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: 7774491
• 负责人: Russell R. Reid
• 金额: $ 12.36万
• 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/7774491
• 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）：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鼠类颅面缺陷模型中骨形态发生蛋白对骨愈合的差异作用。该项目基于以下假设：成骨BMP（BMP2、4、6、7、9）具有可定义的，差异的特征，以诱导关键大小的颅面缺陷的愈合能力。为了检验该假设，提出了以下特定目的：1）i。构建表达单个BMP的重组腺病毒（BMP -2，-4，-6，-7，-9），ii。为了确定上述重组腺病毒对干细胞转染的影响，对干细胞中各种增殖和/或成骨分化标记的表达水平III。为了确定各种BMP（2、4、6、7、9）对鼠模型中临界大小缺陷的骨愈合的差异效应。 iv。表征在非负载轴承（钙钙）和承重（下颌）颅面修复区域的这种差异作用； 2）v。为了产生用BMP进行体外刺激的钙化/下颌成骨细胞的原发性培养物，以刺激具有各种BMP的孤立成骨细胞的原代培养物，并测量每个BMP在早期和晚期分化的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/beta Catenin信号传导，并检查体外和体内途径抑制的影响。 viii。通过SI-RNA技术靶向SMAD4信号，并检查体外和体内Smad4抑制作用的影响。这些具体目标将通过以下实验设计来解决：1）用BMP（AD-BMP）编码的腺病毒转染间充质干细胞； 2）用AD-BMPS感染间充质干细胞，并在体外测试这些干细胞以进行成骨分化； 3）将AD-BMP诱导的间充质干细胞转移到临界大小的鼠颅颅缺陷中，并通过Micro-CT评估缺陷闭合； 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都敲低。 公共卫生相关性：从出生，创伤或癌症切除的大缺陷几乎需要无限的骨骼来源，这是自动供体部位无法提供的而没有严重发病率的。提供大量骨骼的一种方法是转移可能诱导骨再生的工程多能干细胞。本文中的该项目与颅面骨骼骨骼缺陷的患者相关。