Scleraxis-directed stem cell differentiation for ligament tissue engineering

描述（由申请人提供）：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存在根本性的差距。指导前体细胞分化为成熟韧带成纤维细胞的策略是功能性韧带组织发育的关键，尚未满足的需求。提供一个能够引导祖细胞沿着韧带谱系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，作为发现新的方法来改善韧带损伤的治疗手段。本应用的目的是开发一种策略，以增强间充质干细胞沿韧带谱系分化。关键信号可以是细胞内（转录因子和信号蛋白）或环境（机械应变和细胞周围的三维环境）。中心假设是转录因子scliraxis的过度表达将为间充质干细胞发育为成熟的韧带成纤维细胞提供适当的信号环境，从而有助于韧带工程。因此，拟议的研究与NIH的使命有关，即促进创新研究战略，以提高国家改善健康的能力。该假设将通过追求两个特定目标来验证：(1)确定一种机制，通过这种机制，体外培养的干细胞将被诱导沿着韧带谱系分化；(2)建立机械应变条件，以增强硬化修饰干细胞的分化。在第一个目标中，一个小鼠间充质干细胞系将被设计成过度表达硬化，细胞将在静态应变（张力）条件下在三维胶原凝胶中培养。细胞分化将通过基因表达和韧带表型标记的蛋白质生产来决定。在第二个目标中，过度表达硬化轴的间充质干细胞将在三维胶原凝胶中在可变的循环机械应变条件下培养。将评估韧带表型（细胞形态、基因表达和蛋白质产生）和细胞活力的诱导。这项创新研究的重点是利用一种新的转录因子来解决韧带工程中最具挑战性的一个方面：生产功能合适的细胞外基质复合材料。这项研究具有重要意义，因为它有望提高对干细胞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。

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.