载锌纳米氟磷灰石晶体对人工釉质龋抗酸及抗菌性研究

Dental caries is a pathological process of localized destruction of tooth tissue by micro-organisms. The disease is initiated via the demineralization of the tooth mineral (apatite) by organic acids produced from fermentable carbohydrate by dental plaque cariogenic bacteria. Calcium phosphate mineralizing solutions can mineralize the caries lesion by generating hydroxyapatite. Nano-hydroxyapatite incorporating fluoride are desirable materials by greatly increasing the mineralizing efficiency and inhibiting enamel dissolution, but without antibacterial ability. Zinc (Zn) ions has been shown to have antibacterial property, inhibiting plaque formation and gingival inflammation. In our preliminary studies, coating consisting of nano-hydroxyapatite incorporating zinc and fluoride ions (Zn-FHAp) provided dentin surfaces less susceptible to acid dissolution and have antibacterial properties. The concentration of zinc ion not only affected the antibacterial property, but also the type and amount of calcium phosphate crystals forming on the tooth surfaces. The objective of the proposed study is to determine the efficacy of Zn-FHAp treatment on sound enamel and enamel with artificial caries lesion on minimizing susceptibility to acid dissolution and bacterial growth and colonization. Artificial caries lesions will be created and treated with Zn-FHAp. The untreated and treated tooth surfaces will be characterized using MicroCT, scanning electron microscopy (SEM), energy dispersive system; x-ray diffraction and Fourier Transform Infrared spectroscopy. Dissolution properties will be determined in acidic buffer (0.1M KAc, pH 6, 37℃) by monitoring the calcium ions released with time. Confocal Laser Scanning Microscopy, SEM and Bioquant measurements will be used to evaluate the growth and metabolism of bacteria in biofilm model on the Zn-FHAp treated enamel surfaces. This study may demonstrate that Zn-FHAp coating have mineralizing, acid resistance, and antibacterial effects and may be potentially useful as a treatment strategy against caries formation and progression.

目前再矿化治疗的主要缺陷是缺乏抗菌作用，本课题组前期研制的载锌纳米氟磷灰石（Zn-FHAp）矿化液在无龋牙体模型上具备有效矿化牙本质小管及抑制变形链球菌对牙本质粘附性能，并证实其主要成分锌离子可降低口腔菌斑生物膜的活性。我们推测Zn-FHAp对牙釉质龋具有填充脱矿区缺陷和微孔、抗酸及抗菌作用。为深入了解Zn-FHAp对人工釉质龋抗酸及抗菌性的影响及作用机理，本课题拟建立人工釉质龋模型，采用沉淀法制备Zn-FHAp矿化液对人工釉质龋进行再矿化处理，显微CT和电感耦合等离子体分析Zn-FHAp对脱矿牙釉质晶形、结晶度及抗酸性影响；进一步利用人工口腔系统模型，采用激光共聚焦扫描显微镜及BioquantNova图像系统分析Zn-FHAp对菌斑生物膜早期形成过程中结构及细菌活性的影响。本课题将阐明Zn-FHAp在防龋过程中抗酸及抗菌性作用及机理，为Zn-FHAp矿化液临床应用于龋病防治提供理论依据。

研究背景：再矿化治疗是防治龋病的有效手段，理想的再矿化液应同时具有良好的再矿化性、抗酸及抗菌性。再矿化材料包括氟化物、羟基磷灰石、纳米羟基磷灰石(Nano- hydroxyapatite, nHAp)、磷酸钙、碳酸氢钠、酪蛋白磷酸肽-无定形非晶体磷酸钙等。其中氟化物具有重要的生物学活性，而锌离子是影响微生物和细胞正常生长的重要微量营养元素之一。结合氟离子再矿化作用及锌离子抗菌作用，采用沉淀法研制出载锌纳米氟磷灰石矿化液，探索早期龋病防治的新方法。主要研究内容：1.配制不同锌、氟离子浓度的矿化液，比较其并观察其在体外对牙釉质的矿化效果，筛选出性能理想的牙釉质矿化液，研究锌氟磷灰石纳米晶体对口腔菌斑生物膜细菌生长的作用和影响。2.比较牙釉质表面沉积载锌氟磷灰石纳米晶体后对变形链球菌细菌的附着和繁殖的影响，分析锌、氟离子在抗菌防龋过程中所起的抑菌作用。3. 采用CLSM，对口腔原位菌斑生物膜在不同浓度的锌离子作用下细菌聚集和活性进行检测。4. 分析比较载锌纳米氟磷灰石晶体与草酸钾、改良的草酸溶液OX/Ca及OX/P对牙本质小管钙化封闭的性能。重要结果：1.扫描电镜观察到经矿化液处理后的牙釉质切片表面有聚集的微晶沉积。X 射线衍射仪分析牙釉质表层沉积的羟基磷灰石晶体显示，含氟离子组和同时含有锌、氟离子组矿化液与含锌离子组或对照组相比，晶粒增大、晶体完整性增高及沉积量增多。载锌氟磷灰石纳米晶体处理的牙釉质表面红外光谱仪图谱分析，结果显示在1102、1065、1027 cm-1 处呈现多个P-O（磷酸组）吸收峰。2.在细菌粘附和繁殖实验显示，载锌磷灰石纳米晶体和氟磷灰石纳米晶体组粘附的细菌数量也低于磷灰石晶体组。3．对口腔原位菌斑生物膜在不同浓度的锌离子作用下细菌聚集和活性进行检测结果显示2.5、5.0、10 、20 mmol/L ZnCl2 均可减少细菌膜厚度及降低菌斑指数。2.5 mmol/L ZnCl2能有效降低外层生物膜细菌的活性，5 mmol/L ZnCl2 可同时减少菌斑生物膜外层和中间层细菌活性。4. 载锌纳米氟磷灰石晶体封闭牙本质小管的性能明显高于草酸钾、改良的草酸溶液OX/Ca及OX/P。科学意义 载锌氟磷灰石纳米晶体矿化液可以在牙釉质表面形成具有保护功能的载锌氟磷灰石晶体层，有效抑制变形链球菌在牙釉质表面的附着和繁殖，具有较强的抗酸性，暗示其具有明显的抗龋性