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5mm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7d),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。
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. （1）MSCs were cultured in vitro, purified, expanded and marked by Brd-U, then cultured with heterogeneous BMG in vitro to prepare MSC/BMG composite. （2）Sixty 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. （3）A 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 3^rd generation were marked by Brd-U at density of 1×10^9 L^-1）.（4）The 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. （1）Radiographic 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）. （2）The 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.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第15期2801-2805,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
辽宁省教育厅科研项目(2004C039)~~
