Targeting the AMPK pathway to enhance dentin repair with novel metformin-releasing dental cements

项目总结 成人恒牙暴露可影响牙本质-牙髓复合体的正常结构和功能 牙髓在深度龋齿去除、创伤或意外修复手术后的重要牙髓刺激牙本质 修复，保持牙髓活力，避免更具侵入性和昂贵的手术，重要的牙髓疗法依赖于直接盖髓 探员们。这些矿物主要由无机水硬性硅酸钙水泥组成，其中矿物三氧化物集合体。 (MTA)通常被认为是黄金标准。尽管它的治疗价值被广泛接受，但目前还不清楚哪种具体的 牙髓干细胞分化调控修复性牙本质形成的信号机制 (DPSCs)分化为成牙本质细胞样细胞。此外，与MTA相关的常见缺点包括设置时间长和高 成本。因此，通过新的、更实惠的生物活性配方来加强牙本质修复， 分子靶向负责其合成的牙髓细胞的物理机械特性可以转化为真正的 有益且极具成本效益的治疗结果。我们提供了第一个证据来支持 由磷酸钙骨水泥/壳聚糖(CPCC)和二甲双胍(Met)组成的新型生物材料，引发了 DPSCs成牙本质细胞分化标志物表达和矿物质合成显著上调。大都会是 一种广泛使用、安全、低成本的口服抗糖尿病药物，以及AMP激活的蛋白激酶的有效激活剂 (AMPK)信号通路，细胞生物能量学的主要传感机制。这些有希望的初步数据意味着 甲硫氨酸可以在局部给药配方中安全地改变用途，通过分子作用增强修复性牙本质 瞄准AMPK。在拟议的研究中，我们试图通过开发一种新的Met-CPCC盖髓来最大化牙本质修复 具有类似MTA的机械和流动性能的助剂，但在 设定时间和成本。这一创新配方依赖于蛋氨酸诱导AMPK激活和成牙本质细胞分化 在DPSCS和CPCC中，为羟基磷灰石的形成提供碱性、离子构建块。为此，我们将测试 中心假说：活髓暴露后牙本质修复显著增强甲硫氨酸释放的CPCC 生物活性盖髓剂通过AMPK活化和输送矿化的组织构建离子。在体外和在 活体研究将通过两个具体目标扩展我们的初步发现。目标1将检验以下假设：在DPSCS中，一部小说 MET-CPCC盖髓剂以AMPK依赖的方式诱导牙源性反应。在目标2中，我们将测试 Met-CPCC盖髓剂显著促进牙本质修复并增加硬度和弹性的假说 牙髓暴露大鼠牙本质损伤模型中新生牙本质的模数。这项提议的长期目标是 以AMPK为靶点的新型生物活性Met牙科材料促进修复性牙本质形成 激活，产生新的机制和改进的治疗方法，广泛适用于修复和再生 牙科。

PROJECT SUMMARY The normal structure and function of the dentin-pulp complex in adult permanent teeth can be affected by the exposure of a vital pulp following deep caries removal, traumatic injuries, or accidental restorative procedures. To stimulate dentin repair, preserve pulp vitality and avoid more invasive and costly procedures, vital pulp therapy relies on direct pulp-capping agents. These are mainly composed of inorganic hydraulic calcium-silicate cements, where mineral trioxide aggregate (MTA) is often considered the gold standard. Despite its well-accepted therapeutic value, it remains unclear which specific underlying signaling mechanisms orchestrate reparative dentinogenesis through the differentiation of dental pulp stem cells (DPSCs) into odontoblast-like cells. Also, common drawbacks associated with MTA include long setting times and high cost. Thus, enhancing dentin repair through novel, substantially more affordable bioactive formulations with improved physico-mechanical properties that molecularly target the pulp cells responsible for its synthesis could translate into truly beneficial and highly cost-effective therapeutic outcomes. We provide the first evidence supporting the development of a novel biomaterial formulated with calcium phosphate cement/chitosan (CPCC) and metformin (Met), that triggered a significant upregulation in the expression of odontoblastic differentiation markers and mineral synthesis in DPSCs. Met is a widely used, safe and low-cost oral anti-diabetic biguanide drug, and potent activator of the AMP-activated protein kinase (AMPK) signaling pathway, a master sensing mechanism of cellular bioenergetics. These promising preliminary data imply that Met could be safely repurposed within locally delivered formulations to enhance reparative dentin by molecularly targeting AMPK. In the proposed studies, we seek to maximize dentin repair by developing a new Met-CPCC pulp-capping agent with similar mechanical and flowability properties like MTA but with a substantial, several folds of reduction in setting time and cost. This innovative formulation relies on Met to induce AMPK activation and odontoblastic differentiation in DPSCs, and CPCC to provide the alkaline, ionic building blocks for hydroxyapatite formation. To that end, we will test the central hypothesis that dentin repair following vital pulp exposure is significantly potentiated by a Met-releasing CPCC bioactive pulp-capping agent through AMPK activation and delivery of mineralized tissue-building ions. In vitro and in vivo studies will expand our initial findings through two specific aims. Aim 1 will test the hypothesis that in DPSCs, a novel Met-CPCC pulp-capping agent induces odontogenic responses in an AMPK-dependent manner. In Aim 2, we will test the hypothesis that Met-CPCC pulp-capping agent significantly enhances dentin repair and increases the hardness and elastic modulus of new dentin in a rat dentin injury model with pulp exposure in vivo. The long-term goal of this proposal is to potentiate reparative dentinogenesis with novel biologically active Met-containing dental materials targeting AMPK activation, yielding new mechanisms and improved treatments that are widely applicable to restorative and regenerative dentistry.