剂等方法可完整去除软骨中的细胞成分,保留胶原纤维等细胞外基质。更多还原

【Abstract】 AIM:To investigate the methods of making idcal scaffold materials for acellular articular cartilage.METHODS:From December 2005 to August 2006,the experiment was carried out in the laboratory of Institute of Orthopaedics,the Second Hospital of Lanzhou University.The acellular rabbit articular cartilage was prepared by lyophillization and chemical subtraction.Under aseptic condition,the articular cartilage of Chinchilla rabbits was harvested to cut into 3.5 mm by 3.5 mm,and 0.2-2.0 mm in thickness,following freeze drying 12 hours.After persistent oscillation for 48 hours within mixture of Triton X-100,Tris-HCl(10 g/L) and protease inhibitor phenylmethylsulfonyl fluoride,the cartilage was douched by double distilled water,and digested by DNase I enzyme and RNase A enzyme,then eluted in 10 g/L Triton X-100,and Tris-HCl.①The acellular matrix articular cartilage was macroscopically observed;②The acellular matrix articular cartilage was embedded by paraffin,and subjected to HE staining and Massion trichrome staining;③The acellular matrix articular cartilage was observed under scanning electron microscope.RESULTS:①Macroscopic observation indicated that the normal articular cartilage was white or helveous,after acellular matrix,the articular cartilage was grayish-white,semitransparent,bloom,while remained the resembled appearance and structure of normal articular cartilage.②HE staining results showed that the chondrocytes disappeared,and no blue-stained nuclear material or nuclear patch were found,only red-stained extracellular matrix was left.Massion trichrome staining observation showed that collagenous fibers were the main components in acellular articular cartilage.③Under the scanning electron microscope,the cartilage lacuna of the acellular matrix articular cartilage showed honeycomb appearance,and no cytoblast or cellular organ were found;the remnant cavity was porous.CONCLUSION:Lyophillization and chemical subtraction could completely remove the cell components of the articular cartilage,and retain the extracellular matrix component such as collagenous fibers.更多还原