dentine bonding agents are bonding agents that consist of 3 parts : ( i ) an acid that removes the smear layer accumulated on the dentine and that helps increase the microporosity of the enamel ( etching step ) , ( ii ) a substance that enhances the flow of bonding agents into the dentine and increasing the microporosity of the enamel by modifying dentine as a bonding substrate ( priming step ) , and ( iii ) the bonding agents ( bonding step ) . traditionally , the use of dentine bonding agents required the use of all three of these steps . recently , however , dentine bonding agents have been developed that reduce the process to only one or two steps , resulting in faster and more convenient application . selection of a dentine bonding agent requires the consideration of the bonding properties ( i.e. , how effectively the bonding agent can bond the dental restorative material to the dentine ) and the cytotoxicity of the dentine bonding agents.12 in a previous study , the cytotoxicity of three dentine bonding agents was determined using the agar overlay technique.3 the results showed that the toxicity of g - bond and clearfil s bond diffusing into the agar medium was moderate whereas that of clearfil se bond x was high . the study also found that the three dentine bonding agents yielded the same lysis index ( i.e. , 1 ) . this result revealed that the three dentine bonding agents have low toxicity according to the iso / fdis 7405:2008 ( e ) standard , which is based on an in vitro evaluation of the biocompatibility of medical devices used in dentistry.4 however , the agar overlay technique only allowed a preliminary cytotoxicity evaluation and did not reveal the percentage of surviving cells . it is advisable to evaluate the biological properties of dentine bonding agents that are in close contact with dentine prior to using them in patients . according to the iso 7405:1997 standard , an in vitro evaluation of the biological properties of dentine bonding agents ( e.g. , cytotoxicity evaluation through a cell - culture test ) is the primary method used to evaluate the cytotoxicity of such agents.5 originally , researchers used cell - culture tests to evaluate the biological properties of dental materials.6 the agar overlay method and the millipore filter method were introduced to simulate the material - cell contact interaction.78 however , the agar and filter methods still did not simulate dentine in vivo clinically . in 1977 , the model cavity system was introduced to better simulate the clinical situation.9 the test material was separated from the cells by either a synthetic filter or a dentine slice . an in vitro test system using dentine for the evaluation of toxicity has been reported.1012 in these studies , the toxic products of materials that diffused through the dentine were diluted in at least 2.5 ml of cell culture medium before contacting the target cells . however , in a clinical situation , the leachable toxic product will have a direct effect on the cells of the pulp , which lie close to the dentine . in 1996 , a dentine barrier model for the cytotoxicity evaluation of dental cement was reported.13 this model is based on a commercially available device and is considered to be an important prerequisite for a standard test . in this model , the cells were seeded on the pulp side of the dentine and were placed above the cell culture medium . our study used a dentine model and three - dimensional cell cultures that have been previously described by ulker and sengun.14 cell perfusion was limited by the blood circulation inside the tooth cavity and was equal to 20 - 82.4 ml / min/100 grams of tissue.15 it has been reported that perfusing the chamber with 5 ml medium / h did not decrease toxicity but can lead to increased cell death or cell disruption.16 therefore , in the present study , the perfusion conditions included a cell feed flow rate of 2.0 ml / h . at this flow rate this difference may be because this rate mimics the blood flow within the pulp chamber , where cytotoxic leaching occurs . the aim of this study was to evaluate the cytotoxicity of three self - etching dentine - bonding agents ( g bond , clearfil s bond and clearfil se bond x ) with a three - dimensional cell culture and perfusion conditions that were not used in the previous study.3 the hypothesis tested was that different self - etching dentine bonding agents have different cytotoxic profiles . g bond , clearfil s bond and clearfil se bond x were the experimental materials . the negative and positive controls were president ( coltene ag , alstatten , switzerland ) and vitrebond ( 3 m medica gmbh , germany ) , respectively . polyamide ( diameter 8 mm ) was etched with 0.1 m acetic acid for 30 min , washed with sterile water 3 times and sterilized by autoclaving ( hirayama , tokyo , japan ) before the experiment . the mesh was coated in fibronectin ( 0.03 mg / ml in water ) , and the mesh was left to dry for 2 h in a bio - hazard safety cabinet . the target cells used in this experiment were tcpc sv40 ( bovine fibroblast pulp - derived cells transfected with simian virus 40 large t - antigen).16 a 1.25-ml volume of mem - alpha ( minimum essential media , gibco , new york , usa ) , including 20% fetal calf serum , was added to each well of a 6-well tissue culture plate . then , a millicell membrane ( minucells and minutissue , bad abbach , germany ; size 30 mm , pore size 0.45 m ) was inserted in each well . the plate was incubated at 37o c with 5% co2 and 100% humidity for 48 h. then , each mesh was separately placed into the wells of a 24-well tissue culture plate . the cells on the meshes were fed with 1 ml of mem - alpha media , 20%fcs containing 50 g / ml of ascorbic acid , and the medium was changed every other day . after growing for 14 days in the incubator , the three - dimensional cell cultures on the meshes were ready for use in the experiment ( figure 1 ) . a dentine disc that was 500  25 m thick and that was close to the pulp cavity was longitudinally