Cell phenotype-controlled mechanical signaling of MSCs

描述(申请人提供)：功能组织工程学(FTE)寻求加强组织工程(TE)修复，使用来自正常组织的体内负荷和应变，以使结构在手术前适应其机械环境。FTE特别适用于高负荷的组织，如人类膝盖的半月板。在这一新颖的应用中，我们将开发一种实时评估技术，以缩短创建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、相互排斥的比色指示物的小鼠MSCs来源。我们将构建结合人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，并将其分别注射到小鼠囊胚中。将进行单独的转基因系交配，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获得间充质干细胞。目标2“开发测试平台，控制传递到三维细胞种植结构的机械信号，并提供生物和机械事件的实时反馈。最终完成先进的多样本测试系统的设计，以对细胞种子样本施加精确校准的压缩和/或拉伸应变，并能够实时记录其中的比色变化。目标3”评估受控应变状态下半透明凝胶中小鼠骨髓间充质干细胞的基因表达。在诱导MSCs向软骨细胞表型转化后，测试以下假设：1)对细胞凝胶结构施加压缩应变将保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，并从青色荧光软骨细胞向黄色荧光成纤维细胞转变，以及3)对已暴露于压缩应变并已表达CFP的MSC-凝胶结构的拉伸应变将产生向成纤维细胞的去分化，导致组织成分和颜色的混合。这项技术可以极大地减少TE纤维软骨开发阶段的时间/费用。

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.