STTP-PAA双向调控混合层原位再矿化改善树脂牙本质粘接耐久性的研究

Hydrolytic degradation of adhesive resin and enzymatic degradation of exposed collagen fibrils within the bonded interface are known as the key factors contrubuting to bond failure.Clearly, improving the durability of resin-dentin bonds is a significant health issue that affects the quality of life of individuals. Using sodium tripolyphosphate(STTP) and polyacrylic acid (PAA) as dual biomimetic analogs of non-collagenous proteins(NCPs), we successfully achieved remineralization of demineralized dentin, with evidence of intrafibrillar apatite nanoplatelets deposited in an ordered manner observed in natural mineralized collagen fibrils.In this proposal, we aim to examine the binding characteristics as well as mechanism of STTP to type I collagen of dentin, to establish a clinical relevant in-situ biomimetic remineralization model of hybrid layer by developing a biomimetically modified MTA plus resin liner.This will enable STTP, PAA, calcium and hydroxyl ions to diffuse into hybrid layer,resulting in biomimetic remineralization and self-repair of poorly resin-infiltrated collagen matrices. The C-terminal telopeptides (ICTP) released by MMP degradation of hybrid layers will be detected with ELISA to quantitate enzymatic activity on the naked type I collagen fibrils.Representative remineralized specimens will be evaluated with transmission electron microscopy (TEM)and confocal laser scanning microscopy (CLSM) for changes in the ultrastructure of hybrid layers and ability of apatite backfilling of water-filled defects in hybrid layers.Nano-dynamic mechanical analysis (nano-DMA) and microtensile strength testing will be used to quantitatively probe the mechanical behavior of biomimetic remineralized dentin hybrid layers. The success of the proposed vertically-diffusing, in-situ remineralization protocol in resisting the degradation of resin-dentin bonds may warrant the role of biomimetic remineralizaiton technology in imrpoving the integrity of hybrid layers, and benifiting clinicians by providing a means for extending the durability of resin-dentin bonds via a biomimetic scheme.

混合层树脂水解析出和裸露胶原酶解变性是临床树脂充填体脱落的主要原因，如何重建其结构和功能并改善树脂牙本质粘接耐久性是亟待解决的问题。本课题组前期研究表明，多聚磷酸钠和聚丙烯酸能模拟非胶原蛋白对生物矿化的双向调控作用，诱导脱矿牙本质胶原纤维内再矿化，重现天然矿化胶原磷灰石晶体的尺寸及迭序排列特征。本研究拟进一步探讨多聚磷酸钠与I型胶原的分子作用机理，通过无定形磷酸钙纳米粒度分析及单层重组胶原矿化诱导实验，优化含多聚磷酸钠和聚丙烯酸的仿生改性MTA Plus树脂洞衬剂，改建混合层原位再矿化模型。应用酶联免疫吸附法探讨仿生矿化对I型胶原代谢的抑制作用，透射电镜和激光共聚焦扫描电镜定性分析微观形态学变化，纳米动态力学分析与微拉伸测试定量评价粘接界面的硬度和微拉伸强度等生物力学指标改变，深入揭示混合层矿化调控机理及其在改善树脂牙本质粘接耐久性中的意义，为仿生再矿化策略的最终临床应用提供充分科学依据。

本课题计划在前期研究基础上，探讨多聚磷酸钠与聚丙烯酸模拟非胶原蛋白对生物矿化的双向调控作用、牙本质胶原纤维内再矿化的作用机理，优化混合层原位再矿化模型，深入揭示混合层矿化调控机理及在改善树脂牙本质粘接耐久性中的意义，为仿生再矿化的临床应用提供充分的科学依据。本研究已基本按原计划完成，目前取得的主要成果有：⑴随初始STTP浓度的升高，DDP对STTP的吸附量也逐渐增加，浓度为90mg/mL时吸附量基本达平衡。⑵Cl-与PAA竞争胶原纤维上的正电荷结合位点，并影响后者与胶原纤维的吸附。⑶纳米级 CPP-ACP 颗粒可诱导 I 型胶原纤维间矿化，但尺度不足以渗入胶原原纤维内。生物活性树脂可诱导实现纤维内矿化。⑷设计并制备硅酸盐水门汀树脂，固化后作为矿化所需的Ca²﹢、OH-可释源。⑸用Ｉ型胶原建立单层重组胶原纤维的TEM模型，以三氧化矿物聚合物的活性成分硅酸盐水门汀和PBS作为Ca²+和PO4³-离子源，用STTP模拟NCPs C末端对胶原的结合与促成核作用，PAA模拟NCPs N末端对ACP的稳定作用，成功诱导磷灰石晶体在胶原内的有序沉积，再现与天然矿化胶原结构类似的横纹特征。⑹在前期构建重组Ｉ型胶原纤维TEM模型的基础上，在圆形盖玻片表面成功构建了用于SEM观察分析的胶原微模型。⑺从微观形态学角度阐明STTP协同PAA介导非经典成晶途径在I型胶原矿化诱导中的作用及机理：①磷酸化胶原矿化模板的形成。通过STTP处理将胶原基质转化为磷酸化、带负电荷的矿化模板。②PAA稳定ACP的形成，通过毛细作用渗透胶原支架。③钙离子静电吸附于磷酸修饰的胶原分子上，相互结合最终转化为成核前磷酸钙团簇。晶核在消耗ACP前驱相的情况下自发性生长，形成单个磷灰石片状晶体。⑻将仿生矿化与牙本质粘接结合，以STTP作为治疗性底剂，硅酸盐水门汀树脂为洞衬剂，构建临床相关的混合层原位再矿化模型。⑼采用显微CT非损伤性观察并定量分析拟仿生再矿化对人工龋牙本质的作用，证实在PVPA和PAA的协同诱导下，可实现树脂渗透不良人工龋牙本质胶原纤维内和纤维间再矿化，维持粘接界面稳定。