Differential Effects of BMPs on the Healing of Craniofacial Defects

摘要 一种诱人的骨再生方式包括使用多能性 由成骨因子诱导的间充质干细胞。我们的总体目标是 骨形态发生蛋白对小鼠骨愈合影响的差异性研究 颅面缺损模型。这个项目是基于这样一个假设，即存在一个 关于成骨BMP(BMP2、4、6、7、9)的可定义的、不同的轮廓 诱导修复临界大小的颅面缺损的能力。为了检验这一假设， 具体目标如下：1)i.构建重组腺病毒 表达单个BMP(BMP-2、-4、-6、-7、-9)；为了确定干细胞的作用 用上述重组腺病毒转染腺病毒后的表达水平 干细胞中的各种增殖和/或成骨分化标志物，III。至 测定不同BMP(2、4、6、7、9)对小鼠骨愈合的不同影响 小鼠模型中的临界尺寸缺陷。四、来描述不同地区的这种差异效应 非承重区(颅骨)和承重区(下颌)颅面修复； 2)V.建立颅骨/下颌骨成骨细胞的原代培养，用于体外培养 骨形态发生蛋白刺激，即用骨形成蛋白刺激原代培养的成骨细胞 不同的BMP，并测量每个BMP在早期诱导成骨的能力 分化后期；3)靶向规范的Wnt/β连环蛋白信号 通过si-RNA技术，检测体外和体内途径抑制的效果 活着。。通过si-RNA技术靶向Smad4信号转导并检测 Smad4在体外和体内均有抑制作用。这些具体目标将由 实验设计如下：1)将骨髓间充质干细胞 携带BMPs的腺病毒(Ad-BMPs)；2)骨髓间充质干细胞感染 Ad-BMPs及其干细胞的体外成骨分化检测 重组腺病毒骨形态发生蛋白诱导的骨髓间充质干细胞移植入临界大鼠体内 应用Micro-CT评价颅面骨缺损及愈合情况；4)RT-PCR检测 BMP诱导干细胞成骨中重要的下游信号元件；5) 在体外和体内，siRNA击倒这些关键的信号元件。

ABSTRACT An attractive modality for bone regeneration involves the use of pluripotent mesenchymal stem cells that are induced by osteogenic factors. Our overall goal is to investigate differential effects of bone morphogenetic proteins on bone healing in murine craniofacial defect models. This project is based on the hypothesis that there is a definable, differential profile of the osteogenic BMPs (BMP2, 4, 6, 7, 9) in regards to their capacity to induce healing of critical-sized craniofacial defects. To test this hypothesis, the following specific aims are proposed: 1) i. To construct recombinant adenoviruses expressing individual BMPs (BMP-2, -4, -6, -7, -9), ii. To determine the effect of stem cell transfection with the recombinant adenoviruses above on the expression levels of various proliferation and/or osteogenic differentiation markers in the stem cells, iii. To determine the differential effects of the various BMPs (2, 4, 6, 7, 9) on bone healing of critical sized defects in murine models. iv. To characterize this differential effect in areas of non-load bearing (calvarial) and areas of load-bearing (mandibular) craniofacial repair; 2) v. To generate primary cultures of calvarial/mandibular osteoblasts for in vitro stimulation with BMP, vi.To stimulate primary cultures of isolated osteoblasts with the various BMPs and measure the capacity of each BMP to induce osteogenesis in early and late stages of differentiation; 3) vii. To target canonical Wnt/beta catenin signaling via si-RNA technology and examine the effects of pathway inhibition both in vitro and in vivo. viii. To target SMAD4 signaling via si-RNA technology and examine the effects of SMAD4 inhibition both in vitro and in vivo. These specific aims will be addressed by the following experimental design: 1) Transfection of mesenchymal stem cells with adenovirus encoded with BMPs (Ad-BMPs); 2) Infection of mesenchymal stem cells with Ad-BMPs and testing of these stem cells in vitro for osteogenic differentiation; 3) Transfer of Ad-BMP induced mesenchymal stem cells into critical-sized murine craniofacial defects and evaluation of defect closure via micro-CT; 4) RT-PCR of downstream signalling elements important in BMP-induced stem cell osteogenesis; 5) siRNA knockdown of these critical signalling elements both in vitro and in vivo.