可注射性组织工程软骨源性微载体微观结构及其生物相容性观察.docVIP

可注射性组织工程软骨源性微载体微观结构及其生物相容性观察 　 作者：卢强，张莉，彭江，许瑞江，黄靖香，郭全义，卢世璧 【摘要】 ［目的］改进软骨源性微载体的制备方法。对其微观结构特征及其与骨髓间充质细胞的生物相容性进行观察，探索新的可注射性组织工程软骨的制备方法。［方法］将新鲜的猪关节软骨在液体中粉碎，梯度离心后制备成150～300 μm的颗粒，去细胞处理后采用常规组织学方法观察软骨微粒的空间结构及化学组成，采用扫描电镜观察软骨源性微载体的形态特征，随后体外获取扩增骨髓间充质细胞，与软骨微粒复合，然后采用旋转式生物反应器扩增，构建可注射性组织工程软骨细胞。［结果］本研究制备的软骨微粒呈圆形或椭圆形，表面呈毛刷状结构，主要成分是Ⅱ型胶原和GAG，而毛刷的主要成份Ⅱ型胶原、骨髓间充质干细胞不仅与微载体结合良好，还能够在其表面大量扩增。［结论］与传统的微载体不同，软骨源性微载体与细胞复合后，不需要再将细胞消化，避免了软骨细胞外基质的损失，可以作为可注射性组织工程软骨的理想材料和方法。 【关键词】 微载体； 软骨； 骺损伤； 组织工程 Abstract: ［Objective］To improve the preparative method of catilage derived microcarrier and evaluate the micromechanism and biocompatibility of this metarial for injectable tissue engineering cartilage. ［Methods］Fresh porcine articular cartilage were obtained and shattered in the iso-osmia liquid in 4℃. After gradient centrifugation, 150-300 μm size cartilage micelles were gathered and were subjected to 1% Triton X- 100 once and physiological saline twicely. The structure of specimens were observed and assessed by inverted phase contrast microscopy,environmental scanning electron microscope.And the composition of these specimens were stained with haematoxylin-eosin, safranin-O, toluidine blue and immunohistochemistry of collagen type Ⅱ.The microcarriers were seeded with rabbit bone mesenchymal stem cells (BMSCs) and cocultured with Rotary Cell Culture System (Rotary Cell Culture System, RCCS TM).［Results］The cartilage particles had fiocculus apparence.There were numerous villus on the surface of these cartilage micelles, which were stained positive with the immunohistochemistry of collagen type Ⅱ.The inner part of these cartilage micelles were stained positively with safranin-O and toluidine blue. After coated with BMSCs and cultured in the RCCSTM, the cells grew well on the surface of the cartilage micelles and the latter could be disperse again after blowed with pipette.［Conclusion］The cartilage micelles, which have large surface area and good boicompatibility,is a new kind of microcarrier for inject