Mechanisms regulating afferent innervation in the dental pulp

项目摘要/摘要 由于牙齿暴露在环境刺激下，牙齿神经对牙齿的保护和使用至关重要。 在有机体的整个生命过程中。牙齿主要由来自牙齿的感觉神经纤维支配 三叉神经节(TG)通过传递伤害性刺激来保护牙齿器官。牙髓(DP)分泌 神经营养因子在牙发育过程中引导轴突穿透和萌发的实验研究 一种高度管制的方式。研究表明，分泌的磷蛋白，骨桥蛋白(OPN)，促进 神经元的迁移、增殖和存活[2-6]。该项目的长期目标是了解 间充质--促进和维持牙齿感觉神经的神经元信号。整体而言 目的探讨DP在牙齿发育和再生过程中对神经支配的调节作用。 我们的中心假设是，牙齿间充质中的TGFBR2通过OPN调控旁分泌信号，从而引导 牙齿感觉神经支配。我们实验室建立了一种TGFBR2有条件的小鼠模型 在使用Osterix启动子驱动的Cre重组酶(Tgfbr2cko)的成牙本质细胞产生间充质细胞中缺失。 这些小鼠出生后存活下来，但骨骼和牙齿有明显的缺陷[7，8]。我们做了一次核糖核糖核酸- 使用对照和突变的出生后第7天DP进行SEQ分析，发现神经元维护和 发育基因受到最高的调控，包括骨桥蛋白。免疫荧光图像显示 在Tgfbr2cko小鼠中，整个DP的神经支配减少。DP和初级TG的初步实验 当TG细胞与DP共同培养时，轴突出芽增加。在这些数据的指引下， 我们将通过以下两个具体目标来测试我们的假设：1)测试TGFBR2是必要的假设 以促进感觉神经支配；2)检验来自DP的OPN信号引导感觉的假设 神经支配。在这两个目标中，我们建议将表达高水平YFP的TG(Thy1-YFP)16JRS小鼠 通过神经系统[9]来优化神经元的可视化。在第一个目标下，一口井- 特征性轴突生长试验将首先用于将转化生长因子信号转导的三叉神经节神经元与DP共培养 在民主党中被操纵。在第一个目标的第二部分，我们将使用活体牙齿损伤模型和 研究Tgfbr2cko和WT小鼠的神经元再生。在第二个目标下，我们将同样地共同培养 含骨桥蛋白缺失DP细胞的TG神经元+/-转化生长因子_1及缺失细胞的重组 目的：探讨发育迟缓的神经发生。我们将对OPN-/-进行牙齿损伤检测 以检查驱动神经元再生的机制。这项拟议的研究具有重要意义，因为 这有望促进和扩大对DP细胞如何通过轴突保护牙齿器官的理解 引导机制。这些信息将有助于我们更好地理解 牙齿中间充质-神经元的相互作用，可以作为未来预防，治疗， 和再生策略在牙髓学和改善牙齿的保存。

Project Summary/Abstract Since teeth are exposed to environmental stimuli, tooth innervation is crucial to their protection and usage throughout the life of an organism. The tooth is primarily innervated with sensory nerve fibers from the trigeminal ganglion (TG) that protect the tooth organ by relaying noxious stimuli. The dental pulp (DP) secretes neurotrophic factors to guide axonal penetration and sprouting within the tooth during postnatal development in a highly regulated manner. Research has shown that secreted phosphoprotein, osteopontin (OPN), promotes neuronal migration, proliferation, and survival [2–6]. The long-term goal of this project is to understand the mesenchymal-neuronal signals that promote and maintain sensory innervation of the teeth. The overall objective is to determine the role of DP in regulating tooth innervation during development and regeneration. Our central hypothesis is that Tgfbr2 in the dental mesenchyme governs paracrine signaling via OPN to guide tooth sensory innervation. Our laboratory has established a mouse model in which Tgfbr2 is conditionally deleted in odontoblast-producing mesenchyme using an osterix promoter driven Cre recombinase (Tgfbr2cko). These mice survive postnatally but with significant defects in bones and teeth [7,8]. We performed an mRNA- Seq analysis using control and mutant postnatal day 7 DP and found that neuronal maintenance and developmental genes were most highly regulated, including OPN. Immunofluorescent images indicated reduced innervation throughout the DP in Tgfbr2cko mice. Preliminary experiments with DP and primary TG nerves demonstrated increased axonal sprouting when TG cells were cultured with DP. Guided by these data, we will test our hypothesis with the following two specific aims: 1) test the hypothesis that Tgfbr2 is necessary to promote sensory innervation; 2) test the hypothesis that OPN signaling from the DP guides sensory innervation. In both aims, we propose to cross Tg(Thy1-YFP)16Jrs mice, which express a high level of YFP throughout the nervous system [9] to optimize visualization of the neurons. Under the first aim, a well- characterized neurite outgrowth assay will first be used to co-culture TG neurons with DP where Tgf signals are manipulated in the DP. In the second part of the first aim, we will use an in vivo dental injury model and investigate neuronal regeneration in Tgfbr2cko and WT mice. Under the second aim, we will similarly co-culture TG neurons with OPN-deleted DP cells +/- TGF1 and Tgfbr2-deleted cells supplemented with recombinant OPN to investigate developmental neurogenesis in the DP. We will perform the dental injury assay on OPN-/- mice to examine the mechanisms driving neuronal regeneration. The proposed research is significant because it is expected to advance and expand understanding of how DP cells protect the tooth organ via axonal guidance mechanisms. Such information will enhance our understanding of the complex interplay of mesenchymal-neuronal interactions in the tooth that could serve as a basis for future preventative, therapeutic, and regenerative strategies in endodontics and improve the preservation of teeth.