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: 8310813
• 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/8310813
• 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; RNA; 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; skeletal; trafficking

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.

抽象的 一种有吸引力的骨再生方式涉及使用多能 由成骨因子诱导的间充质干细胞。我们的总体目标是 研究骨形态发生蛋白对小鼠骨愈合的不同影响 颅面缺损模型。该项目基于以下假设： 成骨 BMP（BMP2、4、6、7、9）的可定义差异特征 诱导临界尺寸颅面缺损愈合的能力。为了检验这个假设， 提出以下具体目标： 1) i．构建重组腺病毒 表达单个 BMP（BMP-2、-4、-6、-7、-9），ii．确定干细胞的效果 转染上述重组腺病毒后，其表达水平 干细胞中的各种增殖和/或成骨分化标记物，iii．到 确定各种 BMP（2、4、6、7、9）对骨愈合的不同影响 小鼠模型中的临界尺寸缺陷。四.为了表征区域中的这种差异效应 非承重（颅骨）和承重（下颌）颅面修复区域； 2) v. 生成颅骨/下颌成骨细胞的原代培养物用于体外 用 BMP 刺激，vi. 用 各种 BMP 并测量每种 BMP 在早期诱导成骨的能力 和分化后期； 3) 七．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 体内。八.通过 si-RNA 技术靶向 SMAD4 信号传导并检查 SMAD4 体外和体内抑制。这些具体目标将由 实验设计如下： 1) 间充质干细胞转染 编码 BMP 的腺病毒 (Ad-BMP)； 2) 间充质干细胞的感染 Ad-BMP 以及这些干细胞的体外成骨分化测试； 3） 将 Ad-BMP 诱导的间充质干细胞转移到临界大小的小鼠体内 通过显微 CT 进行颅面缺损和缺损闭合评估； 4) RT-PCR BMP 诱导干细胞成骨过程中重要的下游信号元件； 5） siRNA 在体外和体内敲低这些关键信号元件。