三种比色法评价4种牙科金属材料对L-929细胞的毒性

背景：采用不同方法评价材料的细胞毒性可能会得出不同的实验结果。目的：采用3种比色法评价镍铬合金、钴铬合金、3铬13及纯钛等牙科金属材料对小鼠成纤维细胞（L-929细胞）的细胞毒性。方法：以镍铬合金、钴铬合金、3铬13及纯钛4种牙科金属材料的浸提液分别作用于体外培养的L-929细胞24，72h。以体积分数10％小牛血清＋高糖DMEM培养液培养的L-929细胞为阴性对照组，以0.7％丙烯酰胺＋体积分数10％小牛血清＋高糖DMEM培养液培养的L-929细胞为阳性对照组，分别采用MTT、CCK-8及结晶紫3种比色法检测上述材料的细胞毒性。结果与结论：①4种材料浸提液中培养的细胞形态正常，胞内结构清晰，随着培养时间延长细胞大量增殖，与阴性对照组细胞形态无明显差异。阳性对照组细胞数量明显减少，形态完整性受破坏，形成大量细胞碎片。②培养24h时，CCK-8比色法检测中钴铬合金组的细胞相对增殖率低于阴性对照组沪〈0．05），MTT及结晶紫比色法检测中钴铬合金组的细胞相对增殖率与阴性对照组比较差异无显著性意义（P〉0．05）；培养72h时，MTT比色法检测中4种牙科金属材料组细胞相对增殖率低于阴性对照组（P〈0．01），CCK-8及结晶紫比色法检测中4组的细胞相对增殖率与阴性对照组比较差异无显著性意义（P〉0．05），但材料细胞毒性均为0—1级。表明上述4种牙科金属材料细胞毒性均在临床应用的允许范围内，具有良好的生物安全性。

Evaluating cytotoxicity of four different dental metal materials toward L-929 cells using three assay systems

BACKGROUND： Different methods to evaluate cytotoxicity of the same material may produce different results. OBJECTIVE： To evaluate the cytotoxicity of nickel-chromium alloy, cobalt-chromium alloy, 3Cr13 stainless steel and pure titanium toward L-929 cells by using three assay systems. METHODS： The L-929 cells were cultivated in vitro in the extracts of four different dental metal materials （nickel-chromium alloy, cobalt-chromium alloy, 3Cr13 stainless steel and pure titanium） at 24 hours and 72 hours separately. The L-929 cetls, cultured in high-glucose Dulbecco＇s modified Eagle＇s medium supplemented with 10% fetal calf serum, served as the negative control group. And cells, cultured in high-glucose Dulbecco＇s modified Eagle＇s medium supplemented with 10% fetal calf serum and 0.7% acrylamide, served as the positive control group. The detection methods for the cytotoxicity included MTT, cell counting kit-8 and crystal violet cell proliferation assay system. RESULTS AND CONCLUSION： （1） Microscopy showed that the L-929 cells, cultivated in the extracts of four different dental materials, were normal and their intracellular structure was clear. They proliferated well with extension of incubation time. There were no significant differences between the test and negative control groups at all times. However, the number of cells in the positive control group significantly reduced as compared with the negative control group. The morphological integrity destroyed and a lot of cell debris formed. （2） After culture for 24 hours, the cell counting kit-8 assay revealed that the relative growth rate of the cobalt-chromium alloy group was significantly lower than that of the negative group （P 〈 0.05）, while the MTT and crystal violet assay revealed that there were no significant differences in cell viability between the cobalt-chromium alloy and negative control group （P 〉 0.05）. After culture for 72 hours, the M＇I-F assay revealed that the relative growth rate of four dental metal materials groups was significantly lower than that of the negative group （P 〈 0.01）, while the cell counting kit-8 and crystal violet assay revealed that there were no significant differences in cell viability between the four dental metal materials and negative control group （P 〉 0.05）. The cytotoxicity of the tested dental metal materials was grade 0-1 which meets the safety standard. These indicate that these four dental metal materials have good biological safety.