基因修饰的生物可降解人工骨修复骨缺损的血管化研究

目的评价转染骨形成蛋白2（bone morphogenetic protein 2，BMP-2）基因的骨髓间充质干细胞（marrow mesenchymal stem cells，MSCs）复合生物可降解人工骨修复兔桡骨缺损的血管化过程，观察BMP-2基因对促进移植骨血管化的影响。方法利用携带BMP-2基因的腺病毒载体（adenovirus carrying BMP-2 gene，Ad—BMP-2）转染MSCs及复合人工骨制备。取新西兰大耳白兔60只制成双侧桡骨中段1．5cm骨缺损模型，随机分为4组，每组15只（30侧）。各组采用不同材料植入缺损，A组：Ad—BMP-2转染MSCs＋聚乳酸／聚己内酯（polylactic acid／polycaprolactone，PLA／PCL）；B组：β-半乳糖酐酶基因的重组腺病毒转染MSCs＋PLA／PCL；C组：未转染MSCs＋PI，A／PCL；D组：单纯PLA／PCL。分别于术后4、8和12周各组处死5只动物，行X线片、微血管分布、组织学、透射电镜观察，并行血管内皮生长因子（vascular endothelial growth factor，VEGF）表达检测及微血管计数。结果A组4周时见移植骨内片状成骨影，有较多新生血管长入，支架孔隙内充满软骨痂，功能活跃的成骨细胞围绕微血管生长，VEGF表达及微血管数均明显高于其它各组，且差异有统计学意义（P〈0．01）；8周时移植骨内成骨逐渐增多，微血管迂曲扩张并相互连接，软骨痂转变为小梁骨；12周时皮质骨连续，髓腔再通，微血管呈规则地纵向排列。B、C组成骨能力较弱，血管再生缓慢，12周时骨缺损得到初步修复，微血管沿新生骨小梁孔隙分布。D组各时间点新生血管少见，12周时骨端硬化，缺损区被纤维组织填充。结论BMP-2基因转染可通过上调VEGF表达，间接诱导移植骨血管化，促进种子细胞成活，加速新骨形成。

VASCULARIZATION IN TRANSPLANTATION OF GENE MODIFIED TISSUE ENGINEERED BONE FOR REPAIRING BONE DEFECT

Objective To study the vascularization of the composite of bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), Ad-Lacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (P<0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defected area was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by up-regulating VEGF expression, indirectly induces vascularization of grafts,promotes the living of seed cells, and thus accelerates new bone formation.