PAMAM-多肽复合体介导口腔微环境中牙体硬组织仿生矿化的研究

The interface of dental hard tissue, which involves several kinds of organic and inorganic factors, such as healthy or injured dental tissue, dental biofilm and salivary acquired pellicle, is a very complicated microenvironment. Capabilities, such as specific binding ability to dental surface, effectively inhibiting the adhesion of dental biofilms and resisting extreme challenges under the oral microenvironment, are very important for biomimetic mineralization systems to achieve good protective and restorative effects. Being inspired by salivary acquired pellicle, the aim of this project is to establish a biomimetic mineralization system which can specifically bind to dental surface. First, molecular simulation and hydroxyapatite (HAP) binding assay will be employed to design and synthesize a series of polypeptides, which can specifically bind to HAP. Then, the specific polypeptide will be combined to an optimal PAMAM selected according to our previous research to form a multifunctional PAMAM-peptide complex. Meantime, an oral microenvironment mineralization model will be established. All kinds of oral factors, such as the components, flow rate and pH of saliva and oral bacteria will be considered. Subsequently, the binding capacity to dental surface, the protection to healthy dental hard tissue and the restoration to injured dental hard tissue of the complex will be investigated under the oral microenvironment mineralization model. In addition, the binding mechanism and optimal mineralization conditions for the complex will be explored. At last, the dual protection and restoration of the complex will be comprehensively evaluated in an animal caries model and a human in situ model. This project is very promising to develop new strategy and technology for the prevention and treatment of injured dental hard tissues.

牙体硬组织界面是健康/损伤牙体组织、生物膜、唾液蛋白膜等有机/无机物共存的微环境。在该环境中能否竞争生物膜，抵御苛刻口腔环境，增强牙面吸附能力是仿生矿化体系作用的关键。受唾液获得性膜启迪，本课题拟构建特异性吸附于牙面的仿生矿化体系，针对上诉问题展开研究。首先以获得性膜中特异性吸附牙面的功能氨基酸为基础，通过分子模拟和体外羟磷灰石吸附实验，设计合成筛选具有特异性吸附能力的多肽，并结合课题组前期PAMAM仿生矿化的研究结果，将多肽引入目标PAMAM分子结构的外部基团，构建特异性吸附牙面的PAMAM-多肽复合体；并通过调节唾液成分、细菌种类，模拟唾液流速流量、pH值，建立口腔微环境矿化模型。再借助该模型研究复合体对牙体吸附、健康牙体保护和损伤牙体修复的条件、效果和机制。最后通过大鼠龋病模型和口腔原位硬组织模型评价复合体保护/修复的效果。本研究将为牙体硬组织损伤性疾病的仿生防治提供新思路和新技术。

牙体硬组织疾病是最常见的牙病，以龋病、牙本质敏感症为代表，是人类口腔并影响全身健康的重大问题。现行的技术主要通过氟、钙、磷等再矿化处理保护/修复牙体硬组织，采用复合树脂、玻璃离子、陶瓷等高分子材料修复缺损的牙体硬组织。但是，由于微环境中细菌生物膜、唾液蛋白膜、细菌代谢酸等作用，现有的技术保护有限，失败率较高。为解决这一科学问题，本课题受口腔中唾液获得性膜蛋白启迪，围绕构建特异性吸附于牙体硬组织的仿生矿化体系展开研究。课题开展前期通过对唾液获得性膜蛋白吸附羟基磷灰石（HA）的原理及影响因素的研究分析，成功设计并合成了特异吸附HA的7肽，通过一系列体外实验验证了其吸附HA的能力，并进一步发现其可在吸附界面上诱导矿化的发生。因此调整思路，利用龋病动物模型评价了7肽直接防龋的功效。由于牙体硬组织界面中细菌生物膜及其代谢产物对仿生矿化的不利影响，在成功构建7肽-PAMAM复合材料并验证其吸附HA的性能后，进一步揭示了其具有长效抗菌作用。另外，基于对富酪蛋白吸附HA机制的分析，筛选出其具有二级结构的15肽吸附功能域，运用计算机分子模拟技术揭示了其在类唾液微环境中的吸附行为和机制。在此基础上，成功构建15肽-PAMAM复合材料并通过一系列实验验证了其吸附HA的能力，探究了其发挥矿化模版作用，诱导牙釉质再矿化的效果。结合临床粘接修复的应用场景，改变策略，引入纳米无定形磷酸钙粘接剂，在模拟口腔酸冲击环境中，成功实现了牙釉质再矿化。本课题探索了牙体硬组织保护和损伤修复的新途径，为牙体硬组织损伤性疾病的防治提供了新思路和新技术。