去抗原牛松质骨支架复合骨形态发生蛋白2基因在骨缺损修复过程中的血管化反应

背景：体外构建的组织工程骨是细胞与材料的复合体，再血管化是组织工程骨植入体内后关系其能否充分发挥成骨作用，有效修复骨缺损的关键环节。目的：观察以去抗原牛松质骨支架复合骨形态发生蛋白2（bone morphogenetic protein2，BMP-2）基因修复大段骨缺损过程中对血管化的影响。设计、时间及地点：随机分组设计、对照动物实验，于2003—09／2004—12在吉林大学中日联谊医院中心实验室完成。材料：选用清洁级新西兰大耳白兔60只。选取市售新鲜牛肱骨上端松质骨制备去抗原牛松质骨支架（Bovine Cancellous Bone，BCB）。携带人BMP-2和β-半乳糖酐酶基因的重组腺病毒（Ad-BMP-2和Ad—Lacz）及重组人BMP-2（rh-BMP-2）由美国Dr．Oliver及吉林大学病理教研室高航博士馈赠。方法：新西兰大耳白兔60只分离培养兔骨髓基质干细胞，经BMP-2腺病毒载体转染后复合BCB移植修复兔双上肢桡骨1．5cm的缺损。按随机数字表法分为5组，每组12只，植入经不同处理的人工骨，Ad—BMP-2转染细胞＋BCB组，未转染细胞＋rh-BMP-2＋BCB组，Ad-Lacz转染细胞＋BCB组，未转染细胞＋BCB组，单纯BCB组。通过紧密缝合肌膜和筋膜固定移植骨。主要观察指标：术后4，8，12周X射线检查观察骨缺损区的新骨形成情况，微血管墨汁灌注观察血管分布情况，透射电镜观察成骨细胞和血管生成情况，苏木精一伊红染色、爱辛蓝染色和VonKossa染色观察微血管与骨形成关系，血管内皮生长因子免疫组织化学染色测定染色灰度值、CD34免疫组织化学染色特异性标记血管内皮细胞，进行微血管计数。结果：纳入兔60只均进入结果分析。X射线检查和组织学观察显示AD-BMP-2转染细胞＋BCB组和未转染细胞＋rh—BMP-2＋BCB组骨形成及血管生成情况优于其他3组。微血管墨汁灌注显示术后4周，两组每一个骨小梁孔隙中有一支新形成的小血管，骨间血管的密度在周边区域较高，随着向移植骨中央呈向心性减少。透射电镜观察，两组术后4周，可见较多功能活跃的成骨细胞及新生血管芽；术后12周，成熟的板层骨形成，新生血管结构完好。血管内皮生长因子表达检测和微血管计数测定显示AD—BMP-2转染细胞＋BCB组血管内皮生长因子相对含量明显高于其他各组，差异有显著性意义（P〈0．01）。微血管计数明显高于其他各组，差异有显著性意义（P〈0．01）。结论：BMP-2基因可通过上调血管内皮生长因子表达，间接诱导移植骨血管化，比应用rh—BMP-2更有优势。

Vascularized reaction of antigen-extracted bovine cancellous bone stent combining with bone morphogenetic protein 2 during reparative process of bone defect

BACKGROUND: Tissue engineered bone constructed in vitro is a compound of cell and material; additionally, revascularization plays a key role in effectively repairing bone defect after transplantation of tissue engineered bone.OBJECTIVE: To evaluate the influence of antigen-extracted bovine cancellous bone stent combining with bone morphogenetic protein 2 (BMP-2) on vascularized reaction during reparative process of bone defect.DESIGN, TIME AND SETTING: Randomized grouping design and controlled animal study, which was performed at the Central Laboratory, China-Japan Friendship Hospital Affiliated to Jilin University.MATERIALS: Sixty rabbits of clean grade were selected in this study. Superior cancellous bone of bovine humerus was used to establish bovine cancellous bone stent. Recombinant adenovirus carrying human BMP-2 (Ad-BMP-2) and β -galactosidae gene (Ad-Lacz), and recombinant human BMP-2 (rhBMP-2) were graciously presented by Dr. Oliver and Pro. Gao, Department of Pathology of Jilin University.METHODS: Marrow mesenchymal stem cells were extracted from 60 rabbits, and then they were transfected with BMP-2 adenovirus vector to repair 1.5-cm defects of radial bone of both upper extremities by combining with bovine cancellous bone transplantation. Rabbits were randomly divided into 5 group with 12 in each group: Ad-BMP-2 transfected cells+bovine cancellous bone group, non-transfected cells+rh-BMP-2+bovine cancellous bone group, Ad-Lacz transfected cells +bovine cancellous bone group, non-transfected cells + bovine cancellous bone group, and bovine cancellous bone group. Transplanted bone was fixed by tightly suturing tunica muscularis and anadesma.MAIN OUTCOME MEASURES: New bone formation was observed by X ray at 4, 8, and 12 weeks after surgery; microvascular ink perfusion was used to observe vascular distribution; transmission electron microscope was used to observe osteoblasts and vascularization; hematoxylin-eosin (HE) staining, alcian blue staining, and VonKossa staining were used to observe correlation between microvessels and bone formation; immunohistochemical staining of vascular endothelial growth factor was used to detect gray value; immunohistochemical staining of CD34 was used to specifically label vascular endothelial cells for microvascular amount.RESULTS: Sixty rabbits were included into the final analysis. X-ray and immunohistochemical examinations demonstrated that bone formation and vascularization in the Ad-BMP-2 transfected cells+bovine cancellous bone group and non-transfected cells+rh-BMP-2+bovine cancellous bone group were superior to those in other three groups. After four weeks, microvascular ink perfusion indicated that a branch of small vessels was formed in pore of trabecular bone. Vascular density was higher in the peripheral domains but lower in the central regions. Transmission electron microscope suggested that a lot of osteoblasts and new vascular buds with active function were observed four weeks after surgery. Mature lamellar bone was formed 12 weeks after surgery, and structure of new vessels was complete. Detection of vascular endothelial growth factor expression and microvascular amount indicated that content of vascular endothelial growth factor in the Ad-BMP-2 transfected cells+bovine cancellous bone group was significantly higher than that in other four groups (P<0.01), and microvascular amount was also significantly higher than that in other four groups (P<0.01).CONCLUSION: BMP-2 gene can indirectly induce vascularization of transplanted bone through up-regulating vascular endothelial growth factor expression, which is superior to rh-BMP-2.