Objectives: The purpose of this study was to find proper dentin substitute for standardized dentin barrier test and perform the cytotoxicity test of commercial bonding agents with the proper substitute. Materials and methods: The three-dimensional cells attached to dentin disc or millipore filters as the dentin substitute were tested in a dentin barrier test by perfusion. MTT assay was performed as an evaluation method for the cell survival rate. The cytotoxicity test of serial phenol dilution by bovine dentin disc was done to determine a standard toxic material, and the test of this proper phenol by using various millipore combinations was performed to find the suitable dentin substitute. Also, the cytotoxicity test of bonding agents was performed by this standardized substitute. The cell viability was expressed as percentages of untreated group. Results: Phenol concentration of 0.05 % was selected as the standard toxic material. The different combinations of millipore filters-two sheets of 0.45 μm, two sheets of 0.22 μm, and the combination of 0.65, 0.45, and 0.22 μm-showed similar cytotoxicity to natural dentin discs by 0.05 % phenol (p > 0.05). The millipore combination of 0.65, 0.45, and 0.22 μm that had structural similarity to natural dentin discs was used as the substitute for cytotoxicity test of bonding agents. The toxic level of Adper Prompt L-Pop using the selected substitute was significantly the highest among four kinds of dentin bonding agents (p < 0.05). Also, the dentin barrier test by the substitute showed constant results compared with the one by the natural dentin disc. Conclusions: The millipore filter combination of 0.65, 0.45, and 0.22 μm could be used as the substitute for the cytotoxicity test of materials applied to dentin. Clinical relevance: Dentin barrier test by standardized substitutes would be helpful for considering the potential toxicity of dentin bonding agents prior to clinical adaptations and reducing the variations of natural bovine dentin that has individually different characteristics.
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