The Role of Scleraxis and Mechanical Loading on Enthesis Maturation

巩膜和机械负荷对附着点成熟的作用

• 批准号: 8839713
• 负责人: MEGAN Leigh KILLIAN
• 金额: $ 3.78万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-28 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839713
• 关键词: Adipose tissue; Adolescent; Affect; Automobile Driving; BMP-12; Biological Factors; Birth; Botulinum Toxins; Brachial plexus structure; Cells; Collagen; Cues; Defect; Denervation; Development; Differentiation and Growth; Disease; Embryo; Embryonic Development; Engineering; Excision; Experimental Models; Extracellular Matrix; Fibroblasts; Fibrocartilages; Genes; Goals; Growth Factor; Health; Homeostasis; Impairment; In Vitro; Joints; Knock-out; Lead; Life; Link; Mechanics; Mediating; Mesenchymal Stem Cells; Methods; Modeling; Molecular; Motion; Mus; Muscle; Musculoskeletal Development; Nature; Neonatal; Nerve; Orthopedics; Outcome; Paralysed; Physical condensation; Play; Property; Rehabilitation therapy; Role; Shoulder; Skeleton; Small Interfering RNA; Stem cells; Stress; Structure; Tendinopathy; Tendon structure; Testing; Time; Tissue Engineering; Tissues; Transforming Growth Factor beta; Vaginal delivery procedure; bone; fetal; fibrillogenesis; functional outcomes; gene therapy; improved; in vivo; in vivo Model; interest; mechanical drive; mineralization; novel strategies; repaired; response; scleraxis; stem cell differentiation; supraspinatus muscle; tendon development; transcription factor

DESCRIPTION (provided by applicant): The development of a functional attachment between tendon and bone (the "enthesis") is critical for transmitting muscle forces to bone for joint motion. The formation of this attachment is driven by mechanical and biologic factors. Murine models of have demonstrated that removal of muscle load dramatically impairs the development of the enthesis. Furthermore, deletion of scleraxis (Scx), a transcription factor necessary for the development of force-transmitting tendons, leads to defects in enthesis formation. However, the mechanisms driving the mechanosensitivity of enthesis development are not known. Our global hypothesis is that Scx necessary for enthesis development and is driven by mechanical cues during. The first aim of this study is to determine the temporal influence of Scx on neonatal enthesis maturation using an inducible knockout murine model. Expression of Scx will be limited to development up to embryonic day 15.5, post-natal day 1, or post-natal day 7 and developmental adaptations and maturation of the enthesis will be assessed throughout juvenile development. It is expected that deletion of Scx during embryonic development will have a greater effect on enthesis maturation compared to post-natal deletion, and molecular and functional outcomes will be correlated with duration of Scx expression during maturation. These findings will establish the importance of Scx in enthesis development as well as determine its temporal role in post- natal musculoskeletal development. The second aim of this study is to determine the necessity of mechanical loading on Scx expression during development. Using in vivo and in vitro methods, we plan to mimic unloading and overloading, respectively, as well as assess the mechanosensitivity of Scx and its role in maturation and function of the enthesis. Using previously validated models of in vivo unloading and tissue-engineered methods for in vitro dynamic loading, we will track Scx-expressing cells as well as potential differentiation of ASCs into tenocytes. The effect of perturbed loading on the molecular and morphological response of Scx-expressing cells will be correlated with adaptations in structure and function using mechanical tests. These findings will determine the role of mechanical loading in Scx expression as well as the necessity of Scx in mechanically- induced tenogenesis. Lastly, the potency of Scx in load-induced tenogenesis following modulation with growth factors such as BMP-12 will be confirmed using BMP-12 siRNA. Findings from these studies will establish the importance of Scx on post-natal enthesis development, the mechanoresponsiveness of Scx in multi-tiered experimental models, and ultimately lead to new approaches for investigating musculoskeletal development and tissue engineered strategies for mechanosensitive orthopaedic tissues.

描述（由申请人提供）：肌腱和骨骼之间功能性附着（“附着点”）的发展对于将肌肉力传递到骨骼以进行关节运动至关重要。这种依恋的形成是由机械和生物因素驱动的。小鼠模型已经证明，去除肌肉负荷会极大地损害附着点的发育。此外，巩膜轴 (Scx) 的缺失，Scleraxis 是 力传递肌腱的发育导致附着点形成缺陷。然而，驱动附着点发育的机械敏感性的机制尚不清楚。我们的总体假设是 Scx 对于附着点发育是必需的，并且是由机械线索驱动的。本研究的第一个目的是使用诱导敲除小鼠模型确定 Scx 对新生儿着丝点成熟的时间影响。 Scx 的表达将仅限于胚胎第 15.5 天、出生后第 1 天或出生后第 7 天的发育，并且将在整个幼年发育过程中评估发育适应和附着点的成熟。预计与出生后缺失相比，胚胎发育期间 Scx 的缺失将对附着点成熟产生更大的影响，并且分子和功能结果将与成熟期间 Scx 表达的持续时间相关。这些发现将确定 Scx 在附着点发育中的重要性，并确定其在出生后肌肉骨骼发育中的时间作用。本研究的第二个目的是确定开发过程中机械加载对 Scx 表达的必要性。我们计划使用体内和体外方法分别模拟卸载和过载，并评估 Scx 的机械敏感性及其在附着点成熟和功能中的作用。使用先前验证的体内卸载模型和用于体外动态加载的组织工程方法，我们将追踪表达 Scx 的细胞以及 ASC 分化为肌腱细胞的潜在能力。扰动负载对表达 Scx 的细胞的分子和形态反应的影响将与使用机械测试的结构和功能的适应性相关。这些发现将确定机械负荷在 Scx 表达中的作用以及 Scx 在机械诱导的肌腱形成中的必要性。最后，将使用 BMP-12 siRNA 确认 Scx 在用 BMP-12 等生长因子调节后在负载诱导的肌腱形成中的效力。这些研究的结果将确定 Scx 对出生后附着点发育的重要性、Scx 在多层实验模型中的机械响应性，并最终导致研究肌肉骨骼发育和机械敏感骨科组织的组织工程策略的新方法。