A Peptide-Based Biomineralization Strategy for Tooth Repair

项目概要/摘要 我们寻求开发一种生物矿化方法来生长仿生釉质样层， 与天然牙釉质和牙本质的无缝化学附着。这种结构化的生物材料将防止 并将用作增强的牙科修复材料，用于治疗非龋齿。 龋性宫颈病变（NCCL）。我们报道了我们正在申请专利的由壳聚糖和 釉原蛋白（CS-AMEL）可促进釉质样层的再生并使牙本质矿化。在这里，我们将 利用基于釉原蛋白启发的肽的仿生策略。使用肽的优点是 翻译/临床目的在于短肽更容易使用以及更经济的事实 具有临床实用价值。对于肽而言，获得监管批准的途径也可能更容易。我们 假设我们合理设计的肽壳聚糖水凝胶（Amel-P-CS）将刺激 釉质样矿化层在牙本质/釉质界面，并将促进引导的 牙本质胶原隔室，从而增强与牙本质中的有机内容物的结合。下列具体 提出的目的：目的I）研究釉原蛋白的组装及其形成磷灰石矿物的潜力- 衍生的肽P26和P32，然后将它们应用于壳聚糖水凝胶。我们将使用CD，冷冻TEM， 显微拉曼光谱和原位原子力显微镜来研究肽的二级和三级结构， 在体外胶原存在和不存在的情况下，它们对磷灰石矿化的影响。（二）发展 优化了含P26和P32肽的壳聚糖水凝胶（Amel-P-CS）的处方， Amel-P-CS水凝胶重建具有增强的机械性能的釉质样层的潜力， 坚固的附着于蚀刻的釉质表面。脱矿釉质表面的人磨牙冠切片 将用于目的III）检查Amel-P-CS水凝胶重建牙釉质样层的潜力， 增强的机械性能和与脱矿牙本质表面的牢固附着。我们将进一步 检查水凝胶在牙本质胶原内成核和生长磷灰石晶体的潜力 这有助于增强与牙本质中的有机物的结合。人磨牙牙冠切片 将使用脱矿质牙本质表面。目的IV）检查Amel-P-CS水凝胶在治疗中的功效。 在离体模型中修复牙釉质和牙本质暴露的人工宫颈损伤。我们将使用整个 拔出的牙齿进行pH循环方案。总之：如果拟议目标的各项目标得以实现， 我们将提供一种技术（在牙托上提供的水凝胶），该技术：a）将提供增强的仿生 釉质样涂层材料，B）有效地重建由于NCCL损伤而丧失的牙齿结构，和c）将 预防牙本质过敏和蛀牙的发展。

PROJECT SUMMARY / ABSTRACT We seek to develop a biomineralization approach to grow a biomimetic enamel-like layer that will have a seamless chemical attachment to natural enamel and dentin. Such a structured biomaterial will prevent progression of tooth decay and will be utilized as an enhanced dental restorative material for treating non- carious cervical lesions (NCCL). We reported that our patent-pending hydrogels composed of chitosan and amelogenin (CS-AMEL) can promote regrowth of an enamel-like layer and remineralize dentin. Here, we will utilize an amelogenin-inspired peptide-based biomimetic strategy. The advantage of using peptides for translational/clinical purposes lies in the fact that short peptides are easier to use as well as more economical and practical for clinical application. The pathway to regulatory approval may also be easier for peptides. We hypothesize that our rationally designed peptide chitosan hydrogel (Amel-P-CS) will stimulate growth of an enamel-like mineralized layer at the dentin/enamel interface and will promote guided remineralization of the dentin collagen compartments, thereby enhancing bonding to the organic content in dentin. Following specific aims are proposed: Aim I) To investigate the assembly and apatite mineral-forming potential of amelogenin- derived peptides P26 and P32 prior to their application in the chitosan hydrogel. We will use CD, Cryo-TEM, micro Raman spectroscopy and in situ AFM to investigate the peptides’ secondary and tertiary structures and their influence on apatite mineralization in the presence and absence of collagen in vitro. Aim II) To develop and optimize the formulation of P26 and P32 peptide-containing chitosan hydrogels (Amel-P-CS) and examine the potential of Amel-P-CS hydrogels to rebuild an enamel-like layer with enhanced mechanical properties and robust attachment to etched enamel surface. Human molar crown slices with demineralized enamel surfaces will be used. Aim III) To examine the potential of Amel-P-CS hydrogels to rebuild an enamel-like layer with enhanced mechanical properties and robust attachment to a demineralized dentin surface. We will further examine the potential of the hydrogels to nucleate and grow apatitic crystals within the dentin collagen compartments, thereby enhancing bonding to the organic content in dentin. Human molar crown slices with demineralized dentin surfaces will be used. Aim IV) To examine the efficacy of Amel-P-CS hydrogels in repairing artificial cervical lesions in ex vivo models where enamel and dentin are exposed. We will use whole extracted teeth subjected to a pH-cycling regimen. In summary: If the goals of proposed aims are achieved, we will deliver a technology (hydrogel delivered on dental trays) that: a) will provide enhanced biomimetic enamel-like coating material, b) effectively rebuild dental structures lost due to NCCL lesions, and c) will prevent dentinal hypersensitivity and progression of tooth decay.