牙齿修复材料细胞毒性试验新方法的研究

Cytotoxicity test in vitro is widely used in evaluating the biocompatibility of dental restorative materials. Currently three cytotoxicity test methods have shown poor correlation with the cytotoxicity of dental materials in usage test in teeth.The principal cause of the poor correlation is the absence of dentin barrier between the test materials and cultured cell. However, dentin disc has great variance in permeability that influences the results greatly and nowadays it is difficult to get large quantities of human dentin disc，which block the application of dentin disc in standard test. In this study,artificial discs sintered from HAP were used as dentin barrier in a new device for testing the cytotoxicity of dental restorative materials in vitro,which demonstrate not identical or similar permeabilityhas to human dentin and can be controlled strictly. This new device is modified from multiple well plate for cell culturing, such as 96-well or 48-well cell culture plate, and is composed of culture wells, inserts and covers. In each well, there is an insert that use porous permeable hydroxyapatite ceramic disc as its bottom and the insert acts as a dental cavity. The material being tested is placed on the top side of the bottom of the insert, and when placed in position, the bottom of the insert is located in the low part of the culture well that is filled with culture fluid and target cells. By controlling the volume of the culture fluid, only the bottom of the insert is immersed in culture fluid. In this way, the toxic substance from the tested material will diffuse through ceramic disc and into the culture fluid and acts on the target cells. Meanwhile, culture fluid/water will diffuse through ceramic disc and into the tested material and remove soluble constituents from the material, which then will diffuse back into he culture fluid and acts on the target cells. In this item, the researches include:(1) Preparation of HAP discs with identocal or similar permeability to dentin;(2)Preparation of a new cell culture device; (3)Comparation of the cytotoxicity of common dental restorative tested with dentin barrier and HAP disc barrier;(4)correlation study between the cytotoxicity test using HAP discs as barrier and dental restorative test in animal.

体外细胞毒性试验是评价牙齿修复材料生物相容性的重要项目之一，用现有的细胞毒性标准试验方法测定牙齿修复材料，所得结果与动物试验及临床观察结果的相关性很差, 主要原因是培养细胞与被测材料之间缺乏在体那样的牙本质屏障。由于离体牙本质渗透性变异很大, 难于获得大量的渗透性一致的牙本质屏障片，而渗透性又严重影响试验结果，所以用离体牙本质作为屏障来进行细胞毒性试验难以广泛推广和标准化。本项目拟建立一种可大通量进行的、容易标准化的体外细胞毒性试验新方法，该方法使用人工合成的羟基磷灰石陶瓷片做为牙本质屏障，该陶瓷片具有可控的、稳定的、与牙本质相近的渗透性。以该陶瓷片为基础，研制一种体外细胞毒性测定装置，该装置模拟牙齿修复材料与牙髓组织的接触状态，以提高牙齿修复材料体外细胞毒性试验结果与在体试验结果的相关性，使体外细胞毒性试验更具有预测评价意义, 提高牙齿修复材料体外生物相容性的评价水平。

牙齿充填材料是口腔补牙材料，每种充填材料进入临床应用前需要进行生物相容性试验，以确保其安全性。体外细胞毒性试验是评价生物相容性的重要项目之一，现有的体外细胞毒性试验方法存在着牙齿充填材料试验结果与临床观察效果相关性较低的问题,影响了材料生物相容性评价的准确性。造成这一结果的重要原因之一是目前的体外细胞毒性试验方法中细胞与受试材料之间缺少在体那样的牙本质屏障。而采用人或者动物离体牙本质作为体外细胞毒性试验的屏障，存在来源非常有限、难于推广的问题。为此本项目的目标是制备一种人工合成羟基磷灰石微渗陶瓷屏障片，替代人离体牙本质，以此屏障片为基础研制一种牙齿充填材料体外细胞毒性试验新装置，该装置能模拟在体牙齿修复材料与牙髓组织细胞的接触状况，使体外细胞毒性试验结果更能反映材料的在体细胞毒性。为此本项目研究了①溶剂定向冷冻结晶法制备具有定向直通小管结构的HA陶瓷屏障片、HA造粒粉及预烧造粒粉模压成型-烧结制备HA陶瓷屏障片、纳米HA粉模压成型-烧结制备HA陶瓷屏障片制备技术，研究了制备技术条件对制备的HA陶瓷屏障片通透性的影响，筛选出最佳的HA粉体、模压成型压力及烧结温度与时间；②研制了新型牙齿充填材料体外细胞毒性试验新装置，并且对装置进行了持续的改进，研究了新装置用于细胞毒性试验的最佳试验条件和影响细胞毒性试验结果的因素，建立应用新装置进行细胞毒性的方法及细胞毒性的评定标准。.本项目取得如下成果：①制备出具有自主知识产权的微渗HA陶瓷片，建立了这种陶瓷的制备技术。该陶瓷具有微小的通透性，通过控制模压成型压力和烧结温度可以使通透值达到牙本质的通透值范围内，这种微渗陶瓷在离子选择电极分析中的参比电极头中也有潜在的广泛应用；②研究出具有自主知识产权的新型牙齿充填材料体外细胞毒性试验装置，该装置模拟了在体补牙材料与牙髓组织细胞的接触状态，为牙齿充填材料的体外细胞毒性试验提供新的方法与装置。