Scleraxis-directed stem cell differentiation for ligament tissue engineering

描述(申请人提供)：在我们对祖细胞分化为肌腱和韧带成纤维细胞所需的信号的理解上存在着根本的差距。引导前体细胞分化为成熟韧带成纤维细胞的策略在功能性韧带组织的发展中是一个关键的、尚未得到满足的需求。提供一个能够沿着韧带谱系引导祖细胞分化的信号环境是韧带工程领域面临的主要挑战。长期目标是进一步了解正常韧带生物学，作为发现改善韧带损伤治疗的新方法的一种手段。这项应用的目的是开发一种策略，促进间充质干细胞沿韧带谱系的分化。关键信号可以是细胞内的(转录因子和信号蛋白)或环境(细胞周围的机械应变和三维环境)。核心假说是，转录因子sclaxis的过表达将为间充质干细胞向成熟的韧带成纤维细胞提供合适的信号环境，对韧带工程有用。因此，这项拟议的研究与NIH促进创新研究战略以提高国家改善健康的能力的使命相关。这一假说将通过追求两个具体目标来检验：(1)确定体外生长的干细胞将被诱导沿着韧带谱系分化的机制，(2)建立促进硬化轴修饰干细胞分化的机械应变条件。在第一个目标中，将设计一种小鼠间充质干细胞系来过度表达硬化轴，并将细胞培养在三维胶原凝胶中，在静态应变(拉伸)的条件下进行培养。细胞分化将通过韧带表型标志物的基因表达和蛋白质生产来确定。在第二个目标中，将高表达硬化轴的间充质干细胞培养在三维胶原凝胶中，在不同的循环机械应变条件下。将评估韧带表型(细胞形态、基因表达和蛋白质生产)和细胞活力的诱导。这项创新性的研究集中于利用一种新的转录因子来解决韧带工程中最具挑战性的方面之一：生产符合功能的细胞外基质复合材料。这项拟议的研究意义重大，因为它有望提高人们对干细胞是如何沿着韧带谱系驱动的理解，从而推动韧带工程和再生领域的发展。 公共卫生相关性：拟议的研究集中在细胞分化对组织再生的一个重要领域。这些信息与现有的组织工程技术相结合，将极大地推动功能韧带工程领域的发展。这项拟议的研究具有公共卫生意义，因为了解细胞分化为韧带成纤维细胞的关键将改善组织愈合，并最终提高韧带损伤患者的生活质量。

DESCRIPTION (provided by applicant): A fundamental gap exists in our understanding of the signals required for progenitor cells to differentiate into tendon and ligament fibroblasts. Strategies for directing precursor cell differentiation into mature ligament fibroblasts are a critical, unmet need in the development of functional ligament tissues. Providing a signaling environment capable of directing progenitor cell differentiation along a ligament lineage is a major challenge facing the field of ligament engineering. The long term goal is to further understand normal ligament biology as a means of discovering novel ways to improve the treatment of ligament injuries. The objective of this application is to develop a strategy that enhances mesenchymal stem cell differentiation along a ligament lineage. Critical signals can be intracellular (transcription factors and signaling proteins) or environmental (mechanical strain and three- dimensional environment surrounding the cells). The central hypothesis is that overexpression of the transcription factor scleraxis will provide the appropriate signaling environment for mesenchymal stem cells to progress into mature ligament fibroblasts useful for ligament engineering. Thus, the proposed research is relevant to the NIH's mission to foster innovative research strategies to advance the nation's capacity to improve health. The hypothesis will be tested by pursuing two specific aims: (1) Identify a mechanism by which stem cells grown in vitro will be induced to differentiate along a ligament lineage, and (2) Establish conditions of mechanical strain that will enhance differentiation of scleraxis-modified stem cells. In the first aim, a mouse mesenchymal stem cell line will be engineered to overexpress scleraxis and cells will be cultured in three-dimensional collagen gels under conditions of static strain (tension). Cell differentiation will be determined via gene expression and protein production for markers of a ligament phenotype. In the second aim, mesenchymal stem cells overexpressing scleraxis will be cultured in three-dimensional collagen gels under variable conditions of cyclic mechanical strain. Induction of a ligament phenotype (cell morphology, gene expression, and protein production) and cell vitality will be assessed. This innovative research focuses on utilizing a novel transcription factor to solve one of the most challenging aspects of ligament engineering: production of a function-appropriate extracellular matrix composite. The proposed research is significant, because it is expected to improve understanding of how stem cells are driven along a ligament lineage, thus advancing the fields of ligament engineering and regeneration. PUBLIC HEALTH RELEVANCE: The proposed studies focus on an important area of cell differentiation for tissue regeneration. This information combined with existing tissue engineering technologies will significantly advance the field of functional ligament engineering. The proposed research has public health relevance, because understanding the key to cell differentiation into ligament fibroblasts will improve tissue healing and ultimately the quality of life for people suffering from ligament injuries.