The Origin of Tooth Pain: Linking Polymodal TRP channels on Odontoblasts and Pulp Fibroblasts to Sensory Nerves

全世界有24亿人患有未经治疗的龋齿和疼痛的牙齿过敏症。细菌生物膜相关的酸性代谢产物使牙釉质和牙本质脱矿并诱导牙髓炎症。酸性反流引起的侵蚀性损害是另一个广泛存在的问题。由于过敏引起的牙痛是牙科诊所的一个大问题，因为它可能是痛苦的，而普通的止痛药是无效的。在牙髓中，游离神经末梢的密集网络与免疫细胞、形成牙本质的成牙本质细胞和成纤维细胞密切接触。到目前为止，来自热、机械或生物化学线索的疼痛被认为是由流体动力学诱导的机械感觉过程引起的，其中牙本质小管充当物理刺激与牙髓-牙本质边界处的神经末梢之间的液压连接。然而，我们先前的研究提供了实验证据，证明成牙本质细胞过程是通过TRPC 5通道传导冷痛的传导位点。因此，成牙本质细胞作为初级感觉细胞，是牙痛的起源。基于这些新的发现，本研究项目遵循三个主要目标。我们评估了几个TRPC通道，包括TRPC 1，TRPC 3和TRPC 4，跨成牙本质细胞，成纤维细胞和感觉神经的报告和敲除小鼠模型的专门定位和差异功能。然后，我们建立了一个光遗传学小鼠模型，以评估成牙本质细胞在牙痛起源中的特异性功能。该模型基于DMP 1-Cre、ChR 2和ArchT小鼠。我们使用光来操纵成牙本质细胞通过门牙的舌面，那里较薄的釉质层是透明的。该模型将与下颌神经制备物的细胞外记录和三叉神经节中基于GCaMP的活体钙成像相结合。最后但并非最不重要的是，我们将开发重要的牙髓类器官作为研究神经末梢，成牙本质细胞和成纤维细胞原位之间复杂相互作用的关键工具。我们将使用来自光遗传学小鼠模型的牙髓在完整的牙本质上产生由感觉神经支配的类器官。这些类器官将使用光遗传学操作、膜片钳记录和共聚焦钙成像来表征它们的感觉能力，由我们先前建立的转录组离子通道和来自牙齿初级传入神经元的GPCR谱的信息指导。目的是获得小鼠和人类牙髓的具体离子通道和信号分子谱。在牙齿中，牙髓的功能和感觉特性是高度特化的细胞类型和感觉神经的密切相互作用的结果，并且它们的理解对于新的牙痛治疗策略和旨在恢复牙髓的感觉和再生功能的再生牙髓治疗的未来发展至关重要。

Worldwide, 2.4 billion people suffer from untreated caries and painful tooth hypersensitivity. Bacterial biofilm-related acidic metabolites demineralize enamel and dentin and induce inflammation in the dental pulp. Acidic reflux-induced erosive damage is another wide-spread problem. Tooth ache due to hypersensitivity is a large problem in the dental clinic, because it can be excruciating and common analgesics are ineffective. In the tooth pulp, a dense network of free nerve endings is in intimate contact with immune cells, dentin-forming odontoblasts and fibroblasts. So far, pain from thermal, mechanical, or biochemical cues was believed to arise from a fluid-dynamic induced mechanosensory process where dentinal tubules act as hydraulic link between the physical stimulus and the nerve terminals at the pulp-dentin boundary. However, our previous research delivered experimental evidence for the odontoblast processes as the transduction site for painful cold via TRPC5 channels. Therefore, Odontoblasts act as primary sensory cells and are the origin of tooth pain. Based on these novel findings, this research project follows three main objectives. We assess the specialized localization and differential function of several TRPC channels, including TRPC1, TRPC3 and TRPC4, across odontoblasts, fibroblast and sensory nerves with reporter and knockout mouse models. Then, we establish an optogenetic mouse model to assess the specific function of Odontoblasts in the origin of tooth pain. This model is based on DMP1-Cre, ChR2 and ArchT mice. We use light to manipulate Odontoblasts through the incisor’s lingual surface where the thinner enamel layer is transparent. This model will be combined with extracellular recordings from jaw-nerve preparations and GCaMP-based intravital calcium imaging in trigeminal ganglia. Last but not least, we will develop vital pulp organoids as critical tools to investigate the intricate interplay between nerve endings, odontoblasts and fibroblasts in situ. We will use the tooth pulps from the optogenetic mouse models to generate organoids on intact dentin and innervated by sensory nerves. These organoids will be characterized for their sensory capacity using optogenetic manipulation, patch clamp recordings and confocal calcium imaging, guided by information from our previously established transcriptomic ion channel and GPCR profile from dental primary afferent neurons. The aim is to obtain a concrete ion channel and signaling molecule profile for the mouse and human tooth pulp. In teeth the functionality and sensory properties of the tooth pulp are the result of an intimate interaction of highly specialized cell types and sensory nerves and their understanding is crucial for novel tooth pain treatment strategies and the future advancement of regenerative endodontic therapies aiming at the restoration of sensory and regenerative function of the dental pulp.