Differential Effects of BMPs on the Healing of Craniofacial Defects

BMP 对颅面缺损愈合的不同作用

• 批准号: 7938792
• 负责人: Russell R. Reid
• 金额: $ 12.52万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938792
• 关键词: Address; Adenovirus Vector; Adenoviruses; Area; BMP2 gene; Birth trauma; Bone Morphogenetic Proteins; Bone Regeneration; Calvaria; Cell Line; Cells; Critical Pathways; Data; Defect; Differentiation Antigens; Elements; Engineering; Evaluation; Excision; Experimental Designs; Goals; Healed; In Vitro; Individual; Infection; Literature; MADH4 gene; Malignant Neoplasms; Mandible; Measures; Mediation; Mesenchymal Stem Cells; Modality; Modeling; Morbidity - disease rate; Mus; Natural regeneration; Osteoblasts; Osteocalcin; Osteogenesis; Pathway interactions; Patients; Phenotype; Pluripotent Stem Cells; Protein Family; Recombinants; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Site; Skeleton; Small Interfering RNA; Source; Staging; Stem cells; Technology; Testing; Transfection; Weight-Bearing state; base; beta catenin; bone; bone healing; bone morphogenetic protein 2; bone morphogenetic protein 9; craniofacial; craniofacial repair; experience; healing; in vivo; osteoblast differentiation; osteogenic; promoter; public health relevance; skeletal; trafficking

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.

描述（由申请人提供）：一种有吸引力的骨再生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中骨愈合的差异影响。该项目基于以下假设：成骨 BMP（BMP2、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特征。为了检验这一假设，提出了以下具体目标： 1) i．构建表达单个 BMP（BMP-2、-4、-6、-7、-9）的重组腺病毒，ii．确定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的骨愈合的不同影响。四.描述非承重（颅骨）区域和承重（下颌）颅面修复区域的这种差异效应； 2) v. 生成颅骨/下颌成骨细胞的原代培养物，用于用 BMP 进行体外刺激，vi. 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 在分化早期和晚期诱导成骨的能力; 3) 七．通过 si-RNA 技术靶向经典 Wnt/β 连环蛋白信号传导，并检查体外和体内通路抑制的效果。八.通过 si-RNA 技术靶向 SMAD4 信号传导，并在体外和体内检查 SMAD4 抑制的效果。这些具体目标将通过以下实验设计来实现：1）用BMP（Ad-BMP）编码的腺病毒转染间充质干细胞； 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能力； 3）将Ad-BMP诱导的间充质干细胞转移到临界尺寸的小鼠颅面缺损中，并通过显微CT评估缺损闭合情况； 4) 对BMP诱导干细胞成骨重要的下游信号元件进行RT-PCR； 5) siRNA 在体外和体内敲低这些关键信号元件。 公共健康相关性：出生、创伤或癌症切除造成的巨大缺陷需要几乎无限的骨源，而自体供体部位无法提供这些骨源，从而导致严重的发病率。提供大量骨骼的一种方法是转移可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。