Differential Effects of BMPs on the Healing of Craniofacial Defects

描述（由申请人提供）：骨再生的一种有吸引力的方式涉及使用由成骨因子诱导的多能间充质干细胞。我们的总体目标是研究骨形态发生蛋白对小鼠颅面缺损模型骨愈合的差异作用。该项目基于这样的假设，即成骨BMP（BMP 2，4，6，7，9）在诱导临界尺寸颅面缺损愈合的能力方面存在可定义的差异性特征。为了验证这一假设，提出了以下具体目标：1）i。为了构建表达单个BMP（BMP-2、-4、-6、-7、-9）的重组腺病毒，ii.为了确定用上述重组腺病毒转染干细胞对干细胞中各种增殖和/或成骨分化标志物的表达水平的影响，iii.确定各种BMP（2，4，6，7，9）对小鼠模型中临界尺寸缺损骨愈合的差异作用。四.为了表征在非承重区域（颅骨）和承重区域（下颌）颅面修复中的这种差异效应; 2）v.为了产生颅骨/下颌成骨细胞的原代培养物用于用BMP体外刺激，vi.为了用各种BMP刺激分离的成骨细胞的原代培养物并测量每种BMP在分化的早期和晚期诱导骨生成的能力; 3）vii.通过si-RNA技术靶向经典Wnt/β连环蛋白信号传导，并在体外和体内检查通路抑制的效果。八.通过si-RNA技术靶向SMAD 4信号传导，并在体外和体内检查SMAD 4抑制的作用。这些具体目的将通过以下实验设计来解决：1）用BMP编码的腺病毒（Ad-BMP）转染间充质干细胞; 2）用Ad-BMP感染间充质干细胞并在体外测试这些干细胞的成骨分化; 3）将Ad-BMP诱导的间充质干细胞转移到临界大小的小鼠颅面缺损中并通过micro-CT评价缺损闭合; 4）在BMP诱导的干细胞成骨中重要的下游信号传导元件的RT-PCR; 5）这些关键信号传导元件在体外和体内的siRNA敲低。 公共卫生相关性：由于出生、创伤或癌症切除而造成的巨大缺损几乎需要无限的骨来源，而自体骨供区的骨来源不可能不发生严重的并发症。一种提供大量骨骼的方法是转移可以诱导骨骼再生的工程多能干细胞。本文所描述的项目与颅面骨骼骨骼缺陷的患者相关。

DESCRIPTION (provided by applicant): 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. Public Health Relevance: Large defects from birth, trauma or cancer resection require almost a limitless source of bone, which cannot be supplied by autogenous donor sites without serious morbidity. A means of providing large quantities of bone would be to transfer engineered, pluripotent stem cells that could induce bony regeneration. The project characterized herein has relevance to patients with skeletal deficiencies of the craniofacial skeleton.