In Vitro Cytotoxicity Testing Of Dental Restorative Materials

With the development of science and technology,various new types of dental filling materials have been continuously developed.Before being used in clinic,systematic biocompatibility evaluation of these materials is required,whichincludes in vitro tests,animal tests and clinical trials.In vitro tests,as a group of primary evaluation test of biocompatibility evaluation,have the characteristics of convenience,simplicity,good repeatability,easy control of influencing factors and low cost.In vitro cytotoxicity test is the most commonly used test method in vitro test,and is currently widely used in dental filling materials.Cytotoxicity test methods include extract method,direct contact method,agar diffusion method and filter diffusion method.Each of these methods has both advantages and advantages.The imperfections exist in these methods include:the difference of sample form tested in vitro from that of clinical usage and the difference of contact of sample with cells in vitro testing from that of clinical usage,which results in a different cytotoxicity on one material between in vitro tset,animal experiment and clinical uasge.Therfore it is difficult to predict the biocompatibility of a in vitro tested material.For this reason,this study investigated some of the factors that affect the test results in the commonly used cytotoxicity test methods?the extract method and the agar diffusion method?and compared with the latest dentin barrier method,expecting to determine better test conditions.Meanwhile the application of ceramic barriers in barrier cytotoxicity test was explored.Objectives:?1?To study the influence of the sample form of the tested material in the agar diffusion cytotoxicity test on the test results.?2?To study the influence of the concentration of the extract solution on the test results in the extract solution cytotoxicity test and compare it with the dentin barrier method.?3?Explore the effect of the ceramic barrier's permeability on the results of cytotoxicity tests.Materials and methods:Five clinical frequently used dental filling materials were selected as the test materials and their cytotoxicity to pulp have clearly defined:light-cured composite resin,zinc oxide-eugenol cement,zinc phosphate cement,glass ionomer cement and zinc polycarboxylate cement.A silicone rubber impression material was used as a negative control and phenol solution as a positive control.Test 1.The effect of the sample form of the tested material in the agar diffusion cytotoxicity test on the test results?1?Prepare extract solutions and solid disc samples of the above five materials.?2?.Prepare petri dishes attached with active monolayer L929 cells.Then agar culture medium was added to each petri dish to overlay the cells,followed by staining the cells with neutral red solution.?3?The extract solutions,solid discs and freshly mixed material were placed respectively on the agar medium surface and the cells were cultured for another 24hours.?4?After 24 hours,the cell decolorization zone around the test materials and controls were assessed using an inverted microscope and determine a decolorization index and a lysis index for each test sample in accordance with the criteria specified in ISO standard.Test 2.Comparison of the cytotoxicity test between extract and dentin barrier methodsIn this part,the cytotoxicity of the above 5 materials was determined using the extract method and the dentin barrier method,respectively.In the extract method,100%,50%and25%extract solution of the five materials were prepared.In a common 96-well culture well,L929 cell suspension was added and incubated for 24 hours.Then the culture mediums were aspirated and replaced with 100?L 100%,50%and 25%extract solution respectively.After 24 hours of incubation the cell morphology was evaluated using a microscope,and then the cell viability of each well was measured by MTT assay.Finally the cell proliferation rate was calculated and cytotoxicity was determined.In the dentin barrier test,the human dentin discs 0.5 mm in thickness were cut from extracted human molars and their permeability was determined.Dentin discs with permeability value of 0.416⁰.625?L·min^-1·cm^-2·cm H₂O^-11 were used as barrier discs and were bonded to the bottom opening of Transwell insertions with a nontoxic medical adhesive.A Transwell insertion and suited cuture well make a dentin barrier cytotoxicity test set.L929 cells were inoculated into the culture wells and incubated for 24 hours.Afterwards.Transwell insertion was filled with freshly mixed testing material and was then inserted into the culture wells,so that the dentin barrier disc contacts with the culture medium in the culture well.After 24 hours of incubation,the insert wells were removed and the cell morphology in the culture well was assessed using a microscope and cell viability of each well was measured by MTT assay.Finally the cell proliferation rate was calculated and cytotoxicity was determined.Test 3.Effect of permeability of ceramic barrier on the results of cytotoxicity testsTwo groups of porous HA ceramics discs with permeability of 0.445⁰.660 and0.825¹.250?L·min^-1·cm^-2·cm·H₂O^-11 respectively were used as dentin barrier substitute in the barrier cytotoxicity test set.L929 cells were inoculated into the culture wells.After 24hours of incubation,the insert was filled with freshly mixed testing material and was then inserted into the culture wells,so that the ceramic barrier disc contacts with the culture medium in the culture well.After 24 hours of incubation,the insert wells were removed and the cell morphology was assessed under a microscope.and cell viability was measured by MTT assay.Finally the cell proliferation rate was calculated and cytotoxicity was determined.Results:Test 1:In the agar diffusion test,the influence of sample form of the test material on the cell proliferation rate was material material dependent:presented obvious influence in the materials with medium cytotoxicity but no significant influence in the materials with slight and severe cytotoxicity.Among the three sample form,material extraction showed the greatest cytotoxicity,followed by the freshly mixed sample,while the solid sample had the least cytotoxicity.Test 2:In the extraction method,the concentration of the extraction showed a significant effect on the cell proliferation rate,cell proliferation rate decreasing with the extraction concentration increasing.The cytotoxicity of the three concentration extraction of the tested materials measured by extraction method was significantly higher than that by the dentin barrier method,except the light-cured composite resin,.The cytotoxicity grade was also significantly higher than that by the dentin barrier method.Test 3:For the five tested materials,the cell proliferation rate of the high permeability ceramic barrier group was significantly lower than that of the low permeability ceramic barrier group.For the high-permeability ceramic barrier group,the cytotoxicity of zinc oxide eugenol cement was severe,glass ionomer cement was moderate,and the light-cured composite resin,zinc phosphate cement,and zinc polycarboxylate cement mild.For the low-permeability ceramic barrier group,the cytotoxicity of zinc oxide eugenol cement,zinc phosphate cement,and zinc polycarboxylate cement was slight,and both glass ionomer cement and light-cured composite resin showed no cytotoxicity.Conclusions:1.In the agar diffusion method,the influence of sample form of the test material on the cyctotoxicity results is material material dependent:no influence on the materials with slight and severe cytotoxicity but obvious influence on the materials with medium cytotoxicity.Extraction of the tested material can leach more cytotoxic components,will present greater cytotoxicity.After comparing with dentin barrier test results and clinical facts about the pulp stimulation of the five materials,it can be concluded that the sample forms of solid discs and freshly mixed samples are more suitable for evaluating the cytotoxicity of dental filling materials.2.Extraction cytotoxicity test is simple and quick in operation,subjective influence o is small,but the extract concentration has a significant impact on cell proliferation.Moreover,for most materials,the cytotoxicity measured by the extract method is significantly higher than that of the dentin barrier method.The dentin barrier method produces significantly lower cytotoxicity than the extract method and the agar diffusion method.In order to obtain a cytotoxicity result similar to the dentin barrier method,it is necessary to reduce the concentration of thematerials'extraction.3.In the barrier cytotoxicity test using porous HA ceramics discs as dentin substituate,the ceramic permeability has a significant influence on the results of the material's cytotoxicity.The ceramic discs with a permeability similar to that of dentin may be used as a barrier substituate in a barrier cytotoxicity test to evaluate the cytotoxicity of tooth filling materials.