Elucidating Mechanisms of Mechanosensitivity During Secondary Chondrogenesis

描述（由申请人提供）：在生命的所有阶段，骨骼都经过优化以检测和适应生物力学力。当维持骨骼健康的微妙平衡被疾病或损伤破坏时，个人的生活质量就会迅速恶化。该项目的目标是确定允许骨骼感知和响应机械力的机制。二级软骨是一种高度适应于检测机械力的骨骼组织。次级软骨最初在颌骨骨骼区域发育，以响应胚胎运动期间产生的力。在缺乏适当的机械力的情况下，次级软骨不能形成，也可以在任何一点退化，如在颞下颌疾病（TMD）和下颌固定患者中。虽然次级软骨对颌骨功能至关重要，但人们对诱导和维持次级软骨的分子机制知之甚少。为了解决这个问题，目前的建议采用了一个鸟类模型系统，该系统利用了二级软骨进化方式的物种特异性差异来支持专门的喂养模式。鸭子用它们的下颚来舀取和过滤潮湿的沉积物。甚至在孵化之前，鸭下颌骨内收肌内端就出现了次级软骨，它向外侧插入，从而大大延长了冠突。这在下颌内收肌肌腱和下颌骨之间建立了一个坚固的界面，并传递了抬起下颌所需的强大收缩。相比之下，鸡主要以啄食种子为食，它们的下颌内收肌沿下颌冠突背侧插入，没有任何次级软骨。这些关键的区别在鸭和鸡的胚胎中是明显的，尽管在胚胎颌运动方面没有显著的差异。这表明，物种特异性颌骨结构产生的机械力存在于鸭子而不是小鸡中，导致发育过程中机械敏感信号通路的不同激活。根据已发表的和初步的数据，我们假设成纤维细胞生长因子（FGF）和钙（Ca2+）信号在使下颌内收肌内端检测生物力学力和产生次级软骨方面发挥作用。目的1涉及的实验将确定FGF和Ca2+信号是否为继发性软骨形成所必需。在FGF和Ca2+信号的小分子抑制剂中浸泡的小珠将被植入鸭冠突的上皮下。Aim 2的实验将揭示FGF和Ca2+信号是否足以通过FGF和Ca2+信号激动剂促进鸡的继发性软骨形成。Aim 3的实验将采用鸡鸭嵌合体来确定鸡细胞在鸭环境下是否能够形成次生软骨。了解调节继发性软骨形成的机制将有助于对继发性软骨丧失（如TMD）和创伤后发生的情况进行再生治疗。

DESCRIPTION (provided by applicant): Through all stages of life, the skeleton is optimized for detecting and adapting to biomechanical forces. When the delicate balance that maintains skeletal health is disrupted by disease or injury, an individual's quality of life can deteriorate rapidly. The goal of this project is to identify mechanisms that allow the skeleton to sense and respond to mechanical forces. One skeletal tissue that is highly attuned to detecting mechanical force is secondary cartilage. Secondary cartilage initially develops on regions of bone in the jaw skeleton in response to forces arising during embryonic motility. In the absence of proper mechanical forces, secondary cartilage fails to form, and can also degenerate at any point as in temporomandibular disorders (TMD) and in patients with immobilized jaws. Though secondary cartilage is essential for jaw functionality, little is known about the molecular mechanisms that induce and maintain secondary cartilage. To address this issue, the current proposal employs an avian model system that exploits species-specific differences in the way secondary cartilage has evolved to support specialized modes of feeding. Duck feed by using their jaws to scoop and filter through wet sediment. Even before hatching, secondary cartilage arises in the duck mandibular adductor enthesis, which inserts laterally and thus greatly extends the coronoid process. This creates a robust interface between the tendon of the mandibular adductor muscle and the mandible, and transmits the powerful contractions necessary to lift the jaw. In contrast, chick feed primarily by pecking at seed, and their mandibular adductor muscle inserts dorsally along the coronoid process of the mandible without any secondary cartilage. These key distinctions are apparent in duck and chick embryos, even though there are no significant differences in embryonic jaw motility. This suggests that species-specific jaw architecture generates mechanical forces that are present in duck but not chick, leading to the differential activation of mechanosensitive signaling pathways during development. Based on published and preliminary data, we hypothesize that Fibroblast Growth Factor (FGF) and Calcium (Ca2+) signaling play a role in enabling the mandibular adductor enthesis to detect biomechanical forces and produce secondary cartilage. Aim 1 involves experiments that will determine whether FGF and Ca2+ signaling are necessary for secondary chondrogenesis. Beads soaked in small molecule inhibitors of FGF and Ca2+ signaling will be implanted beneath the epithelium overlying the presumptive duck coronoid process. Experiments in Aim 2 will uncover whether FGF and Ca2+ signaling are sufficient to promote secondary chondrogenesis by using FGF and Ca2+ signaling agonists in chick. Experiments of Aim 3 will employ chick-duck chimeras to determine whether chick cells are competent to form secondary cartilage when in a duck environment. Understanding mechanisms that regulate secondary chondrogenesis will lead to regenerative therapies for conditions involving loss of secondary cartilage such as TMD and those that occur following trauma.