新生牛皮胶原蛋白海绵的制备及其体外细胞相容性

背景：现已证实,人肌腱、牛肌腱、鼠尾、猪皮、新生牛跟腱制备的胶原蛋白海绵有良好的细胞相容性。目的：采用新生牛皮提取胶原蛋白、制备生物医用胶原蛋白海绵,观察其与羔羊肾成纤维细胞的相容性。设计、时间及地点：对比观察实验,于2006-05/2007-02在西北民族大学生命科学与工程学院生物工程与技术国家民委重点实验室完成。材料：出生24h内宰杀的新生尕里巴牛犊皮,岷县黑裘皮羔羊肾原代成纤维细胞。方法：取新生牛犊皮,经脱毛、胃蛋白酶＋冰醋酸联合处理、盐析、透析、冻干后制备胶原蛋白海绵。将海绵与羔羊肾F3代成纤维细胞混悬液共培养,分别设胶原海绵材料组、阴性对照组（生理盐水）、阳性对照组（橡胶塞浸提液）。主要观察指标：应用倒置相差显微镜及JVC数码摄像系统观察、拍摄与记录细胞形态、生长情况。培养20,35d,对共培养物进行AO染色,观察细胞在胶原海绵中的增殖情况。培养65d后制石蜡切片,行苏木精-伊红染色,观察细胞在胶原海绵中的生长情况。结果：阳性对照组细胞培养24h细胞圆缩,不贴壁,3d后全部死亡。胶原海绵材料组与阴性对照组细胞形态均正常,细胞贴壁生长良好。随着培养时间的延长,海绵孔隙逐渐变小,细胞数量增加,形态变小,海绵外观从柔软、混浊变得挺拔、透明。AO染色显示培养20,35d的胶原海绵-羔羊肾成纤维细胞共培养物中有大量细胞存在,并且在胶原蛋白空隙中有大量细胞成团簇状生长。苏木精-伊红染色显示有大量蓝色细胞核和新生的红色胶原纤维。结论：制备的新生牛皮胶原蛋白海绵对岷县黑裘皮羔羊肾成纤维细胞有良好的相容性。

Preparation and in vitro cytocompatibility of collagen sponge from newborn bovine skin

BACKGROUND:It is confirmed that collagen sponge prepared from human tendon,bovine tendon,rat tail,pig skin and newborn bovine tendon have good cytocompatibility.OBJECTIVE:To extract collagen from newborn bovine skin,prepare the collagen sponge for biomedical application,and observe the biocompatibility and cytocompatibility of collagen sponge with lamb fibroblasts.DESIGN,TIME AND SETTING:Controlled study was performed in the Key Laboratory of Bioengineering of State Ethical Committee,Life Science and Engineering College,Northwest University for Nationalities from May 2006 to February 2007.MATERIALS:Newborn Galiba bovine within 24 hours and black fur lamb kidney fibroblasts were used.METHODS:Newborn bovine skin was harvested to prepare the collagen sponge with a series of procedures,including depilation,pepsin+glacial acetic acid,salting-out,dialysis and freeze drying.The obtained collagen sponge was inoculated with fibroblast suspension,which were divided into collagen sponge group,negative control group (saline) and positive control group (rubber bung leaching liquor).MAIN OUTCOME MEASURES:Inverted phase contrast microscope and JVC digital camera system were used to observe the cell morphology and growth.Acridine orange dyeing was used to observe the proliferation of cell in collagen sponge at 20 and 35 days of culture.Hematoxylin-eosin staining was used to observe the growth of cells in collagen sponge at 65 days of culture.RESULTS:The cells of positive control group were not adhesive and all died three days later.Those of collagen sponge group and negative control group were normal and adhesive.With the prolong of culture time,the sponge pore decreased gradually,sponge appearance became eminent and transparent,the cell increased in number but decreased in morphology.Acridine orange dyeing at 20 and 35 days of culture showed that a large amount of cells appeared in the co-culture of collagen sponge with lamb kidney fibroblast,and pack of cell clumps grew.Abundant blue nuclei and newborn red collagen fiber were found by hematoxylin-eosin staining.CONCLUSION:The collagen sponge from newborn bovine skin has a good biocompatibility with lamb kidney fibroblast cell of black fur,and no cytotoxicity appears.