融合肽(钛无机结合肽-富组蛋白5衍生抗菌肽)涂层抑制钛表面细菌生物膜形成的机制和应用研究

Biofilm formation caused by oral bacteria adhered to the surface of the titanium implant may lead to peri-implantitis and eventually contribute to failure of the implant treatment, which is one of the difficulties oral medicine currently faced with. Present studies have shown that antimicrobial coatings of titanium surfaces (including inorganic/organic antibacterial agents and antibiotics) are able to avoid bacterial adhesion and prevent biofilm formation. However, several problems still remain, such as cytotoxic, unstable drug loading amount and sustained release time, antibiotic resistance and etc. Thus, antimicrobial peptides(AMPs) are now considered as a kind of ideal anti-biofilm material for its broad-spectrum activity, lower concentration of the onset, less possibility of inducing drug resistance and other advantages. Nevertheless, the current procedure of fixing method, covalent cross-linking, is relatively complicated and is unable to maintain the desired conformation which is indispensible for the antibacterial activity of antimicrobial peptides. To solve these problems, we decide to link the peptide (minTBP1) that can specifically bind to titanium surfaces and the antimicrobial peptides derived from histatin5(dHis5) with a proper linker. These chimeric peptides are expected to elaborate strong antimicrobial ability on titanium surfaces. The goals of our study are as follows: (1)to elucidate the designing rules of chimeric peptides by vesicle models and computer simulations;(2)to explain their binding and antimicrobial mechanisms by biofilm models and animal models;(3)to summarize their rules of inhibiting biofilm formation and promoting osteointegration.

钛种植体表面形成生物膜将引起种植体周围炎，并最终导致种植失败。研究表明钛表面抗菌涂层(装载无机、有机抗菌剂或抗生素)可以抵御细菌粘附阻止生物膜形成，但也存在有细胞毒性、载药量和缓释时间不稳定、产生抗生素耐药性等问题。抗菌肽凭借其广谱抗菌、低浓度起效、低诱导耐药性等优势被认为是理想的抗菌剂。但是，目前用共价交联方法在钛表面固定抗菌肽的过程复杂，不易使抗菌肽保持抗菌活性所需的构象。针对这些问题，我们将一种能与钛表面特异性结合的多肽-钛无机结合肽1(minTBP1)与人唾液中存在的富组蛋白5(His5)的衍生抗菌肽连接，得到具有钛结合端和抗菌端的双功能融合肽，并将其吸附于钛表面达到抗菌作用。本研究将通过囊泡模型和计算机模拟阐明融合肽的设计规律，通过钛表面生物膜模型和动物模型进一步解释融合肽与钛表面结合及抗菌机制，总结其在种植体微环境下抑制生物膜形成和促进成骨的规律，最终获得新型种植体抗菌涂层。

目的：细菌附着及生物膜形成是种植体相关感染的首要病因。生物膜一旦形成则很难消除，其长期存在影响种植体-骨界面形成骨结合，最终导致种植手术的失败。钛种植体表面抗菌涂层修饰成为近年来研究热点。本研究设计通过将抗菌肽与钛结合肽以柔性/刚性连结肽相连接设计新型融合肽，并以融合肽修饰金属钛表面以获得抑菌的生物性能，旨在抑制种植术后生物膜的形成，以期降低种植体周围炎和种植体周围黏膜炎的发生率。材料与方法：通过直接连接及柔性（GGGGS）/刚性（PAPAP）连接肽连接抗菌端（JH8194: KRLFRRWQWRMKKY）和钛结合端（minTBP-1: RKLPDA）构建3种融合肽，并以合成的多肽修饰钛金属表面。通过石英晶体微天平（QCM）、原子力显微镜（AFM）以及X射线光电子能谱（XPS）表征多肽在金属钛表面的吸附学行为，并以激光共聚焦显微镜观察该表面对牙龈卟啉单胞菌的抑菌作用。结果：与无linker的融合肽相比，以linker连接抗菌端和钛结合端构建的融合肽显示出更强的结合性能（p<0.05）。各多肽在钛表面随浓度增加的吸附学行为符合单一Langmiur吸附模型，其随时间变化的吸附学行为符合双项Langmiur吸附模型。同时，有linker的融合肽显示出较柔性者更强的抗菌性能。结论：linker和无机结合肽可以明显发挥融合肽的抵抗专性厌氧菌和结合金属的性能，能够构建有效的双功能融合肽，从而为临床上有效抑制牙种植术后种植体周围生物膜的形成提供了理论基础。同时，本研究也为连结肽的恰当选择和融合肽的合理设计提供了实验依据。