【作者】 田鲲；

【导师】 陈治清；

【作者基本信息】 四川大学 ， 口腔临床医学， 2007， 博士

【摘要】 背景：开发具有与牙体组织结构相似的生物材料一直都是口腔医学界的梦想。但牙釉质是高度矿化、高度有序组装的天然纳米复合材料，一直被认为太复杂而难以模拟构建，因此，通过人工手段控制磷酸钙类矿物的生长并有序排列是目前口腔仿生材料研究的重点和难点之一。目前关于牙体硬组织的体外合成的研究较少，较突出的有早期氟处理技术，亚稳态含钙、磷的矿化液，及纳米磷灰石晶体或含蛋白的纳米磷灰石晶体在牙釉质表面沉积等报道。这些方法技术隐含了仿生构建牙釉质的设计思想，但是沉淀的磷灰石晶体在形态、大小、组装上都与釉质结构有较大差距；有学者利用牙乳头细胞及组织工程的方法实现了少量牙釉质的再生，但仍存在应用上的障碍。因此，用非生物方法在体外模拟牙釉柱形成是现阶段牙釉质修复和牙釉质仿生材料研究的一个重要方向，近年来备受各科学者的关注。据本课题组了解，在牙体组织原位进行类牙羟基磷灰石生长的研究还少有报道。在这样的研究背景下，本实验依据生物矿化的有机基质调控理论，运用有序高分子作为矿化模板，体外原位模拟牙体组织仿生合成羟基磷灰石，初步获得了有序排列的晶体结构。首先，对惰性的牙釉质表面进行低温等离子改性，用氨气、氢气和氧气作为反应气体，活化牙釉质羟基磷灰石中的羟基，并引入部分氨基和羧基。其次，通过碳乙基二亚胺盐酸盐和N-羟基丁二酰亚胺介导缩合反应，将有机高分子明胶、丝蛋白通过肽键交联连接到处理过的牙釉质表面和脱矿的牙本质表面。然后在钙、磷离子溶液（过饱和矿化液和人工唾液）中实现晶体生长，生成羟基磷灰石，最后对新生的羟基磷灰石进行理化和生物学性能的检测。结果显示：低温等离子处理后釉质表面引入了-NH、-NH₂、-NH₃、-COOH等活性基团，-OH活性增加，C元素含量增高。碳乙基二亚胺盐酸盐和N-羟基丁二酰亚胺介导缩合反应将明胶和丝蛋白以肽键键合到牙体组织表面，覆盖原来的-NH₃位点，由于明胶和丝素蛋白的空间三级结构，令原有牙体表面零星分布的与钙、磷离子结合的活性位点变成致密、有序的三维分布。用过饱和矿化液和人工唾液均能实现晶体生长，所得晶体呈矮柱状，30～50nm长，垂直于牙体组织表面，相互平行排列。经理化检测证实新生晶体为羟基磷灰石，与牙体组织的结合强度在20～30N之间，硬度接近正常牙本质。经生物学性能测试证实新生晶体有良好的生物相容性，无细胞毒性，无细菌的特异粘附性。综上所述，本实验初步实现了高分子化合物在牙体组织原位引导羟基磷灰石仿生合成，生成的羟基磷灰石类似于牙体组织釉柱中的晶体形貌，说明三维有机高分子引导的矿化反应是实现牙再生的有巨大潜力的途径之一。更多还原

【Abstract】 The biogenetic formation of mineralized tissues such as enamel is a complex multi-step process including elements or compounds such as potassium, calcium, or phosphate and leading from precursor soft tissue, formed by extracellular matrix proteins （mainly collagen typeⅠ）, to a mineralized tissue, mainly composed by hydroxyl apatite and some residues of the organic extracellular matrix. As adult tooth enamel is not living tissue, it hardly can remineralize after the substantial mineral loss. Many in vitro methods has been developed to produce the artificial enamel lesions for the use in de- or remineralization studies, which include the acidified gels, buffered solutions, exposure to acid vapor, or incubation with natural plaque. In addition, various surface-protective agents have been used to obtain lesions with characteristic surface zones.The formation of Calcium matrix proteins（mainly collagen typeⅠ） in bones and teeth are associated with the nucleation and growth of hydroxyapatite crystals. As the hydrolysate of collagen, gelatin shows this characteristics both in vivo an vitro. Based on the theory of "molecular recognition", we designed a cross-link reaction to get a bounding layer between gelatin/silk protein and tooth tissue to enhance the intensity. This organic molecules model can be used to induce the crystallization of hydroxyapatite, which is usefull to build a tooth-like calcium phosphate/hydroxyapatite under a controllable way in vitro.Human molars were sliced into 2mm thickness disks and etched with phosphoric acid/EDTA to reveal the different orientations of the enamel rod/dentinal tubule. Surface modification of decalcified enamel was conducted by low temperature plasma processing. Peptide bond compound: N,N-（3-dimethylaminopropyl） -N’-ethyl-carbodiimide hydrochloride （EDC） and N-hydroxysuecinimide （NHS） were purchased from Medpep（Shanghai, China）. The molars disks were incubated with EDC and NHS in 0.02M Phosphate Buffer Solution（0.02 M EDC, 0.05M NHS） for 30min at 37℃. Gelatin/silk protein was added into the suspension at concentration of 0.1M, and the mixture was incubated at 37℃for 2 h with gentle shaking. The variation of elements on the surface of sample were investigated by X-ray photoelectron spectroscopy （XPS, Kratos Analytical Ltd., Surface Analysis Product Group, United Kingdom）.Disks of molar after crosslinking were soaked in calcification solution （SCS, 5mM CaCl₂, 5mM NaH₂PO₄, 1.5mM NaHCO₃, pH 6.31） for 24h at 37℃. Other group were soaked in artificial saliva（NaCl 1.71mmol/L, KCL 1.32mmol/L, CaCl₂ 1.02mmol/L, NaH₂PO₄ 0.90 mmol/L, Na₂S 0.004 mmol/L, CO（NH₂）₂ 4.16 mmol/L） for one month, supersede the environmental fluid every day. All the samples were studied by X-ray diffraction （XRD, X’Pert Pro MPD, Philips, Holland）, SEM and XPS. The microhardness of the neonatal crystal were tested through Knoop hardenss testing. Periodontal fibroblast were cultivated with samples to evaluate the biological reaction of the neonatal crystal. Oral pathogenic microbes（Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Actinomyces viscosus, Candida albicans） were co-incubated with neonatal crystal to test the adhesion of these pathogenic microbes to neonatal HA.XPS analysis of gelatin/silk protein monolayer for samples cycle group revealed that the surface organic compositions is higher than that of normal dentin and decalcified dentin surface. We speculated that-NH, -NH₂,-NH₃ and -COOH were grafted to the surface of tooth.The XRD showed that the precipitation was calcium fluoride phosphate and Ca:P was 1.6. At the same time, SEM micrographs of biomaterial showed this composite a continuous structure of columns crystal with size of 10-40nm. Furthermore, there were column crystal with parallel direction inside, as same as the crystal array in the top of enamel rod. The hardness of neonatal crystal close to the normal dentin. The binding affinity between neonatal crystal and normal tooth is 20N.There are well bio-compatibility of neonatal crystal which confirmed by MTT test. There aren’t differential adhesion between oral pathogenic microbes and neonatal crystal.According to the guide of block copolymer solution, the enamel-like calcium phosphate/hydroxyapatite compound material can be mineralized by biomimetic method. The specific functions of the copolymers can be used as a potential effective crystal growth modifiers.更多还原