Testing Of Human Dentin Permeability In Vitro And Its Application

With the development of the stomatology, more and more new dental restorativematerials were developed and applied in clinic. Before applying, one factor should beconsidered is the stimulation to the dental pulp, which relate to the permeability of thedentin contacting with the materials and the biocompatibility of the materials themselves.The dentin hypersensitivity occurs due to the higher dentin permeability. It is difficult tomeasure the dentin permeability in vivo because many factors affect it. To study thepermeability of dentin in vitro and its application in evaluating the cytoxicity of dentalrestorative materials, the experiment was developed as follows:Objectives: To compare the permeability of the dentins located in different regions and underdifferent test conditions in vitro. Then to evaluate the efficacy of desensitizing materialsthrough the permeability change in vitro. To study the influence of dentin permeability onthe material cytotoxicity, the permeability was applied to the cytoxicity test in vitro, whichis meaningful to choose dentin materials.Methods:1. The measurement of permeability of human dentin in vitroOcclusal inner dentin discs, occlusal middle dentin discs, buccal inner dentin discsand buccal middle dentin discs were cut from extracted human mandibular third molarsusing a low-speed saw. Then these dentin discs were polished by SiC paper and cleaned byultrasound and their baseline permeability were measured with perfusion method. Afteretched for different periods, their permeability was measured under different hydraulicpressures and in different perfusion fluid. The dentin tubule density in different regionswas observed under a metallurgical microscope, and the correlation between dentinpermeability and dentin tubule density was analyzed.2. The effect of desensitizing materials on dentin permeabilitySixty occlusal inner dentin discs were cut from extracted human mandibular thirdmolars using a low-speed saw. The surface of these dentin discs was polished by SiC paper,cleaned by ultrasound and dried. The occlusal surface of each disc was etched with350g/Lphosphate acid for30s. Then all dentin discs were randomly divided into three groups andtheir baseline permeability was measured with perfusion method. After application ofthree desensitizing materials, Green Or, Gluma and Sensodyne repair toothpaste, thepermeability were tested respectively, and dentin surface was observed by SEM.3. The application of dentin permeability in cytotoxicity test of dental filling materi-alsThe buccal inner dentin discs with similar permeability were selected from the formerexperiment. A membrane permeable plate with96holes was separated into membranepermeation devices with eight holes (embedded well). The membrane was replaced by thebuccal inner dentin disc which was cemented on embedded well by medical biological glue. To prepare cell culture apparatus with dentin disc as barrier in vitro, the embeddedwell containing dentin discs were inserted in96holes plate. Samples of10mm×10mm×1mm in size were made from five dental filling materials and immersed inRPMI1640culture medium containing100ml/L FBS (the ratio of liquid amount to surfacearea of the sample was10ml/cm2) at37. After24h incubation, each eluate was dilutedto a concentration of50%. The cytotoxicity of each dental material was tested by the waysof culture extract testing and dentin barrier test, and the results of both methods wereevaluated according to cell morphology and cytoactive.Results：1. After being etched, the dentin permeability was significantly greater than the groupnot etched (P＜0.05). In particular, the permeability of dentin etched for30s increasedmost obviously and also had statistical significance (P<0.05) compared with the dentinetched for60s. The permeability tested with100cm hydrostatic pressure was significantlyhigher than that with32cm (P＜0.05). The permeability tested with diluted bovine serumwas significantly lower than that with distilled water and saline (P＜0.05), but statisticalanalysis revealed no significant difference between the permeability tested with distilledwater and saline (P＞0.05). The occlusal dentin presented a significantly higherpermeability than the buccal dentin, and the permeability of the inner dentin wassignificantly higher than that of middle dentin (P＜0.05). The permeability of the dentinwas positively related to the dentin tubular density (P＜0.05).2. The dentin permeability decreased significantly after application of desensitizingmaterials (P＜0.05). An ascending order of the dentin permeability treated with thematerials was as follows: Green Or≈Gluma＜Sensodyne professional repair toothpaste (P＜0.05). SEM showed that all these desensitizing materials occlude the dentin tubular indifferent extent.3. In high concentration extract of ZOE cement group, the cells were almost roundand their density decreased; as to ZOE cement of low concentration, a few cells presentedfusiform but extend little; a few cells turned round and the morphology was abnormal indentin barrier test of ZOE cement. In the same extract group in extract test, the toxicity of these materials was as follows: calcium hydroxide cement≈glass ionomer cement＜resincomposite＜zinc phosphate cement＜ZOE cement (P＜0.05). While in dentin barrier test,the toxicity of these materials was as follows: calcium hydroxide cement≈glass ionomercement＜ZOE cement＜resin composite＜zinc phosphate cement (P＜0.05). The RGR offive materials were higher in dentin barrier test than in culture extract test, which wasespecially high in ZOE cement group, and the RGR in dentin barrier test was (89.1±3.6)%.The cells in ZOE cement group showed severe toxicity in culture extract test according torating scale, but showed slight toxicity in dentin barrier test.Conclusions:1. The smear layer was removed without dentin tubular changed after the dentinsurface was etched with350g/L phosphate acid for30s. The dentin permeability changedwith different regions, the permeability of occlusal dentin was higher than the buccaldentin, the permeability of inner dentin was higher than the middle dentin, and there was astrong correlation between dentin permeability and tubular density. The perfusion fluidinfluenced the permeability measurement.2. The results showed that three desensitizing materials could occlude dentin tubularand significantly reduce the permeability. The effect of reducing permeability is bestespecially with Green Or, Gluma. Observation of materials blocking dentinal tubules bySEM presented deviation. The permeability measurement in vitro can be quantitative, easyto operate and have more accurate results. The quantitative testing of dentin permeabilityin vitro could be used as one of the means of desensitization effect evaluation.3. Due to dentin permeability, substance and chemical composition fromincompletely polymerized materials could diffuse through dentin tubules, causingcytotoxicity. The chemical components of materials diffused into dentinal tubules could bediluted by dentin fluid. Dentin discs with controllable permeability may be used as thebarrier of the cell culture device which has simple manufacture and easy popularization.The dentin barrier testing could simulate the condition of materials and pulp tissue in vivo,by which the results have improved clinical relevance.