Scleraxis-directed stem cell differentiation for ligament tissue engineering

用于韧带组织工程的 Scleraxis 定向干细胞分化

• 批准号: 7881267
• 负责人: Linda Ann Dahlgren
• 金额: $ 21.15万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7881267
• 关键词: Allografting; Area; Autologous Transplantation; Automobile Driving; Biological Assay; Biology; Cell Differentiation process; Cell Line; Cell Proliferation; Cells; Cellular Morphology; Collagen; Collagen Type I; Development; Embryonic Development; Engineering; Environment; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fostering; Gel; Gene Expression; Glycosaminoglycans; Goals; Healed; Health; In Vitro; Joint Capsule; Knee; Ligaments; Mechanics; Mesenchymal; Mesenchymal Stem Cells; Mission; Morbidity - disease rate; Mus; Natural regeneration; Phenotype; Production; Proteins; Quality of life; Research; Risk; Set protein; Signal Transduction; Signaling Protein; Site; Staining method; Stains; Stem cells; Technology; Tenascin; Tendon structure; Testing; Time; Tissue Engineering; Tissues; Transplanted tissue; United States; Western Blotting; Work; anterior cruciate ligament rupture; decorin; disease transmission; healing; improved; innovation; ligament injury; mammalian COMP; musculoskeletal injury; novel; overexpression; precursor cell; programs; promoter; public health relevance; regenerative; scaffold; scleraxis; stem cell differentiation; tissue regeneration; transcription factor

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.

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信号的理解存在根本性差距。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中关键的、未满足的需求。提供能够引导祖细胞沿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，以此发现改善韧带损伤治疗的新方法。该应用的目的是开发一种增强间充质干细胞沿韧带谱系分化的策略。关键信号可以是细胞内的（转录因子和信号蛋白）或环境的（机械应变和细胞周围的三维环境）。中心假设是转录因子scleaxis的过度表达将为间充质干细胞提供适当的信号环境，使其进展为可用于韧带工程的成熟韧带成纤维细胞。因此，拟议的研究与 NIH 的使命相关，即促进创新研究策略，以提高国家改善健康的能力。该假设将通过追求两个具体目标进行检验：（1）确定体外生长的干细胞被诱导沿韧带谱系分化的机制，以及（2）建立机械应变条件，以增强巩膜的分化-修饰干细胞。第一个目标是，小鼠间充质干细胞系将被改造为过度表达巩膜，并且细胞将在静态应变（张力）条件下在三维胶原凝胶中培养。细胞分化将通过韧带表型标记的基因表达和蛋白质产生来确定。在第二个目标中，过度表达巩膜的间充质干细胞将在循环机械应变的可变条件下在三维胶原凝胶中培养。将评估韧带表型的诱导（细胞形态、基因表达和蛋白质产生）和细胞活力。这项创新研究的重点是利用一种新型转录因子来解决韧带工程中最具挑战性的方面之一：生产功能合适的细胞外基质复合材料。拟议的研究意义重大，因为它有望提高对干细胞如何沿着韧带谱系驱动的理解，从而推进韧带工程和再生领域的发展。 公共健康相关性：拟议的研究重点关注组织再生的细胞分化的重要领域。这些信息与现有的组织工程技术相结合将显着推进功能韧带工程领域的发展。拟议的研究具有公共卫生相关性，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，并最终改善韧带损伤患者的生活质量。