Differential Effects of BMPs on the Healing of Craniofacial Defects

描述(申请人提供)：一种诱人的骨再生方式包括使用由成骨因子诱导的多潜能间充质干细胞。我们的总体目标是研究骨形态发生蛋白对小鼠颅面骨缺损模型骨愈合的不同影响。该项目基于这样的假设，即成骨BMPs(BMP2、4、6、7、9)在诱导临界大小的颅面缺损愈合方面存在可定义的、不同的特征。为了验证这一假设，提出了以下具体目标：1)1)构建表达单个BMP(BMP-2、-4、-6、-7、-9)的重组腺病毒；3.检测上述重组腺病毒对干细胞中各种增殖和/或成骨分化标志物表达水平的影响。目的：探讨不同BMP(2，4，6，7，9)对小鼠严重骨缺损愈合的不同影响。四、在颅面修复的非承重区(颅骨)和承重区(下颌)描述这种不同的作用；2)建立颅骨/下颌骨成骨细胞的原代培养细胞，用于BMP的体外刺激，即用不同的BMP刺激分离的成骨细胞的原代培养，并测量每种BMP在分化早期和晚期诱导成骨的能力；3)vii.通过si-RNA技术靶向规范的Wnt/β连环蛋白信号转导通路，并检测体内和体外信号通路抑制的效果。八.通过si-RNA技术靶向Smad4信号转导，检测Smad4在体内外的抑制作用。这些特定的目标将通过下列实验设计来实现：1)携带BMPs的腺病毒(Ad-BMPs)转染间充质干细胞；2)Ad-BMPs感染间充质干细胞并在体外检测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评估缺损的闭合情况；4)RT-PCR检测在BMP诱导的干细胞成骨中起重要作用的下游信号元件；5)在体外和体内下调这些关键信号元件的siRNA。 公共卫生相关性：出生、创伤或癌症切除造成的巨大缺陷需要几乎无限的骨来源，如果没有严重的发病率，自体供骨部位是无法提供的。提供大量骨骼的一种方法是转移可以诱导骨骼再生的工程多能干细胞。在此描述的项目与头面部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.