Vascularized reaction of antigen-extracted bovine cancellous bone stent combining with bone morphogenetic protein 2 during reparative process of bone defect
- VernacularTitle: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
- Author:
Wei YU
;
Jianjun LI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2008;12(23):4559-4562
- CountryChina
- Language:Chinese
-
Abstract:
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.
