混合阳离子交联剂甲基丙烯酸海藻酸盐三维培养软骨细胞  

Three-dimensional Culture of Chondrocyte Using Methacrylic Alginate Gel Beads Cross-linked with Mixed Metal-cation

作　　者：汪洋[1] 冯玉霞[1] 樊星[2] 任利玲[1] 

机构地区：[1]兰州大学口腔医学院,兰州730000 [2]延安大学附属医院,延安716000

出　　处：《生物医学工程学杂志》2015年第3期599-604,共6页Journal of Biomedical Engineering

基　　金：兰州大学中央高校基本科研业务费专项资金资助项目(LZUJBKY-2010-141);国家自然科学基金资助项目(81300860)

摘　　要：本研究旨在建立一种更适合软骨细胞生长的三维培养体系。首先通过酰胺键反应将甲基丙烯酸引入海藻酸盐长链形成甲基丙烯酸海藻酸盐(MA),然后将MA和混合阳离子Ba2+∶Ca2+=5∶5进行光交联反应,形成内部结构为贯通式凝胶网络(IPN)的凝胶小球(MA凝胶小球)。将P1代软骨细胞包裹在MA凝胶小球中连续培养三周,在培养的不同时间点取适量凝胶小球进行倒置显微镜观察,冰冻切片后HE染色和免疫组化染色,扫描电镜观察第21d细胞在支架中的情况;通过Alamar blue法检测软骨细胞的增殖情况;二甲基亚甲兰法定量检测糖胺多糖(GAG)的含量。结果显示MA和混合阳离子Ba2+∶Ca2+=5∶5发生光交联反应形成的MA凝胶小球,与对照组藻酸钙相比,P1代软骨细胞在MA凝胶小球内增殖更明显,细胞外基质GAG的分泌也更多,扫描电镜下观察到软骨细胞可以良好地黏附在支架中生长,免疫组化染色呈阳性。以上结果证实软骨细胞在MA凝胶小球中的增殖和细胞外基质的分泌量优于藻酸钙组,并且能维持软骨细胞的表型,是一种更好的软骨细胞三维培养载体。This study was to explore a better three-dimensional（3-D）culture method of chondrocyte.The interpenetrating network（IPN）gel beads were developed through a photo-cross linking reaction with mixed barium ions and calcium ions at the ratio of 5∶5with the methacrylic alginate（MA）,which was a chemically conjugated alginate with methacrylic groups.The second generation of primary cartilage cells was encapsulated in the MA gel beads for three weeks.In the designated timing,HE stain,Alamar blue method and Scanning electron microscopic were used to determine the cartilage cells growth,proliferation and the cell distribution in the scaffolds,respectively.The expression of typeⅡ collagen was investigated by an immunohistochemistry assay and the glycosaminoglycan content was quantitatively evaluated with the spectrophotometry of 1,9dimethylene blue assay.Compared to the alginate control group,the deposition of glycosaminoglycan was significantly upregulated in IPN-MA gel beads with higher cell proliferation.The secretion of extracellular matrix and proliferation of chondrocyte in methacrylic alginate gel beads were higher than that in Alginate beads.Cells were able to attach,to grow well on the scaffolds under scanning electron microscopy.The result of immunohistochemistry staining of collagen typeⅡ was positive,confirming the maintenance of chondrocyte phenotype in methacrylic alginate gel beads.This study shows a great potential for three-dimensional culture of cartilage.

关 键 词：甲基丙烯酸海藻酸盐 三维培养 软骨细胞 阳离子交联剂 光交联反应 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]