Establishment of Human Fibroblasts deribed from Dental Pulp Tissues of Primary Teeth and Permanent Teeth, and Development of Systems of Cytotoxicity Tests for Dental Materials

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1)Separation of Human Fibroblasts Derived from Pulp Tissue and Transfection of Simian Virus 40 large T antigen cDNA For this research, some pulp tissues were used. In order to get single cell suspension, after cutting the pulp tissue into small pieces, digestion was accomplished by using PBS containing 0.1% trypsin and 0.02% EDTA.Then second digestion was caried out by using PBS containing 2mg/ml collagenase. The cells were subcultured few times, and then transfected by electroporation. Stable transfected cells were selecled, and further subcultured to obtain the population doubling level (PDL).2)Establishment of Fibroblasts' Clones derived from human Pulp Tissue and Analysis of Phenotypes of Clones We established cell clones which were subcultured more than 100 PDL.All clones showed the phenotype of normal immortalized cells ; because the cells neither showed the colony forming abilities in agarose nor induced tumor formation when used in nude mice, it could be considered that they are not tumor cells.3)Cytotoxity Test through the Dentin Slices for Dental Materials, Resin (AP-X) using Established Clones Using established cell clones, we investigated whether dental resin through the dentin tubles would express the cell toxity. We also compared the responses to dental materials of clones derived from primary and permanent teeth pulps. Survival cell number, synthesis of Interleukin-1beta (ILL beta), determination of apoptosis were analyzed after cytotoxity test. Unporimerised resin could express toxic effects on all clones through the dentin tubles, and did not show any difference in response to these materials among primary and permanent teeth clones. This system of toxic test can show whether or not dental materials express toxic effects through the dentin tubules. Moreover, various analysis can be carried out by using this method, so that this system is vary useful.