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）旨在使用来自正常组织的体内负荷和应变来增强组织工程（TE）修复，以在手术前使结构适应其机械环境。FTE对于高负荷组织（如人膝关节中的滑膜）尤其相关。在这个新的应用中，我们将开发实时评估技术，以缩短创建TE产品的开发周期。我们将在真实的时间内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鼠间充质干细胞，我们将解决以下目标：目标1：创建具有成纤维细胞和软骨细胞程序的双重、互斥比色指示剂的鼠MSC的来源。将构建结合联合收割机人I型胶原启动子（黄色荧光蛋白-YFP）和CD-RAP的软骨特异性启动子元件（青色荧光蛋白-CFP）的表达基因，并分别注射到小鼠胚泡中。将单独的转基因系进行交配，并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MSC。目标2”开发测试平台，控制传递到三维细胞接种结构的机械信号，并提供生物和机械事件的实时反馈。完成先进的多样本测试系统的设计，将精确校准的压缩和/或拉伸应变应用于接种细胞的样本，并实时记录其中的比色变化。目的3”在受控的应变状态下，评估小鼠MSC在半透明凝胶中的基因表达。将MSC诱导为软骨细胞表型后，测试以下假设的应用：1）对细胞-凝胶构建体的压缩应变将维持指示软骨特异性启动子元件的CFP，2）拉伸应变将诱导去分化和从青色荧光软骨细胞向黄色荧光成纤维细胞的转变，和3）对已经暴露于压缩应变并已经表达CFP的MSC-凝胶构建体的拉伸应变将产生对成纤维细胞的去分化，导致组织组分和颜色的混合组合物。这项技术可以大大减少TE纤维软骨开发阶段的时间/费用。