基于多模态成像技术监测基因修饰支架中血管生成及修复临界性骨缺损的研究  

Investigation of Angiogenesis in Genetically Modified 3D-PLGA/nHAp Scaffolds by Using Multimodality Imaging Techniques and Promotes Critical Sized Bone Defects Repair in vivo

作　　者：李健[1] 孙语韬 刘成波[2] 林日强[2] 郑炜[2] 任培根[1] 

机构地区：[1]中国科学院深圳先进技术研究院转化医学研究与发展中心,深圳518055 [2]中国科学院深圳先进技术研究院生物医学光学与分子影像研究室,深圳518055

出　　处：《集成技术》2018年第1期11-24,共14页Journal of Integration Technology

基　　金：广东省公益基础研究项目(2015A020212030);深圳市科技计划项目(JCYJ20160531185738467;JCYJ20170413165503382);中国科学院深圳先进技术研究院优秀青年创新基金(Y5G0101001)

摘　　要：基因修饰促血管化的支架是促进骨再生的有效方法之一,它可以将目的基因转移到内源性细胞中实现生长因子原位、持续表达,诱导内源性细胞的增殖、迁移和分化,从而促进骨组织再生。该文以慢病毒介导基因修饰多孔PLGA/n HAp复合支架诱导血管生成为研究对象,采用静电吸附和低温冷冻方法制备基因修饰多孔PLGA/n HAp复合支架;以小鼠顶骨临界性骨缺损为模型,利用多模态双光子及光声显微成像技术在体、实时、连续监测,研究骨修复过程中基因修饰多孔支架诱导血管化的动态过程。体外实验结果显示,慢病毒颗粒从支架上的持续释放长达5天,缓释出的病毒颗粒可以有效转染293T细胞并表达PDGF-BB因子。体内实验结果表明,慢病毒介导基因修饰多孔PLGA/n HAp复合支架,可以实现PDGF-BB因子原位表达,促进体内局部及系统性干细胞等细胞迁移,加快血管诱导生成并提高骨缺损部位骨组织的再生能力。同时,在该研究中,成功使用并比较了多模态双光子及光声显微成像技术在体、实时、连续监测3D骨组织支架内血管形成的动态变化过程,并验证了基因修饰对于提高3D打印支架的生物学反应性的作用。该研究为研究不同支架对血管生成作用的监测与鉴定提供了新的技术手段。Genetic modification of biological scaffold to enhance angiogenesis is an effective method for bone regeneration. In this study, a 3 D-PLGA/n HAp scaffold containing pdgfb-expressing lentiviral vectors to enhance angiogenesis for calvarial critical bone defect repair was designed. The modified scaffolds（LVpdgfb/PLGA/n HAp） could continuously release bioactive LV-pdgfb particles for up to 5 days in vitro. In scaffold implanted critical calvarial bone defect mouse model, how the genetically modified 3 D scaffolds affect the angiogenesis and bone formation was studied by two-photon and photoacoustic imaging, micro CT and histomophological methods. Eight weeks post-implantation, blood vessel areas in LV-pdgfb/PLGA/n HAp scaffolds were significantly higher than in PLGA/n HAp scaffolds at each observed time point. In accordance with the angiogenesis process, micro CT analysis indicated that the repairment of the critical-calvarial defects in LV-pdgfb/PLGA/n HAp group dramatically improved compared to the other groups, including bone mineral density（BMD）, the ratio of bone volume to tissue volume（BV/TV）, trabecular number（Tb.N）. In this study, we verified and compared the application of two state of the art non-invasive in vivo imaging techniques in imaging of neo-vasculature inducing in 3 D bone artifact, and demonstrated that lentivirus-mediated pdgf-bgene modified scaffolds could be a promising tool to build vascularized tissue engineering bone to repair a large bone defects in murine model.

关 键 词：临界性骨缺损 多模态双光子成像 光声成像 基因修饰 3D打印支架 血管生成 

分 类 号：TG156[金属学及工艺—热处理]