骨髓间充质干细胞复合异种骨基质明胶修复大鼠桡骨缺损*★
田志逢，秦书俭，王瑞芳，柏永刚，谷学静
课题背景：目前骨组织工程中对种子细胞与支架材料复合体修复骨缺损的研究较多，但仍无构建复合体的成熟技术，本课题已取得的成果：①骨髓间充质干细胞与骨基质明胶复合体体外构建时细胞的理想浓度；②复合体构建的骨缺损的修复效果。
应用要点：实验结果证实异种骨基质明胶与骨髓间充质干细胞复合构成的复合体，对骨节段性缺损有较好的修复作用，而且其抗原反应不明显，但在实验中发现材料吸收过快，如果进一步能改善异种骨基质明胶的性状，此材料与骨髓间充质干细胞的复合体可以为临床提供较好的植骨材料。
偏倚或不足：在实验室骨缺损模型制备的过程中，为了减少手术对实验动物的创伤，虽然严格按照骨缺损临界值要求造模，但每只动物骨缺损的位置仍不太一致，降低了实验的完整性。�
摘要
目的：在骨组织工程学领域内，主要集中种子细胞、支架载体和细胞支架复合体的构建3个方面的研究，在干细胞方面大多数学者认为骨髓间充质干细胞较为理想，支架材料虽然种类繁多，但却没有一种理想的载体。实验拟通过观察骨髓间充质干细胞/骨基质明胶复合体植入骨缺损处成骨性细胞的变化以及钙化情况，认识同种骨髓间充质干细胞复合异种骨基质明胶修复大鼠桡骨节段性骨缺损的可行性。
方法：实验于2006-10/2007-08在辽宁医学院解剖学试验室与科学实验中心完成。①骨髓间充质干细胞体外培养、纯化、扩增、Brd-U体外标记后并在体外与骨基质明胶复合培养，制成骨髓间充质干细胞/骨基质明胶复合体。②60只SD大鼠采用随机数字表法分为骨髓间充质干细胞/骨基质明胶复合体治疗组、单纯骨基质明胶治疗组和单纯骨髓间充质干细胞治疗组，每组20只；每组分2，4，8，12周4个时间段，每个时间段5只。 实验过程中对动物处置符合动物伦理学要求。③制备同种异体SD大鼠桡骨骨干5 mm节段性骨缺损模型。骨髓间充质干细胞/骨基质明胶复合体治疗组缺损处植入骨髓间充质干细胞/骨基质明胶复合体，单纯骨基质明胶治疗组缺损处植入单纯骨基质明胶（骨基质明胶由完全培养液同等条件下培养7 d），单纯骨髓间充质干细胞治疗组缺损处植入单纯骨髓间充质干细胞（Brd-U标记的第3代骨髓间充质干细胞，密度1×109个/L）。④大体观察大鼠的术肢活动情况；X射线放射学、组织学对比观察各组大鼠骨缺损的修复情况。
结果：除骨髓间充质干细胞/骨基质明胶复合体治疗组有1例动物因麻醉过深死亡外，其余59只大鼠进入结果分析。①各组术后各时间段骨缺区X射线片表现：骨髓间充质干细胞/骨基质明胶复合体治疗组：2周桡骨缺损处略见骨痂形成，8周可见明显形成骨缺损区的桥接，12周骨缺损区完全被新生骨组织填充，已有骨髓腔出现。单纯骨基质明胶治疗组：12周骨缺损区骨痂形成增多，但未出现桥接，并可见两端有骨质硬化现象。单纯骨髓间充质干细胞治疗组：12周可见两断端有骨质硬化现象，最后形成骨不连。术后 2，4，8，12周复合体治疗组与单纯治疗组放射学检查评价新骨生成差异有显著性意义（P < 0.01）。②组织学检测结果：术后 2，4，8，12周复合体治疗组与单纯治疗组组织学检查新骨生成速度、生成量差异均有显著性意义（P < 0.01）。
结论：骨髓间充质干细胞/骨基质明胶复合体有较强的成骨能力，对节段性骨缺损有较好的修复作用，有望成为骨组织工程中可以选择的植骨材料。
关键词：骨髓间充质干细胞；骨基质明胶；异种；细胞培养； 骨缺损；组织构建
田志逢，秦书俭，王瑞芳，柏永刚，谷学静.骨髓间充质干细胞复合异种骨基质明胶修复大鼠桡骨缺损[J].中国组织工程研究与临床康复，2008，12(15):2801-2805 [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2801(ps).pdf]
辽宁医学院解剖教研室骨研究所，辽宁省锦州市 121001
田志逢★，男，1981年生，河南省濮阳市人，汉族，辽宁医学院在读硕士，主要从事骨组织损伤与修复方向。
zhifeng07@sina.
