课题背景：课题为广东省自然科学基金资助项目(06301417)；广东省卫生厅资助项目(A2006726)。本实验拟通过基因工程与组织工程的有机结合来获得新生骨组织。实验结果表明转染人骨形态发生蛋白2较未转染在体内形成新骨能力明显增强，人骨形态发生蛋白2基因修饰的自体细胞复合支架材料，改善成骨环境促进骨愈合，加速了组织工程化骨的形成。

同行评价：联合应用基因工程和组织工程技术研制具有较强生物诱导活性的组织工程骨是近5年来引人注目的研究方向。文章采用转染人骨形态发生蛋白2基因的组织工程化骨修复骨质疏松症下颌骨缺损，具有一定创新性，对相关领域研究具有一定指导意义和参考价值。

偏倚或不足：实验中虽然人骨形态发生蛋白2基因修饰的组织工程化骨植入绝经后骨质疏松症大鼠下颌骨骨缺损区，增强了其骨再生能力，但8周时的结果显示仍未达到完全的骨性愈合。究竟是在体内相关细胞外环境的改变，还是脂质体介导的基因转移效率不高所致，尚待深入观察。

摘要

背景：近年来基因修饰干细胞与支架材料复合形成组织工程化骨为修复骨缺损提供了骨组织重建的全新思路。
目的：实验拟观察转染人骨形态发生蛋白2基因转染骨髓间充质干细胞复合珊瑚羟基磷灰石支架材料修复骨质疏松症下颌骨缺损的效果。
设计、时间及地点：随机对照动物实验，于2005-06/2006-10在教育部口腔生物医学工程重点实验室完成。
材料：6月龄未孕雌性SD大鼠24只，SPF级，体质量250~300 g，构建绝经后骨质疏松症动物模型。含人骨形态发生蛋白2基因的pcDNA3.1-hBMP-2重组质粒由解放军第三军医大学提供；支架材料珊瑚羟基磷灰石由卫生部口腔生物医学工程重点实验室提供。
方法：24只SD模型大鼠随机数字表法分为实验组和对照组，每组12只。3个月后分别取其骨髓间充质干细胞培养。实验组骨髓间充质干细胞通过脂质体Lipogen介导转染pcDNA3.1-hBMP-2质粒，对照组细胞未作处理。3 d 后将每只大鼠骨髓间充质干细胞与珊瑚羟基磷灰石支架材料复合，再分别对应地植入取材动物的自体下颌骨极限性骨缺损区。
主要观察指标：术后第4，8周组织切片行免疫组织化学染色，观察新骨形成情况。术后8周进行成骨量的半定量检测。
结果：24只SD模型大鼠均进入结果分析。①术后4周实验组在珊瑚羟基磷灰石材料边缘有新生骨质形成，8周时见相互连接的层板状成熟骨基质形成，对照组4周时只在材料边缘有少量骨基质形成，8周时新生骨质数量上明显少于实验组，且材料边缘及中央处有脂肪样结构形成。②对照组术后8周新生骨所占面积百分比为（0.046±0.004）%，实验组为（0.233± 0.015）%，各组成骨量差异具有显著性意义（P < 0.01）。
结论：人骨形态发生蛋白2基因修饰的骨髓间充质干细胞复合珊瑚羟基磷灰石支架形成的组织工程化骨修复骨质疏松症下颌骨的成骨量较无基因修饰明显增加。
关键词：骨形态发生蛋白2；骨质疏松症；细胞分化；基因治疗

唐尤超，王远勤，汤炜.基因修饰骨髓间充质干细胞复合珊瑚羟基磷灰石支架材料修复骨质疏松性下颌骨缺损[J].中国组织工程研究与临床康复，2008，12(14):2601-2605 [www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2601(ps).pdf]

Repairing mandibular defect by gene-modified bone marrow mesenchymal stem cells combined with coral hydroxyapatite scaffold in osteoporotic rats

Abstract

BACKGROUND: In recent years, tissue engineered bone by gene-modified stem cells compounded with scaffold materials in repair of bone defect has provided a novel thought for the bone tissue reconstruction.
OBJECTIVE: To explore the effect on repairing mandibular defect with human bone morphogenetic protein-2 (hBMP-2) gene transfected bone marrow mesenchymal stem cells (BMSCs) compounded with coral hydroxyapatite (CHA) scaffolds in osteoporotic rats.
DESIGN, TIME AND SETTING: A randomized control animal experiment was completed at the Key Laboratory for Oral Biomedical Engineering by the State Ministry of Education from June 2005 to October 2006.
MATERIALS: Twenty-four unpregnant female Sprague-Dawley rats, 6 months old, SPF gradem weighing 250-300 g, were used to establish models of postmenopausal osteoporosis. The pcDNA3.1-hBMP-2 recombinant plasmid carrying hBMP-2 was offered by the Third Military Medical University of Chinese PLA; CHA scaffolds was offered by the Key Laboratory for Oral Biomedical Engineering by the State Ministry of Education.
METHODS: Twenty-four SD rats were divided into experimental group and control group at random, each containing 12 animals. Three months later, BMSCs harvested from osteoporotic rats were transfected by pcDNA3.1-hBMP-2 recombinant plasmid, and control group leaved untreated. Three days later, the transfected and untransfected autogenous BMSCs were seeded into CHA scaffolds, respectively. Then the cell/scaffolds composites were implanted into the critical-size defect area in the mandible bone of osteoporotic rats.
MAIN OUTCOME MEASURES: At the 4th and 8th weeks after implantation, immunohistochemical analysis was performed to evaluate the new bone formation, and the amount of newly formed bone was detected semi-quantitatively at the 8th weeks.
RESULTS: All 24 SD rats were involved in the result analysis.①Newly formed bone was found at the margin of the defect area treated with the BMSCs modified by BMP-2 gene transfer at 4 weeks after implantation. The lamellar bone matrix was visible at 8 weeks. In the control group, only a little bone matrix was observed at the margin of CHA material at 4 weeks after implantation, however, the amount of newly formed bone was much less than the experimental group, and there was some adipose-like tissues at defect margins and central part at 8 weeks after implantation.②The percentage of newly formed bone area was (0.046±0.004)% and (0.233±0.015)% in the control group and the experimental group, respectively. There were significant differences in the amount of newly formed bone between two groups (P < 0.01).
CONCLUSION: Tissue engineered bone formed by hBMP-2 gene-modified BMSCs in conjunction with CHA scaffolds, can allow large bone formation for treatment of mandibular defect in osteoporosis rats.

Tang YC, Wang YQ, Tang W.Repairing mandibular defect by gene-modified bone marrow mesenchymal stem cells combined with coral hydroxyapatite scaffold in osteoporotic rats.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(14):2601-2605
[www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2601(ps).pdf]