Mechanisms regulating afferent innervation in the dental pulp

调节牙髓传入神经支配的机制

• 批准号: 10214592
• 负责人: Sarah Peters
• 金额: $ 24.63万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214592
• 关键词: Adult; Automobile Driving; Axon; Biological Assay; Cells; Cementoclast; Chronic Disease; Coculture Techniques; Complex; Data; Defect; Dental; Dental Pulp; Dental caries; Dental crowns; Dentin; Deposition; Development; Developmental Gene; Embryo; Endodontics; Enterobacteria phage P1 Cre recombinase; Eph Family Receptors; Epithelial; Exposure to; Extracellular Matrix Proteins; Future; Ganglia; Generations; Genetic Transcription; Goals; Image; In Vitro; Injury; Investigation; Knockout Mice; Laboratories; Life; Maintenance; Mediating; Mesenchymal; Mesenchyme; Modeling; Mus; Mutant Strains Mice; NTN1 gene; Natural regeneration; Nerve; Nerve Fibers; Nerve Regeneration; Nervous system structure; Neurites; Neurons; Odontoblasts; Organ; Organism; Pain; Pain management; Paracrine Communication; Pathway interactions; Pattern; Penetration; Perinatal mortality demographics; Phosphoproteins; Plant Roots; RNA; Reader; Recombinants; Regulation; Reporting; Research; Role; Sensory; Signal Transduction; Stimulus; Structure; Structure of trigeminal ganglion; Temperature; Testing; Therapeutic; Tissues; Tooth Germ; Tooth structure; Trigeminal nerve structure; Viral; Visualization; afferent nerve; axon guidance; axonal sprouting; base; bone; experimental study; ganglion cell; improved; in vivo; knock-down; mRNA sequencing; migration; mouse model; mutant; nerve stem cell; nerve supply; neuroblast; neurogenesis; neuron development; neurotransmission; neurotrophic factor; new therapeutic target; osteopontin; overexpression; postnatal; postnatal development; preservation; pressure; promoter; regenerative approach; response; skeletal

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.

项目概要/摘要 由于牙齿暴露在环境刺激下，牙齿神经支配对其保护和使用至关重要 生物体的整个生命周期。牙齿主要受来自牙齿的感觉神经纤维的支配。 三叉神经节（TG）通过传递有害刺激来保护牙齿器官。牙髓（DP）分泌 神经营养因子在出生后发育过程中指导轴突穿透和在牙齿内发芽 高度监管的方式。研究表明，分泌型磷蛋白、骨桥蛋白 (OPN) 可促进 神经元迁移、增殖和存活[2-6]。该项目的长期目标是了解 促进和维持牙齿感觉神经支配的间充质神经元信号。整体 目的是确定 DP 在发育和再生过程中调节牙齿神经支配的作用。 我们的中心假设是牙齿间充质中的 Tgfbr2 通过 OPN 控制旁分泌信号传导以指导 牙齿感觉神经支配。我们实验室建立了Tgfbr2有条件表达的小鼠模型 使用 osterix 启动子驱动的 Cre 重组酶 (Tgfbr2cko) 在成牙本质细胞产生的间充质中删除。 这些小鼠出生后存活，但骨骼和牙齿存在明显缺陷[7,8]。我们进行了 mRNA- 使用对照和突变体出生后第 7 天 DP 进行 Seq 分析，发现神经元维持和 发育基因受到最严格的调控，包括 OPN。免疫荧光图像显示 Tgfbr2cko 小鼠整个 DP 的神经支配减少。 DP 和初级 TG 的初步实验 当 TG 细胞与 DP 一起培养时，神经表现出轴突萌芽增加。在这些数据的指导下， 我们将通过以下两个具体目标来检验我们的假设：1）检验 Tgfbr2 是必要的假设 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。为了实现这两个目标，我们建议对表达高水平 YFP 的 Tg(Thy1-YFP)16Jrs 小鼠进行杂交 整个神经系统 [9] 以优化神经元的可视化。在第一个目标下， 特征性神经突生长测定将首先用于与 DP 共培养 TG 神经元，其中 Tgf 信号 在DP中被操纵。在第一个目标的第二部分中，我们将使用体内牙齿损伤模型 研究 Tgfbr2cko 和 WT 小鼠的神经元再生。在第二个目标下，我们将同样共同培养 TG 神经元，含有 OPN 删除的 DP 细胞 +/- TGF1 和 Tgfbr2 删除的细胞，补充有重组蛋白 OPN 用于研究 DP 中的发育神经发生。我们将对 OPN-/- 进行牙齿损伤检测 小鼠来检查驱动神经元再生的机制。拟议的研究意义重大，因为 预计它将推进和扩大对 DP 细胞如何通过轴突保护牙齿器官的理解 指导机制。这些信息将增强我们对复杂相互作用的理解 牙齿中的间充质-神经元相互作用可以作为未来预防、治疗、 和牙髓再生策略并改善牙齿的保存。