com
通讯作者：秦书俭，教授，博士生导师，辽宁医学院解剖教研室骨研究所，辽宁省锦州市
121001
辽宁省教育厅科研项目
(2004C039)*
中图分类号:R329.471
文献标识码:A
文章编号:1673-8225
(2008)15-02801-05
收稿日期：2007-11-05
修回日期：2007-11-23
(07-50-11-6065/M·A)
Bone marrow mesenchymal stem cells combined with heterogeneous bone matrix gelatin for the repair of segmental radial defects in rats
Abstract
AIM：In bone tissue engineering domain, seed cells, scaffold and cell-scaffold composite are three focuses in the study. Bone marrow mesenchymal stem cells (MSCs) are generally considered ideal seed cells, but there is not an ideal scaffold. In this study, we investigated the feasibility of MSCs combined with heterogeneous bone matrix gelatin (BMG) to repair the segmental radial defects in rats by observing the change and calcification of osteogenic cells.
METHODS: Between October 2006 and August 2007, the experiment was performed at in the Anatomic Laboratory and Scientific Experiment Center of Liaoning Medical University. ①MSCs were cultured in vitro, purified, expanded and marked by Brd-U, then cultured with heterogeneous BMG in vitro to prepare MSC/BMG composite. ②Sixty SD rats were randomly divided into MSCs/BMG group, BMG group and MSCs group with 20 animals in each group. Moreover, each group was subdivided into four time points: 2, 4, 8 and 12 weeks with 5 animals at each time point. The animal disposal was consistent with the ethical requirements. ③A bone defect (5 mm in length) was created at the left radial backbone in each rat. The composite of MSCs/BMG were implanted into MSCs/BMG group; BMG was implanted into BMG group (the BMG was cultivated in the same condition for 7 days); MSCs were implanted into MSCs group (the MSCs of the 3rd generation were marked by Brd-U at density of 1×109 L-1). ④The activity of the operated limb was evaluated by gross observation; radiographic examination and histological analyses were performed to observe the bone defect repair after surgery.
RESULTS: Except one rat died of overdose of anesthesia, the rests all entered the result analysis. ①Radiographic examinations of each group: In the MSCs/BMG group, there were few callus formations in the radial defects in 2 weeks; evident bridges grafting was observed in the defects in the 8th week; the defects were filled with newly formed bone completely and cavitas medullatis appeared in 12 weeks. In the BMG group, there were more callus formations in 12 weeks, but no bridges grafting was observed and there was osteosclerosis in the two ends. In the MSCs group, the osteosclerosis in the two ends in 12 weeks resulted in bone nonunion eventually. There were significant differences in new bone formation between experimental groups and control group in 2, 4, 8 and 12 weeks after operation (P < 0.01). ②The histological analyses tests showed that the rate and quality of new bone formation were significantly different (P < 0.01).
CONCLUSION: The composite of MSCs/BMG has strong osteogenic ability. It can well repair segmental bone defects, and is promising to serve as grafting material in bone tissue engineering.
Tian ZF, Qin SJ, Wang RF, Bai YG, Gu XJ.Bone marrow mesenchymal stem cells combined with heterogeneous bone matrix gelatin for the repair of segmental radial defects in rats.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008; 12(15):2801-2805(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2801(ps).pdf]
Institute of Orthopedics, Department of Human Anatomy, Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
Tian Zhi-feng★, Studying for master's degree, Institute of Orthopedics, Department of Human Anatomy, Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
zhifeng_07@sina.
com
Correspondence to: Qin Shu-jian, Professor, Tutor of doctor, Institute of Orthopedics, Department of Human Anatomy, Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
Supported by: the Scientific Research Program of Education Department of Liaoning Province, No. 2004C039*
Received: 2007-11-05
Accepted: 2007-11-23
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