Biomimetic Growth of Enamel-like Hierarchical Structures

描述（由申请人提供）：拟定研究旨在证明，在存在特定分子的情况下，使用焦磷酸盐（PPi）稳定的高度过饱和溶液可生成酸蚀刻人牙釉质的高度定向牙釉质样纳米和微米结构，这些分子具有引导成熟牙釉质晶体外延生长的能力。这项研究是由PI实验室的最新进展指导的，这些进展为釉基质蛋白调节釉质形成的机制提供了独特的见解，并提供了强有力的初步数据，支持提出的仿生方法用于釉质再生的可行性。尽管在促进口腔卫生和氟化方面做出了巨大努力，但需要进一步研究以实现易于应用、快速生长的用于仿生修复的釉质样生物陶瓷。因此，非常需要开发有效的手段来再生牙齿结构。中心假设是，可以通过调节矿物离子的可用性、晶体生长动力学和晶体取向来实现适当的釉质结构和功能的恢复。从长远来看，更好地了解釉质矿物质形成的机制将有助于开发用于釉质再生的新型仿生和生物相容性修复材料，例如，治疗早期龋齿。釉原蛋白样物质，特别是商业合成的富含亮氨酸的釉原蛋白肽（LRAP）中保留的釉原蛋白的关键功能序列，可以潜在地用于牙釉质结构和性质的再生。本提案的目标将通过完成以下两个具体目标来实现：目标1。确定LRAP和非磷酸化全长釉原蛋白在体外使用焦磷酸盐（PPi）稳定的过饱和磷酸钙溶液引导釉质结构再生的机制和有效性。酸蚀刻的人牙釉质的矿化动力学和外延生长将通过PPi矿化抑制剂的水解来调节，所述水解通过1）牙釉质表面本身和2）碱性磷酸酶（AP）进行。目标2.确定高度过饱和磷酸钙溶液的机制和有效性，该溶液由磷酸化的天然和合成釉原蛋白稳定，使用AP在体外触发釉质结构的再生。全长天然（磷酸化）猪釉原蛋白和LRAP（+P），过饱和磷酸钙溶液的有效稳定剂，将被研究作为PPi的替代品，并检查其额外的潜力，引导再生的酸蚀釉质矿物结构后，通过添加AP去磷酸化。将使用SEM、EDX、FT-IR和掠入射X射线衍射评估形成的矿物的程度、性质和取向。将从机械性能、物理化学性能和矿物附着强度方面评估基线釉质性能的恢复和/或改善。

DESCRIPTION (provided by applicant): The proposed study is designed to prove that highly oriented enamel-like nano- and micro-structures of acid-etched human enamel can be generated using pyrophosphate (PPi)-stabilized highly supersaturated solutions in the presence of specific molecules that exhibit the capacity to guide the epitaxial growth of mature enamel crystals. This study is guided by recent advances in the PI's laboratory that have provided unique insight into the mechanism by which enamel matrix proteins regulate enamel formation and strong preliminary data that support the feasibility of proposed biomimetic approaches for enamel regeneration. Despite tremendous efforts in promoting oral hygiene and fluoridation, further research is needed to achieve an easy-to-apply, fast growing enamel-like bioceramic for biomimetic repair. Thus, there is a great need to develop effective means to regenerate tooth structures. The central hypothesis is that the restoration of proper enamel structure and function can be achieved through the regulation of mineral ion availability, crystal growth kinetics, and crystal orientation. Long-term, a better understanding of the mechanism of enamel mineral formation will aid in the development of novel biomimetic and biocompatible restorative materials for enamel regeneration and, for example, the treatment of early dental caries. Amelogenin-like materials, especially key functional sequences of amelogenin retained within commercially synthesized leucine-rich amelogenin peptide (LRAP) can potentially be used in the regeneration of tooth enamel structure and properties. The goals of this proposal will be achieved through the completion of the following two Specific Aims: Aim 1. To determine the mechanism and effectiveness of LRAP and non-phosphorylated full-length amelogenin to guide the regeneration of enamel structure in vitro using pyrophosphate (PPi)-stabilized supersaturated calcium phosphate solutions. Mineralization kinetics and epitaxial growth of acid-etched human enamel will be regulated by the hydrolysis of the PPi mineralization inhibitor by 1) the enamel surface itself and 2) alkaline phosphatase (AP). Aim 2. To determine the mechanism and effectiveness of highly supersaturated calcium phosphate solutions that are stabilized by phosphorylated native and synthetic amelogenins using AP to trigger the regeneration of the enamel structure in vitro. Full-length native (phosphorylated) porcine amelogenin and LRAP(+P), potent stabilizers of supersaturated calcium phosphate solutions, will be studied as substitutes for PPi and examined also for their additional potential to guide th regeneration of the acid-etched enamel mineral structure upon dephosphorylation by added AP. The extent, nature, and orientation of formed mineral will be assessed using SEM, EDX, FT-IR and grazing incidence X-ray diffraction. The restoration and/or improvement of base-line enamel properties will be assessed with respect to mechanical properties, physico- chemical properties, and strength of mineral attachment.