海藻酸钠-明胶共混体系/成骨细胞凝胶修复兔颅骨极限缺损的CT评估

Application of CT evaluation in repairing the maximum defects of rabbit skull using sodium alginate-gelatin/osteoblast gel

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,在骨修复过程中需要选择相对准确、方便的检测方式,以明确体内成骨过程。目的:利用螺旋CT和三维重建监测海藻酸钠-明胶共混体系/成骨细胞凝胶修复兔颅骨缺损的愈合过程。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15只,由北京海淀区兴隆实验动物养殖场提供。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,螺旋CT为德国SIEMENS公司产品。方法:将15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,细胞终密度为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。15只兔颅骨均制备直径1.5cm的极限缺损,1周后植入凝胶复合体0.5mL进行修复。主要观察指标:缺损修复后4,8,12周利用CT进行薄层扫描和三维重建检查,采用苏木精-伊红染色和Mallory三色染色观察骨组织愈合情况。结果:修复后4周,冠状位CT显示颅骨缺损区可见骨痂形成,三维重建仍显示有缺损;标本苏木精-伊红染色显示缺损为纤维结缔组织,并有软骨样组织形成,Mallory三色染色标本呈淡蓝色。修复后8周,冠状位CT显示缺损边缘圆钝,与凝胶材料有骨性突起连接,缺损处密度增高明显,三维重建显示缺损范围较之前有所减小;标本苏木精-伊红染色后缺损部可见大量含血管成分的纤维结缔组织,有骨样组织结构形成,Mallory三色染色显示有褐红色成熟骨组织形成,其旁边有淡蓝色软骨样组织。修复后12周,冠状位CT显示缺损区基本被骨痂填满,三维重建示缺损基本修复;缺损区域和周围骨组织形成骨性结合,骨小梁粗大,哈弗氏系统成熟。结论:海藻酸钠-明胶共混体系/成骨细胞凝胶以软骨化骨的形式成功修复了兔颅骨极限缺损。在修复过程中,CT冠状位扫描可准确反映出各阶段成骨的过程,但三维重建有一定的局限性,对成骨的判断会产生一定的误差。

BACKGROUND： Various gel systems were served as scaffolds using tissue engineering technique and received more and more interest, while accurate and convenient indications are required to judge the status of in vivo bone formation. OBJECTIVE： To observe the repair of bone defects by CT scanning and 3-dimensional （3D） reconstruction in rabbit calvariums using sodium alginate-gelatin/osteoblast gel. DESIGN, TIME AND SETTING： The material, in vivo animal experiments were conducted at the Beijing Shijitan Hospital and Department of Histology and Embryology, Medical College, Peking University from October 2007 to March 2008. MATERIALS; A total of 15 clean 2-month New Zealand rabbits were supplied by Xinglong Experimental Animal Nursery, Beijing. Sodium alginate dried powder （Sigma, USA）, gelatin dried powder （Lvdao, Hebei, China）, and spiral CT （Siemens, Germany） were used in this study. METHODS： Following numbering, bone marrow was collected from 15 rabbits. Bone marrow stromal stem cells （BMSCs） were isolated by the density gradient centdfugation and in vitro cultured with osteoblast inductor. Osteoblasts following passage were an order of magnitude of 107. Bright pink gelatiniform liquid with mass ratio of sodium alginate and gelatin of 2：3 was prepared. Rabbit osteoblasts at 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 15 rabbits. After I week, cell/scaffold complex （0.5 mL） was implanted. MAIN OUTCOME MEASURES： Bone healing was observed by CT scanning and 3D reconstruction 4, 8, and 12 weeks after surgery. Hematoxylin and eosin staining and Mallory trichrism staining were applied to observe the bone repair. RESULTS： Four weeks following repair, CT showed that callus formation appeared in coronal skull defects. 3D reconstruction still demonstrated some defects. Hematoxylin and eosin staining displayed defects were fibrous connective tissue, with the presence of chondroid tissues. Mallory trichrism staining exhibited light blue samples. Eight weeks following repair, CT showed round blunt defect edges in the coronal position, which had bony pustute connection with gel materials. Density at the defect region significantly increased. 3D reconstruction suggested that defects became small. After hematoxylin-eosin staining, abundant fibrous connective tissue appeared in defect regions, with bony tissues. Mallory trichrism staining revealed that maroon mature bone tissues were found, surrounded by light blue chondroid tissues. Twelve weeks following repair, coronal defect regions were filled with callus. 3D reconstruction showed that defects were repaired. Defect region and surrounding bony tissues had bony connection, with thick bone trabecula and mature Haversian system. CONCLUSION： Sodium alginate-gelatin/osteoblast gel successfully repairs rabbit cranium limited defects. During repair, CT scanning can exactly reflect osteogenic process in the coronal position. However, 3D reconstruction has some limitations, and can obtain some errors to judge osteogenic formation.