原子力显微镜对牙体硬组织的纳米结构分析

Nanometer structural analysis of dental hard tissue by atomic force microscope

目的:运用原子力显微镜观察牙冠部的牙釉质、牙本质和釉牙本质界纳米水平结构,分析其结构与功能的关系。方法:实验于2006-10/12在暨南大学纳米技术实验室完成。实验材料:选取暨南大学附属第一医院口腔外科拔除的健康第三恒磨牙30颗,患者知情同意并自愿捐献。受试牙为健康的、需拔除的第三恒磨牙,牙体完整无损坏,拔除后置于含有麝香草酚的水中冻存。实验方法:沿髓腔的上方横切牙齿,切的方向与平面平行,使牙齿一分为二,保留上半部分,抛弃髓腔及牙根部分。用SiC砂纸抛光,然后用1.00,0.30,0.05μm的氧化铝浆水抛光,超声水中振荡清洗,样品在乙醇液中超声浴5min,然后再次在乙醇中漂洗。实验评估:在成像前,所有样品在超纯水中漂洗,室温下干燥后用原子力显微镜观测,每一样品取5个不同位置成像,观察牙釉质、牙本质、釉牙本质界的表面形貌。结果:①牙釉质的表面形貌:在纳米尺度下,组成牙釉质的羟基磷灰石晶体是由大小较均匀一致的颗粒组成,颗粒的大小为(767.05±76.71)nm,颗粒之间排列得非常致密、有序,具有一定的方向性,形成长条索状结构,即釉柱。釉质表面的平均粗糙度是(16.88±0.18)nm。②牙本质的表面形貌:在纳米尺度下,组成牙本质的羟基磷灰石晶体是呈球形的颗粒,大小较均匀,颗粒较大,为(1120.24±162.34)nm,颗粒排列紧密。可见到牙本质小管的开口,其边缘为管周牙本质,其余部分为管间牙本质,牙本质小管的直径是(1471.13±182.03)nm,牙本质表面平均粗糙度是(20.14±0.16)nm。③釉牙本质界的表面形貌:成束状排列的釉柱,与釉牙本质界垂直,组成釉牙本质界的牙釉质和牙本质的羟基磷灰石晶体相互渗透、彼此重叠,它们之间的连接并非完全致密,而是有一定地腔隙,呈扇贝状的波浪外形,凹面朝向牙釉质,凸面朝向牙本质,牙釉质和牙本质相互交替有规律镶嵌。结论:纳米尺度下,牙釉质的羟基磷灰石晶体排列致密、有序,平均粗糙度小,是其硬度大、脆性高,表面光滑的结构基础;牙本质的羟基磷灰石晶体排列较疏松,有机基质含量多,这样的结构赋予其一定的韧性;釉牙本质界在阻止釉质裂扩散的过程中起到关键的阻挡作用,这种优异的生物学结构和机械特性为研究与牙体硬组织生物学特性相似的充填材料的结构提供一种纳米量级的可视化平台。

AIM： To investigate the nanometer ultrastructures of enamel, dentin and dentin-enamel junction （DEJ）, and analyze the relationship between the structure and function using atomic force microscope. （AFM）. METHODS： The experiments were performed in the Bio-nanotechnology Laboratory of Jinan University between October and December in 2006. Experimental teeth： Totally 30 non-carious intact human molars were freshly extractedfor treatment reasons from the patients admitted in the Department of Stomatology, the First Affiliated Hospital of Jinan University, which were preserved in water containing thymol. Informed consents were obtained from all the patients. Experimental methods： The crown was sectioned with the slicing plane parallel to the occlusal surface. The pulp and root were removed. The surface of each section was polished parallelly to the natural surface using silicon carbide paper and 1.00, 0.30, 0.05 μm aluminium oxide slurry. Samples were cleaned by 5-minute ultrasonication in ethanol before and after polishing. Experimental evaluation： All samples were washed in ultra-pure water and dried in air before AFM imaging. Each sample was observed the surface morphology pf enamel, dentin and DEJ in five different positions. RESULTS：（1）The surface morphology of enamel： The hydroxyapatite crystals of enamel were uniform with the size of （767.05±76.71） nm in nanoscale. The particles were closely touching each other and they arranged in enamel rods of directivity. The roughness of the enamel surface was （16.88±0.18） nm. （2）The surface morphology of dentin： In comparison with enamel, hydroxyapatite crystals of dentin were larger and also uniform, round, arranged and closely contacted, with the size of （1 120.24±162.34） nm. Several opens of dentinal tubule was found with the diameter of （1 471.13±182.03） rim, and the opens were surrounded with peritubular dentin and the other parts were intertubular dentin. The average roughness of the dentin surface was （20.14±0.16） nm.（3）The surface morphology of DEJ： Enamel rod arranged in fasciculation and was perpendicular to DEJ. The hydroxyapatite crystals from enamel and dentin were penetrative and overlapped each other to form a scalloped shape with its convexities directed toward the dentin and concavities directed toward the enamel. CONCLUSION： Uniform hydroxyapatite particles that composed enamel very closely contact each other, and align in enamel rods, with low roughness. Such constitution and structure endues it with the most hardness and brittleness of the calcified tissues. Dentin is a less mineralized tissue, which contains less hydroxyapatite crystals and more organic matrix, can provide teeth with toughness. The arrangement of hydroxyapatite crystals is loose. DEJ plays a crucial role in arresting enamel crack diffusion, and its excellent biological structure and biomechanical properties provide a nanoscale visible plane for investigating new restorative materials that have similar structure and biomechanical properties to the hard tissues of teeth.