Cell phenotype-controlled mechanical signaling of MSCs

MSC 的细胞表型控制的机械信号传导

• 批准号: 6801879
• 负责人: DAVID L BUTLER
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-19 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6801879
• 关键词: bioengineering /biomedical engineering; biological signal transduction; bioluminescence; biomechanics; biotechnology; cell differentiation; chondrocytes; colorimetry; fibroblasts; gene expression; genetically modified animals; immunocytochemistry; laboratory mouse; mesenchyme; phenotype; pluripotent stem cells; polymerase chain reaction; technology /technique development; tissue /cell culture; tissue engineering; tissue support frame

DESCRIPTION (provided by applicant): Functional tissue engineering (FTE) seeks to enhance tissue engineered (TE) repairs using in vivo loads and strains from normal tissues to condition the constructs to their mechanical environment prior to surgery. FTE is particularly relevant for highly loaded tissues such as menisci in the human knee. In this novel application, we will develop a real-time assessment technology to shorten the development cycle for creating TE products. We will screen, in real time, how mechanical stimuli affect gene expression. Using specialized murine mesenchymal stem cells in a mechanical stimulation system, we will address the following aims: Aim 1: Create a source of murine MSCs having dual, mutually exclusive colorimetric indicators for fibroblastic and chondrocytic programs. Expression genes that combine human type I collagen promoter (yellow fluorescent protein-YFP) and cartilage-specific promoter element of CD-RAP (cyan fluorescent protein-CFP) will be constructed and injected separately into mouse blastocysts. Separate transgenic lines will be mated, and MSCs will be harvested from doubly transgenic animals that induce chondrocytic differentiation or fibroblast phenotype. Aim 2" Develop test platforms that control mechanical signals delivered to 3-D cell-seeded constructs and that provide real-time feedback of biological and mechanical events. Finalize designs of advanced, multi-specimen testing systems to apply precisely calibrated compressive and/or tensile strains to cell-seeded specimens and that enable real-time recordings of colorimetric changes within them. Aim 3" Evaluate gene expression for murine MSCs in translucent gels under controlled strain states. After inducing MSCs to chondrocytic phenotype, test the hypotheses that application of: 1) compressive strains to cell-gel constructs will maintain CFP indicative of a cartilage-specific promoter element, 2) tensile strains will induce dedifferentiation and transition from a cyan fluorescent chondrocyte to a yellow fluorescent fibroblast, and 3) tensile strains to MSC-gel constructs already exposed to compressive strains and already expressing CFP will yield dedifferentiation to fibroblasts resulting in a mixed composition of tissue components and colors. This technology can dramatically reduce time/expense during the development phase of TE fibrocartilage.

描述（由申请人提供）：功能性组织工程（FTE）旨在在手术前利用来自正常组织的体内负荷和应变来调节构建物的机械环境，从而增强组织工程（TE）修复。FTE特别适用于高负荷组织，如人类膝盖的半月板。在这个新颖的应用中，我们将开发一种实时评估技术，以缩短制造TE产品的开发周期。我们将实时筛选机械刺激如何影响基因表达。在机械刺激系统中使用专门的小鼠间充质干细胞，我们将实现以下目标：目标1：为成纤维细胞和软骨细胞项目创建具有双重互斥比色指标的小鼠间充质干细胞来源。构建结合人I型胶原启动子（黄色荧光蛋白- yfp）和CD-RAP软骨特异性启动子元件（青色荧光蛋白- cfp）的表达基因，分别注入小鼠囊胚。分离的转基因细胞系将进行交配，从诱导软骨细胞分化或成纤维细胞表型的双转基因动物中收获间充质干细胞。目标2“开发测试平台，控制传递给3-D细胞种子结构的机械信号，并提供生物和机械事件的实时反馈。完成先进的多标本测试系统的设计，以精确校准细胞种子标本的压缩和/或拉伸应变，并能够实时记录其中的比色变化。目的3“在受控菌株状态下，评估半透明凝胶中小鼠间充质干细胞的基因表达。诱导MSCs形成软骨细胞表型后，检验应用：1)压缩应变到细胞-凝胶构建体将维持CFP指示软骨特异性启动子元件，2)拉伸应变将诱导去分化并从青色荧光软骨细胞转变为黄色荧光成纤维细胞，3)拉伸应变到已经暴露于压缩应变和已经表达CFP的msc -凝胶构建体将产生成纤维细胞的去分化，导致组织成分和颜色的混合组成。这项技术可以大大减少TE纤维软骨开发阶段的时间/费用。