关节软骨源性微载体生物相容性及异位成软骨特性观察  

Biocompatibility and chondrogenesis of articular cartilage-derived microcarrier

作　　者：李丙岩 彭江[1] 王鑫[1] 王玉[1] 苟文隆[1] 袁雪凌[1] 徐峰[1] 黄靖香[1] 陈继营[1] 卢世璧[1] 

机构地区：[1]中国人民解放军总医院骨科研究所,北京100853

出　　处：《中华关节外科杂志（电子版）》2013年第6期56-59,共4页Chinese Journal of Joint Surgery(Electronic Edition)

基　　金：国家自然科学基金(31240048);全军十二五重点项目(BWS11J025)

摘　　要：目的 观察改良后软骨源性微载体的微观结构及主要成分,其对软骨细胞增殖的影响及体内异位成软骨特性.方法 将新鲜猪软骨片粉碎后,梯度筛滤后得到直径大200～400 μm的软骨源性微载体,经1%十二烷基硫酸钠（SDS）脱细胞处理后,Hoechst 33258荧光染色观察其DNA残留,采用扫描电镜对其微结构进行观察,通过甲苯胺蓝、番红花O组织学染色和Ⅱ型胶原免疫荧光染色观察微载体的主要成分;体外复合软骨细胞后,通过MTT观察软骨源性微载体对软骨细胞增殖的影响;将复合有软骨细胞的软骨源性微载体包埋于裸鼠皮下观察其异位成软骨能力.结果 改良后的微载体形态近似椭圆形,直径200～400 μm,微丝覆盖其表面,微丝长度40～90 nm;脱细胞处理后Hoechst 33258荧光染色阴性;甲苯胺蓝和番红花O组织学染色及Ⅱ型胶原免疫组化染色阳性;在MTT检测细胞增殖中,第1天以后软骨源性微载体组及条件培养基组的OD值均高于完全培养基组（P〈0.05）;复合有软骨细胞的软骨源性微载体具有异位成软骨能力.结论 本实验成功制备的软骨源性微载体,可作为天然微载体高效扩增软骨细胞,可为组织工程提供优质种子细胞;其具有异位成软骨能力,有望成为软骨组织工程的新型支架.Objective To improve the previous preparation of the articular cartilage-derived microcarrier （CDM） and to detect the composition, influence on the proliferation of chondrocytes in vitro and the capability of chondrogenesis in vivo of this mierocarrier. Methods The fresh cartilage tablets were smashed into cartilage slurry by the dishes grinder. The cartilage slurry was diluted with distilled water and was filtered through meshes. And then the CDMs of 200 - 400 μm in diameter were harvested. The 1% SDS was used to get rid of the cells on the CDMs. Hoechst 33258 staining was used to detect the left DNA. The composition, GAG and collagen Ⅱ were detected by the scanning electron microscope, alcian blue staining, safaranin 0 staining, and collagen Ⅱ immunohistochemistry staining. The influence of CDMs on the chondrocytes proliferation was assessed by MTT in vitro. The CDMs loaded with the chondrocytes were embedded subeutaneously in the nude mice to test the eapability of chondrogenesis. Results The CDMs of 200-400 μm diameter, were harvested successfully by the improved preparation and were oval in shape. The CDMs were covered with microfllaments, 40-90 nm in length. The Hoechst 33258 fluorescent staining was negative after decellularization. The alcian blue staining, safaranin O staining and collagen Ⅱ immunohistochemistry staining were positive. In MTF test, the OD value of the CDMs group and the condition medium group were higher than the complete medium group except the first day （ P 〈 0. 05）. The CDMs loaded with the chondrocytes had the capability of chondrogenesis in vivo. Conclusion In this experiment the successfully harvested CDMs could serve as a natural carrier to proliferate chondrocytes for the tissue engineering and have the potential to become the new natural scaffold in the cartilage tissue engineering.

关 键 词：细胞外基质 软骨细胞 细胞增殖 组织工程 

分 类 号：R684[医药卫生—骨科学]