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

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150～250 μm,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单纯聚乳酸/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.

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

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 β-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 gone by up-regulating vascular endothelial growth factor expression can indirectly induce vascularization of bone graft, promote survival of seed cells, and accelerate bone formation.