采用三维细胞培养的牙本质屏障细胞毒性试验研究

目的：采用牙本质屏障法对4种常用的牙体充填材料和2种牙本质粘接剂进行细胞毒性试验，并与传统的滤膜扩散法进行比较，从而探讨牙本质屏障法的优势。方法：选取丁香油水门汀、磷酸锌水门汀、粘结用玻璃离子水门汀、复合树脂和两种自酸蚀粘接剂（REMI BOND与Adper Easy One）进行试验。牙本质屏障法中将L929细胞立体培养于聚苯乙烯网状支架上，切取人第三磨牙近髓牙本质片，用液压通透装置测量其通透性，选取试验区域内通透值相近的牙本质片进行试验。将长满细胞的网状支架置于细胞培养分隔灌注小室的“牙髓腔”，牙本质片（“牙髓面”）紧贴网状支架放置其上，试验材料和对照与牙本质片的“面”接触24 h后用MTT法测量细胞的光密度值（D_{540 nm}），试验材料与阴性对照光密度值的百分比为材料的相对细胞存活率。使用非参数检验对结果进行统计学分析。滤膜扩散法中材料与有单层细胞生长的滤膜接触24 h后进行细胞染色、评级。结果：近髓牙本质片的平均通透值为0.293 μL/(min·cm²·cmH₂O)（1 cmH₂O=0.098 kPa）。牙本质屏障法中，丁香油水门汀降低细胞存活率至82%，REMI BOND与Adper Easy One分别降低细胞存活率至63%和54%，其余材料对细胞无损伤或损伤很低；滤膜扩散法中，光固化复合树脂为中度细胞毒性、牙科粘接用玻璃离子水门汀为轻度细胞毒性，其余材料均为重度细胞毒性；6种材料在牙本质屏障法中表现的细胞毒性明显低于滤膜扩散法的试验结果。结论：使用牙本质屏障法测得材料与三维培养的细胞接触后的细胞毒性比使用滤膜扩散法时的细胞毒性低，结果与临床实际相关性好。

Dentin barrier cytotoxicity test with three-dimensional cell cultures

Objective:To evaluate the cytotoxicity of four dentin filling materials and two dentin adhe-sives with a dentin barrier test and to compare the results with those in a conventional filter diffusion test in order to investigate the advantages of the dentin barrier test.Methods: Eugenol cement, zinc phos-phate cement, adhesive glass ionomer cement, composite resin and two self-etching adhesives ( REMI BOND and Adper Easy One) were tested.In the dentin barrier test, L929 mouse fibroblasts were three-dimensional cultured in polystyrene meshes.The dentin disks were cut from the human third molars, near the pulp and in parallel with the occlusal surface, and their permeability within the measurement area was evaluated by a hydraulic permeability device.A mesh with the cells was placed in the “pulp cavity” of the chamber and one dentin disk was put on the cell mesh and its “pulp side” was in contact with the mesh.The test materials and controls were in contact with the“occlusal side” of the dentine disks for 24 h.The cell viability was obtained with MTT assay and the results were expressed as a percentage of con-trol tissues.The Mann-Whitney U test was used to make the statistical analyses.In the filter diffusion test, after a 24 h contact between the test materials and the filters with monolayer cells, the filters were dyed and the grades of cytotoxicity were decided.Results:A mean permeability of the dentin disks near the pulp was 0.293 μL/(min· cm2· cmH2O)(1 cmH2O=0.098 kPa);In the dentin barrier test, Eu-genol cement, REMI BOND and Adper Easy One respectively reduced the cell survival rates to 82%, 63%and 54%.Other materials showed no or very low toxic reactions; In the filter diffusion test, the light-curing composite resin was moderately cytotoxic, the dental adhesive glass ionomer cement was mild-ly cytotoxic and the others were severely cytotoxic;All the six materials in the dentin barrier test had low-er cytotoxicity than in the filter diffusion test.Conclusion:The cytotoxicity of the test materials using the
dentin barrier test with three-dimensional cell cultures is lower than that in the filter diffusion test, which has good correlation with the clinical situation.