聚乳酸/聚己内酯生物可降解人工骨复合骨形态发生蛋白2基因转染骨髓基质干细胞修复骨缺损的血管化过程(英文)

Polylactic acid/polycaprolactone in combination with marrow mesenchymal stem cells modified by bone morphogenetic protein 2 for the repair of bone defect during vascularization

学术背景：组织工程骨植入体内后，再血管化是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：评价经骨形成蛋白2基因修饰的骨髓间充质干细胞复合聚乳酸／聚己内酯人工骨修复兔桡骨缺损的血管化过程，观察骨形成蛋白2基因对促进移植骨血管化的影响。设计：随机对照动物实验。单位：实验于2005-01／12在中国医科大学中心实验室完成。材料：聚乳酸／聚己内酯生物可降解材料块由中国科学院长春应用化学研究所提供，孔隙直径150～250μm，孔隙率90％以上；实验动物为3月龄新西兰大耳白兔60只。方法：60只新西兰大耳白兔双侧桡骨中段制成1．5cm骨缺损模型，随机分为4组，每组15只（30侧），植入经不同处理的人工骨。①AD-BMP-2组：转染骨形成蛋白2基因的细胞＋聚乳酸／聚己内酯。②对照基因组：转染β-半乳糖酐酶基因的细胞＋聚乳酸／聚己内酯。③未转染组：未转染细胞＋聚乳酸／聚己内酯。④单纯聚乳酸／聚己内酯组：植入单纯聚乳酸，聚己内酯支架。主要观察指标：于术后4，8和12周行X射线片观察新骨形成情况，立体显微镜观察微血管分布，苏木精．伊红染色观察微血管与骨形成关系，透射电镜观察成骨细胞和血管内皮细胞间的联系，并行血管内皮生长因子表达检测及微血管计数。结果：①AD-BMP-2组术后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，血管内皮生长因子表达及微血管数均明显高于其他各组；术后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；术后12周时皮质骨连续，髓腔再通，微血管呈规则地纵向排列。②对照基因组和未转染组成骨能力较弱，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。③单纯聚乳酚聚己内酯组各时间点新生血管少见，术后12周时骨端硬化，缺损区被纤维组织填充。结论：骨形成蛋白2基因转染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，促进种子细胞的成活，加速新骨形成。

BACKGROUND： Revascularization is necessary for tissue-engineered bone implantation by osteogenesis to effectively repair bone defect. OBJECTIVE： To evaluate marrow mesenchymal stem cells （MSCs） modified by bone morphogenetic protein 2 （BMP-2） in combination with polylactic acid/polycaprolactone （PLA/PCL） to repair rabbit radial bone defect during the vascularization, and to investigate the promotive effects of BMP-2 gene on the vascularization of bone graft. DESIGN： Randomized controlled animal study. SETTING： This study was performed in the Central Laboratory of China Medical University from January to December 2005. MATERIALS： PLA/PCL with 150-250 μm pore diameter and 90% interval porosity was provided by Changchun Applied Chemistry Institute, Chinese Academy of Science. Sixty 3-month-old New Zealand rabbits were selected in this study. METHODS： Sixty rabbits were randomly divided into four groups with 15 rabbits in each group. Subsequently, middle segments of bilateral radial bone were obtained to establish 1.5-cm bone defect models that were implanted with processed artificial bones. Adenovirus carrying BMP-2 （AD-BMP-2） group： Artificial bones were processed with transfected BMP-2 cells plus PLA/PCL; Control group： Artificial bones were processed with adenovirus carrying 13 -gal gene （Ad-Lacz） plus PLA/PCL; Non-transfection group： Artificial bones were processed with non-transfected cells plus PLA/PCL; PLA/PCL group： PLA/PCL alone for transplantation. MAIN OUTCOME MEASURES： Four, eight, and twelve weeks after surgery, X-ray was used to observe new bone formation; stereoscopic microscope to observe distribution of microvessels; haematoxylin-eosin staining to detect the relationship between microvessels and bone formation; transmission electron microscope to investigate the correlation between osteoblasts and vascular endothelial cells, detect vascular endothelial growth factor expression, and calculate the number of microvessels. RESULTS： Four postoperative weeks in the AD-BMP-2 group, numerous microvessels were observed; stent pore was full of cartilage calluses; active osteoblasts grew around microvessels; vascular endothelial growth factor expression and numbers of microvessels were higher and more than those in other groups. Eight postoperative weeks, osteoblasts gradually increased in the bone graft; microvessels circuitously expanded and connected each other; cartilage callus changed into trabecular bone. Twelve postoperative weeks, cortical bones were successive; medullary cavity recanalized; microvessels longitudinally arranged in order. Ability of bone formation in the control group and non-transfection group was weak, and vascular regeneration was slow; 12 postoperative weeks, bone defect was primarily repaired; microvessels were distributed along the pores of newborn bone trabecula. Newborn vessels were hard found in the PLA/PCL group at each time point. Twelve postoperative weeks, bone extremities sclerotized, and defect regions were fully filled by fiber tissues. CONCLUSION： Transfected BMP-2 gene by up-regulating vascular endothelial growth factor expression can indirectly induce vascularization of bone graft, promote survival of seed cells, and accelerate bone formation.