课题背景：课题受四川省科技厅科技攻关资助项目（03GG0092027, 03SG022200321）及国家教育部优秀青年教师教学科研奖励计划资助项目(2003682)资助。在骨组织工程研究领域，对支架材料的研究虽然报道甚多，但一直没有非常理想的选择。纳米陶瓷材料具有小尺寸效应和表面或界面效应，使其呈现出与传统陶瓷显著不同的独特性能。由于比表面积的增加，其引导组织细胞的能力和生物降解性增强。实验旨在探讨纳米双相磷酸钙陶瓷作为替代骨细胞外基质支架材料的可行性。

应用要点：实验将自体骨髓基质细胞与纳米双相磷酸钙陶瓷复合后直接进行颅骨缺损修复，植入后16周有大量新骨生成，X射线片显示陶瓷中央部已有高密度骨形成，表明骨髓基质细胞/纳米双相磷酸钙陶瓷复合物能够有效修复颅骨缺损，可作为支架材料用于组织工程骨组织的构建。

重要的概念：建立极量骨缺损的实验模型是进行骨缺损修复动物实验研究必要的前提。极量骨缺损的目的是最大限度在骨缺损修复的动物实验研究中减少因动物种系、年龄、骨缺损部位不同对骨缺损愈合的混杂因素影响。有学者认为极量骨缺损定义为在某一特定动物的生命周期内，在某一特定的骨上，不能自行愈合的最小骨缺损。另外有学者认为在动物生命周期内愈合小于所造成缺损10%的骨缺损。或者如果1年内修复未达到缺损的10%，就认为此缺损符合极量骨缺损的标准。

摘要
背景：传统生物陶瓷材料作为组织工程支架材料的脆性大，不易加工成形且在体内降解困难，影响新骨的长入和后期的改建，故其应用受到一定限制。
目的：观察纳米双相磷酸钙陶瓷(Biphasic calcium phosphate nanocomposite,NanoBCP)用于组织工程支架修复颅骨缺损的成骨性能。
设计、时间及地点：随机分组、动物对照实验，于2004-09/2005-05在四川大学口腔生物医学工程教育部重点实验室完成。
材料：选择2月龄雄性体健SD大鼠48只，体质量180~200 g，在颅骨上制成直径为8 mm的颅骨全层缺损区为极量骨缺损模型。实验用孔径100~400 μm，含孔率为60%~80%的NanoBCP陶瓷及SD大鼠自体骨髓基质细胞(bone marrow stromal cells,BMSCs)由四川大学口腔生物医学工程教育部重点实验室提供。
方法：按随机数字表法将模型大鼠分为3组，NanoBCP/BMSCs组16只，BMSCs经含地塞米松、抗坏血酸、β-甘油磷酸钠的诱导液培养后与NanoBCP复合植入SD大鼠颅骨缺损区；单纯NanoBCP组16只，仅在相同部位单纯植入NanoBCP支架材料；空白对照组16只不植入任何材料。
主要观察指标：植入后4，16，24周取材，通过X射线片分析、大体和组织学观察评价其成骨性能。
结果：SD大鼠48只均进入结果分析。①植入后4，16周，NanoBCP/BMSCs组新生骨组织逐渐增多，Ⅰ型胶原免疫组织化学染色阳性。植入后24周，NanoBCP/BMSCs组SD大鼠颅骨缺损完全修复，单纯NanoBCP组部分修复，空白对照组未修复。②植入后4，24周，NanoBCP/BMSCs组骨缺损区X射线阻射影像密度逐渐增加，新骨充填，接近于正常骨；单纯NanoBCP组骨缺损区亦见X射线阻射影像，与颅骨邻接区的环形透光影密度逐渐增加，骨缺损中央部密度不均匀。
结论：NanoBCP与BMSCs复合物能够有效地修复颅骨缺损。
关键词：骨髓基质细胞；纳米双相磷酸钙陶瓷；组织工程；支架

王涛，田卫东，李声伟，廖运茂.纳米双相磷酸钙陶瓷组织工程支架复合大鼠自体骨髓基质干细胞修复极量颅骨缺损[J].中国组织工程研究与临床康复，2008，12(14):2606-2610 [www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2606(ps).pdf]

Biphasic calcium phosphate nanocomposite loaded with bone marrow stromal cells for repair of critical cranial defect in rats

Abstract

BACKGROUND:Traditional bioceramics materials, as the carrier for tissue engineering, have been limited to certain application due to their unsatisfactory performances, such as great fragility, difficult shaping process and in vivo degradation, which will affect the new bone formation and later reconstruction.
OBJECTIVE: To observe the osteogenesis ability of biphasic calcium phosphate nanocomposite (NanoBCP) served as tissue engineered carrier for repairing cranial defect.
DESIGN, TIME AND SETTING: The animal controlled experiment was carried out according to random grouping in the Key Laboratory of Oral Biomedical Engineering, Ministry of Education, Sichuan University from September 2004 to May 2005.
MATERIALS: Forty-eight male SD rats, two months old and weighing 180-200 g, were prepared into critical-size defect models in the full-thickness cranial bone. The porosity of NanoBCP scaffold was 60%-80% and the pore size was 100-400 μm. Both NanoBCP and autogenous bone marrow stromal cells (BMSCs) were offered by the Key Laboratory of Oral Biomedical Engineering, Ministry of Education, Sichuan University.
METHODS: All rat models were randomly divided into three groups, and each group contained 16 animals. In NanoBCP/BMSCs group, BMSCs were cultured in inducing liquid containing dexamethasone, antiscorbic acid and β-sodium glycerophosphate, then the defects were filled with the NanoBCP/BMSCs composites; In pure NanoBCP group, the defects were filled only with NanoBCP; and blank control rats were filled with nothing.
MAIN OUTCOME MEASURES: At 4, 16 and 24 weeks after implantation, the osteogenesis activity was evaluated by radiographic analysis, gross observation and histological examination.
RESULTS: All of 48 SD rats were involved in the result analysis.①At 4 and 16 weeks postoperatively, more newly formed bone was found in NanoBCP/BMSCs group, and they were positive for type I collagen immunohistochemical staining. At 24 weeks, cranial defects were completely healed in NanoBCP/BMSCs group, and partly healed in pure NanoBCP group. Blank controls showed no detectable signal of bone defect repair.②At 4 and 24 weeks postoperatively, the density of shadow image in cranial defect areas was increased gradually in NanoBCP/BMSCs group, and newly formed bone was close to normal bone; In pure NanoBCP group, the ring low-density was presented in cranial defects and increased along the periphery of the skull, the central domain showed uneven density image.
CONCLUSION: The NanoBCP/BMSCs composites can repair cranial defects effectively.

Wang T, Tian WD, Li SW, Liao YM.Biphasic calcium phosphate nanocomposite loaded with bone marrow stromal cells for repair of critical cranial defect in rats.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(14):2606-2610
[www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2606(ps).pdf]