Neurobiological control of periodontal homeostasis through microRNA, TGF-beta, and Wnt signaling

牙周组织是一个丰富的神经支配的组织，经历连续的造型和重塑， 牙槽骨成骨细胞、骨细胞和破骨细胞。我们实验室的研究表明， 三叉神经节和Malassez上皮细胞之间的密切空间关系， 上皮细胞网络驻留在非矿化牙周韧带。下牙槽神经 导致牙槽骨强直和ERM减少的横断研究证实， 感觉神经支配对牙周内环境稳定的重要作用。对于本申请，我们具有 在我们的实验室中建立了IAN横断模型，并为增强IAN横断提供了放射学证据。 矿化和强直的大鼠磨牙牙周区。基因表达谱比较 IAN横断和对照组织显示甘丙肽意外地显著增加28倍 (GAL)TGF-β信号分子Smad 2、Smad 3和TGF-β1减少两倍以上， 以及Wnt抑制剂Dkk 1、Dkk 2和Gsk-3β。在同一IAN横断组中，microRNA miR-92 b 通过miRNA谱和RT-PCR证实，表达上调超过两倍。MiR-92b IAN切断后的上调与涉及Wnt和TGF-β作为 可能的miR-92 b靶点促使我们推测GAL影响其成骨下游效应 通过miR-92 b。体外研究表明GAL处理促进PDL的成骨分化 祖细胞和增加矿化，同时减少BMMC的破骨细胞生成。RhoA阻断或 GAL拮抗剂的应用影响PDL细胞骨架结构和基因表达，表明 GAL通过G蛋白偶联受体发挥作用。当应用于动物牙周袋时 患有牙周炎，GAL组织工程构建体完成了20%的牙槽骨增加， 骨水平相比，对照组，导致临床显着增加牙槽骨高度。基于 基于这组有希望的新数据，我们设计了一项研究计划，以确定GAL在响应 IAN横断，确定其对骨骼发生影响的机制，并利用其 适用于骨再生和预防牙周强直。我们的总体目标是 研究计划是验证牙周神经影响牙槽骨稳态的假设 通过GAL-GPCR-miR-92 b-TGF-β/Wnt调控环及其应用 神经分泌肽将刺激Wnt信号传导和新骨形成。我们预计 我们的研究结果将导致创新的神经肽为基础/工程混合方法， 帮助数百万患者恢复牙周健康，并预防相关的情感和功能性疤痕 牙齿脱落和牙列不全

The periodontium is a richly innervated tissue that undergoes continuous modelling and remodeling by alveolar bone osteoblasts, osteocytes, and osteoclasts. Studies from our laboratory have demonstrated a close spatial relationship between trigeminal nerve ganglia and the Epithelial Rests of Malassez (ERM), an epithelial cell network residing within the non-mineralized periodontal ligament. Inferior alveolar nerve (IAN) transection studies resulting in dento-alveolar ankylosis and a reduction in ERM have confirmed the essential role of sensory innervation for periodontal homeostasis. For the present application, we have established the IAN transection model in our laboratory and provided radiographic evidence for enhanced mineralization and ankylosis in the periodontal region of rat molars. Gene expression profiling comparing IAN transected and control tissues demonstrated an unexpected 28-fold significant increase in galanin (GAL) and a more than two-fold decrease in the TGF-β signaling molecules Smad2, Smad3 and Tgf-β1, and the Wnt inhibitors Dkk1, Dkk2 and Gsk-3β. In the same IAN transection group, microRNA miR-92b expression was more than two-fold upregulated, as verified via miRNA profiling and RT-PCR. MiR-92b upregulation after IAN transection in conjunction with bioinformatics data implicating Wnt and Tgf-β as possible miR-92b targets prompted us to speculate that GAL affects its skeletogenic downstream effects through miR-92b. In vitro studies revealed that GAL treatment promoted osteogenic differentiation of PDL progenitors and increased mineralization, while reducing osteoclastogenesis of BMMCs. Block of RhoA or application of GAL antagonist affected PDL cytoskeletal organization and gene expression, indicating that GAL functions through G protein coupled receptors. IWhen applied to periodontal pockets of animals suffering from periodontitis, GAL tissue engineering constructs accomplished a 20% increase in alveolar bone levels compared to controls, resulting in a clinically significant increase in alveolar bone height. Based on this promising new set of data we have designed a research plan to define the role of GAL in response to IAN transection, determine the mechanism underlying its effect on skeletogenesis, and exploit its applicability for bone regeneration and the prevention of periodontal ankylosis. The overall goal of our research plan is to test the hypothesis that periodontal nerves affect alveolar bone homeostasis through a GAL–GPCR–miR-92b–TGF-β/Wnt regulatory loop and that application of GAL neurosecretory peptides will stimulate Wnt signaling and new bone formation. We anticipate that the outcomes of our study will lead to innovative neuropeptide based/engineering hybrid approaches that will restore periodontal health in millions of patients and prevent the emotional and functional scars associated with lost teeth and incomplete dentitions.