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

项目摘要 成年恒牙的牙本质-牙髓复合体的正常结构和功能会受到暴露的 深龋去除、外伤或意外修复程序后的活髓。刺激牙本质 修复，保持牙髓活力，避免更具侵入性和昂贵的程序，活髓治疗依赖于直接盖髓 剂.这些主要由无机水硬性硅酸钙水泥组成，其中矿物三氧化物聚集 (MTA)通常被认为是黄金标准。尽管它的治疗价值被广泛接受，但仍不清楚哪些特异性 牙髓干细胞分化诱导修复性牙本质形成的信号机制 成牙本质细胞样细胞。此外，与MTA相关的常见缺点包括凝固时间长和温度高。 成本因此，通过新型的、实质上更实惠的生物活性制剂增强牙本质修复， 物理机械性能，分子靶向纸浆细胞负责其合成可以转化为真正的 有益的和高成本效益的治疗结果。我们提供了第一个证据支持发展的一个 用磷酸钙骨水泥/壳聚糖（CPCC）和二甲双胍（Met）配制的新型生物材料， DPSC中成牙本质细胞分化标志物和矿物质合成的表达显著上调。Met是 一种广泛使用的、安全且低成本的口服抗糖尿病双胍类药物，以及AMP活化蛋白激酶的强效激活剂 （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表明， Met可以在局部递送的制剂中安全地重新利用，以通过分子方式增强修复性牙本质， 目标是AMPK在本研究中，我们试图通过开发一种新的Met-CPCC盖髓术来最大限度地修复牙本质 具有与MTA类似的机械和流动性性能，但具有显著的，几倍的降低， 设置时间和成本。这种创新的配方依赖于Met诱导AMPK活化和成牙本质细胞分化 在DPSC和CPCC中，以提供用于羟基磷灰石形成的碱性离子结构单元。为此，我们将测试 中心假设是，在活髓暴露后牙本质修复显著增强的Met释放CPCC 通过AMPK激活和矿化组织构建离子的递送，提供生物活性盖髓剂。体外和 体内研究将通过两个具体目标扩展我们的初步发现。目的1将检验以下假设：在DPSC中， Met-CPCC盖髓剂以AMPK依赖性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设 牙髓暴露的大鼠牙本质损伤模型中新生牙本质的模量。该提案的长期目标是 以AMPK为靶点的新型生物活性含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，广泛适用于恢复和再生 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.