Differential Effects of BMPs on the Healing of Craniofacial Defects

摘要 骨再生的一种有吸引力的方式涉及使用多能 间充质干细胞是由成骨因子诱导的。我们的总体目标是 骨形态发生蛋白在小鼠骨愈合中差异作用研究 颅面缺损模型。这个项目是基于一个假设，即有一个 成骨BMP（BMP 2，4，6，7，9）在其生物学特性方面的可定义的差异性特征 诱导临界尺寸颅面缺损愈合的能力。为了验证这个假设， 具体目标如下：一，构建重组腺病毒 表达个体BMP（BMP-2、BMP-4、BMP-6、BMP-7、BMP-9），ii.确定干细胞的作用 用上述重组腺病毒转染， 干细胞中的各种增殖和/或成骨分化标志物，iii.到 确定各种BMP（2，4，6，7，9）对骨愈合的不同作用， 小鼠模型中的临界尺寸缺陷。四.为了描述这种差异效应， 非承重区（颅骨）和承重区（下颌骨）颅面修复; 2)v.产生用于体外培养的颅骨/下颌骨成骨细胞的原代培养物 用BMP刺激，vi. 各种BMP，并测量每种BMP在早期诱导骨生成的能力。 和分化的晚期阶段; 3）vii.靶向经典Wnt/β连环蛋白信号传导 通过si-RNA技术，并检查体外和体内途径抑制的效果， vivo.八.为了通过si-RNA技术靶向SMAD 4信号传导，并检查 体外和体内SMAD 4抑制。这些具体目标将由 以下实验设计：1）转染间充质干细胞， BMP编码的腺病毒（Ad-BMP）; 2）用BMP感染间充质干细胞 Ad-BMPs和体外测试这些干细胞的成骨分化; 3） Ad-BMP诱导的骨髓间充质干细胞在小鼠体内的移植 颅面缺损和通过micro-CT评价缺损闭合; 4）RT-PCR， 在BMP诱导的干细胞成骨中重要的下游信号传导元件; 5） 在体外和体内siRNA敲低这些关键信号元件。

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.