The Role of Scleraxis and Mechanical Loading on Enthesis Maturation

描述（由申请人提供）：肌腱和骨之间的功能性附着（“附着点”）的发展对于将肌肉力传递到骨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，肌肉负荷的去除显著损害了附着点的发育。此外，删除巩膜轴（Scx），一种转录因子所必需的， 力传递肌腱的发展，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必要的附着点的发展，是由机械线索期间。本研究的第一个目的是使用诱导型基因敲除小鼠模型确定Scx对新生儿附着点成熟的时间影响。Scx的表达将限于发育至胚胎第15.5天、出生后第1天或出生后第7天，并将在整个幼年发育过程中评估发育适应和附着点的成熟。预计在胚胎发育过程中Scx的缺失将对附着点成熟产生比出生后缺失更大的影响，并且分子和功能结果将与成熟过程中Scx表达的持续时间相关。这些发现将确立Scx在附着点发育中的重要性，并确定其在纳塔尔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在发育过程中对Scx表达施加机械负荷的必要性。使用体内和体外方法，我们计划分别模拟卸载和过载，以及评估Scx的机械敏感性及其在附着点成熟和功能中的作用。使用先前验证的模型，在体内卸载和组织工程方法在体外动态加载，我们将跟踪Scx表达细胞以及潜在的分化成腱细胞的ASC。扰动载荷对Scx表达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证实Scx在用生长因子如BMP-12调节后的负荷诱导的肌腱形成中的效力。这些研究的结果将确立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反应性，并最终导致研究肌肉骨骼发育和机械敏感性骨科组织的组织工程策略的新方法。

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.