仿生肽引导纳米无定形磷酸钙定向有序再矿化牙体硬组织

The remineralization of the dental hard tissues is of great clinical value to the prevention and treatment of enamel/dentine caries，root caries and dentine hypersensitivity. With traditional methods using fluoride, the remineralization of teeth is carried out by formation of fluorapatite and through the epitaxial growth of the residual crystals. Due to the lack of ability of guiding the formation mineral crystals, the conventional methods cannot result in oriented and ordered remineralization of dental hard tissues, which are different from the natural ones. Although nano-amorphous calcium phosphate (nano-ACP), as precursor of hydroxyapatite (HAP), is the ideal choice for biomimetic remineralization, it is difficult for nano-ACP to accomplish self-assembly and bind to dental hard tissues. According to the biomimetic mineralization strategy from the non classical crystallization theory, we try to link a C-terminal peptide from amelogenin and the peptides that can specially bind to HAP or collagen with a proper linker, to obtain a biomimetic chimeric peptide including both guiding motif and binding motif to dental hard tissues, which can guide nano-ACP to remineralize enamel and dentine orderly. In this study, the biomimetic peptides would be optimized continuously through the in vivo and in vitro mineralization studies to obtain the shortest but the most functional chimeric peptide to induce ordered and oriented remineralization. This novel method would make the remineralized dental hard tissues approach natural healthy ones with respect to mechanical and structural properties. Additionally, the rationales of the design and mineralization of the biomimetic peptides would be clarified.

牙体硬组织再矿化对预防和治疗牙釉/本质龋、牙根面龋和牙本质过敏有重要临床价值。传统的氟化物再矿化方法只能凭借残余晶体的取向外延生长和形成氟磷灰石再矿化牙体硬组织，缺乏引导定向有序矿化的能力，形成矿物晶体的结构和排列与自然牙体硬组织不同。作为生物矿化过程中羟基磷灰石的前体: 纳米无定形磷酸钙是理想的再矿化材料，但缺乏定向有序排列自组装和与牙体硬组织特异性结合的能力。本研究利用基于非经典结晶理论的仿生矿化策略，将来源于釉原蛋白的C端多肽和能与羟基磷灰石或胶原特异性结合的多肽连接，得到具有引导功能端和牙体组织结合端的双功能仿生融合肽，用于引导纳米无定形磷酸钙定向有序再矿化脱矿牙釉/本质。本课题将通过体外和体内的矿化研究，不断优化仿生肽的设计，最终获得长度较小且能发挥定向有序矿化能力的仿生肽；使再矿化的牙体硬组织在机械力学性能和矿化结构上都达到或接近自然健康组织，并阐明仿生肽的设计和矿化作用规律。

牙齿再矿化是微创牙科的重要治疗技术之一，仿生再矿化对预防和治疗早期釉质龋、牙本质龋和牙本质过敏有着重要临床价值。天然牙体硬组织的形成需要蛋白质和无机矿物盐协同配合，无定形磷酸钙纳米颗粒（Amorphous calcium phosphate, ACP)被包裹在有机结构中并在蛋白质的引导下有序排列。随着有机蛋白质被降解，ACP在一定时间和空间有序的成核、生长和组装，最终转化成为HAP晶体。. 本研究首先创新性地合成了一条仿生多肽序列，其组成包括了釉原蛋白的关键序列和特异性羟基磷灰石连接序列。应用次氯酸钠处理羧甲基壳聚糖（Carboxymethyl Chitosan，CMC）稳定的ACP，并引入仿生多肽来模拟体内釉原蛋白对ACP的生物矿化的影响。实验结果显示NaClO快速降解CMC/ACP纳米复合物以模拟基质金属蛋白酶和激肽释放酶等釉原蛋白降解酶的作用，并在仿生多肽诱导下，纳米颗粒快速而特异性地在釉质表面形成再矿化牙釉质，其表面机械性能（硬度及弹性模量）与天然牙釉质接近。为进一步优化仿生肽并获得定向有序再矿化能力的釉原蛋白类似物，采用甘氨酸模拟釉原蛋白达到定向有序再矿化的效果。本实验研究了一种接近生理条件下（37℃）在ACP的参与下，甘氨酸发挥调控作用在牙釉质深层和表面直接形成类牙釉质结构的羟基磷灰石层而达到修复牙釉质的目的的方法，证明了甘氨酸能引导ACP定向有序排列，并且利用这种方法修复以后的牙釉质具有与天然牙釉质类似的柱状结构。. 本研究合成的仿生多肽及进一步优化后的仿生肽能够促进ACP快速向羟基磷灰石晶体转化并特异性的连接在缺损牙体硬组织表面，再矿化形成的晶体结构和机械强度与天然牙相似，临床上可以进一步用于牙体硬组织龋的直接快速治疗和预防，并符合微创口腔医学的要求。