可注射性骨组织工程载体海藻酸钠-明胶共混体系的致畸突变试验

Aberration test of injectable tissue engineered bone carriers with algin-gelatin blend system

背景:利用组织工程学技术将各种凝胶系统作为支架材料对骨缺损进行修复日益受到重视。所选择的支架材料必须具有无毒、无致畸的特点。目的:观察海藻酸钠-明胶共混体系/成骨细胞凝胶修复兔颅骨缺损的愈合过程中对染色体畸变的影响。设计、时间及地点:材料学体内动物实验,于2007-10/2008-03在北京世纪坛医院和北京大学医学部组织胚胎学教研室完成。材料:清洁级2月龄新西兰纯种大白兔12只,随机分为2组:实验组8只,雌性4只,雄性4只,对照组4只均为雌性。海藻酸钠干粉为美国Sigma公司产品,明胶干粉为河北绿岛公司产品。方法:将12只兔编号后抽取骨髓,密度梯度离心法分离骨髓基质干细胞,加入成骨细胞诱导液进行体外培养,诱导分化的成骨细胞经传代增殖为107数量级。制备海藻酸钠与明胶质量比为2∶3的透明粉红色胶状液体,引入兔成骨细胞,细胞终浓度为5×109L-1,与CaCl2溶液混合,形成果冻样海藻酸钠-明胶共混体系/成骨细胞凝胶。12只兔颅骨均制备直径1.5cm的极限缺损,1周后实验组植入凝胶复合体0.5mL进行修复,对照组不植入任何材料。主要观察指标:缺损修复后3个月取心脏血观察细胞遗传学的变化。结果:①每只实验动物观察100个中期分裂细胞,均未见染色体型畸变。对照兔在400个中期分裂相中见4个,单体畸变率为1%。实验兔在800个中期分裂相中见12个,单体畸变率为1.5%。两组均在正常范围内。②每只实验动物做2个染色体核型分析,染色体数目2n=44,对照兔核型为44,XX,正常雌性;雄性实验兔为44,XY,未见异常;雌性实验兔为44,XX,未见到异常。结论:海藻酸钠-明胶共混体系/成骨细胞凝胶在细胞遗传学角度上是安全的。

BACKGROUND： Utilizing tissue engineering technique, various gel systems are served as scaffolds to repair bone defect. The scaffolds should have features of nontoxic and no teratoiogical effects to the body. OBJECTIVE： To observe the effect of sodium alginate-gelatin/osteoblast gel on chromosomal pattern aberration in rabbits. DESIGN, TIME AND SETTING： The in vivo material animal experiments were conducted at the Beijing Shijitan Hospital and Department of Histology and Embryology, Peking University Health Science Center from October 2007 to March 2008. MATERIALS： A total of 12 New Zealand rabbits, aged 2 months, with clean grade, were randomly divided into 2 groups. The experimental group contains 4 female and 4 male rabbits, and the remaining 4 females were served as the control group. Sodium alginate dried powder were purchased from Sigma, USA, and the gelatin dried powder were supplied by Ludao Company, Hebei, China. METHODS： Following numbering, bone marrow was collected from 12 rabbits. Bone marrow stromal stern cells （BMSCs） were isolated by the density gradient centrifugation, and then in vitro cultured with osteoblast inductor. Osteoblasts following passage were an order of magnitude of 10^7. Bright pink gelatiniform liquid with mass ratio of sodium alginate and gelatin at ratio of 2：3 was prepared. Rabbit osteoblasts with final concentration of 5× 10^9/L were mixed with CaCl2 solution to form fruit jelly-shaped sodium alginate-gelatin/osteoblast gel. Critical-sized calvarial defects were created in diameter of 1.5 cm in 12 rabbits. After 1 week, cell/scaffold complex （0.5 mL） was implanted to repair the bone defect in the experimental group. There was no treatment in the control group. MAIN OUTCOME MEASURES： The change of chromosomal pattern was observed at 3 months following reparation. RESULTS： No Chromosome somatotype aberration was found in 100 metaphases in the experimental group. From 400 metaphases of the control group, 4 abnormal cells were found, with 1% chromatid-type aberration ratio. Meantime, 12 abnormal cells in 800 metaphases of the control group were found, with 1.5% chromatid-type aberration ratio. The numerical value was within the normal range. Chromosome karyotype analysis： the chromosome number of each experimental rabbit was 2n=44, karyotype of the control rabbit was 44, XX, which was normal female; or 44, XY, normal male, no abnormal was found. The female rabbit in the experiment group was 44, XX, no abnormal was seen. CONCLUSION： From the cytogenetoxicity point of view, sodium alginate-gelatin/osteoblast gel is safe in repairing bone defects.