sectioned from a bovine incisor . a dentine disc with a diameter of 67 mm under the cementoenamel junction was cut for the experiment . the pulp side of each disc was etched with 50% citric acid for 30 s , soaked in normal saline and autoclaved before the experiment ( figure 2 ) . a commercial cell culture chamber ( minucells and minutissue , bad abbach , germany ) was used for the in vitro model.16 the chamber was separated into a pulp side and a cavity side by the dentine disc mentioned above . the pulp side of the disc was placed over the cultivated cell mesh , and a stainless steel clamp held the 2 compartments together . cells in the mesh were fed with mem - alpha media , 20%fcs that was contained in the lower part of the chamber . the model was placed on a hot plate ( 37  2 c ) , and culture medium was perfused through the lower part of the chamber . each dentine bonding agent as well as the negative and positive control was tested in 5 cell culture chambers . all experiments were repeated in triplicate . the perfusion pump ( ismatec uk co. weston - super - mare , england ) was connected and adjusted to a perfusion rate of 0.2 ml / h for 24 h before application of the test material ( figure 3 ) . a cotton pellet soaked with culture medium after 24 h of perfusion , the perfusion rate of the medium was adjusted to 2 ml / h to simulate blood flow in the pulp . the dentine in the cavity side was cleaned with sterile water and dried with gently blown air . three self - etching bonding agents as well as the president and vitrebond controls were applied to the dentine according to the manufacturers recommendations . the enzymatic activity of target cells was analyzed using the mtt ( 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyl tetrazolium bromide ) assay after 24 h of test material exposure . the mesh was removed from the stainless - steel holder of the perfusion chamber and immediately inserted into 0.5 ml of freshly prepared mtt solution ( 1 well/1 mesh ) in a 48-well tissue culture plate . the plates were incubated for 2 h. mitochondrial dehydrogenase in living cells converts the yellow water - soluble tetrazolium salt 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5 diphenyltetrazolium bromide into dark blue formazan crystals that are stored in the cytoplasm of the cells . then , the mtt solution was removed , and the mesh was washed twice with 0.5 ml of phosphate - buffered saline solution . dimethyl sulfoxide ( dmso ; 250 l ) was added to each well to dissolve the formazan . the plate was agitated on a shaker for 30 minutes to enhance the dissolution of the formazan . a 200-l aliquot was drawn from each well and transferred to a 96-well tissue culture plate . the mean optical density of the negative control group was set to represent 100% viability . the results for the experimental groups and the positive control were expressed as the percentages of the negative control . the statistical analysis was performed by applying the non - parametric mann - whitney test ( p<.05 ) . g bond , clearfil s bond and clearfil se bond x were the experimental materials . the negative and positive controls were president ( coltene ag , alstatten , switzerland ) and vitrebond ( 3 m medica gmbh , germany ) , respectively . polyamide ( diameter 8 mm ) was etched with 0.1 m acetic acid for 30 min , washed with sterile water 3 times and sterilized by autoclaving ( hirayama , tokyo , japan ) before the experiment . the mesh was coated in fibronectin ( 0.03 mg / ml in water ) , and the mesh was left to dry for 2 h in a bio - hazard safety cabinet . the target cells used in this experiment were tcpc sv40 ( bovine fibroblast pulp - derived cells transfected with simian virus 40 large t - antigen).16 a 1.25-ml volume of mem - alpha ( minimum essential media , gibco , new york , usa ) , including 20% fetal calf serum , was added to each well of a 6-well tissue culture plate . then , a millicell membrane ( minucells and minutissue , bad abbach , germany ; size 30 mm , pore size 0.45 m ) was inserted in each well . the plate was incubated at 37o c with 5% co2 and 100% humidity for 48 h. then , each mesh was separately placed into the wells of a 24-well tissue culture plate . the cells on the meshes were fed with 1 ml of mem - alpha media , 20%fcs containing 50 g / ml of ascorbic acid , and the medium was changed every other day . after growing for 14 days in the incubator , the three - dimensional cell cultures on the meshes were ready for use in the experiment ( figure 1 ) . a dentine disc that was 500  25 m thick and that was close to the pulp cavity was longitudinally sectioned from a bovine incisor . a dentine disc with a diameter of 67 mm under the cementoenamel junction was cut for the experiment . the pulp side of each disc was etched with 50% citric acid for 30 s , soaked in normal saline and autoclaved before the experiment ( figure 2 ) . a commercial cell culture chamber ( minucells and minutissue , bad abbach , germany ) was used for the in vitro model.16 the chamber was separated into a pulp side and a cavity side by the dentine disc mentioned above . the pulp side of the disc was placed over the cultivated cell mesh , and a stainless steel clamp held the 2 compartments together . cells in the mesh were fed with mem - alpha media , 20%fcs that was contained in the lower part of the chamber . the model was placed on a hot plate ( 37  2 c ) , and culture medium was perfused through the lower part of the chamber . each dentine bonding agent as well as the negative and positive control was tested in 5 cell culture chambers . the perfusion pump ( ismatec uk co. weston - super - mare , england ) was connected and adjusted to a perfusion rate of 0.2 ml / h for 24 h before application of the test material ( figure 3 ) . a cotton pellet soaked with culture medium was placed on the dentine of the cavity side . after 24 h of perfusion , the perfusion rate of the medium was adjusted to 2 ml / h to simulate blood flow in the pulp . the cotton pellet in the cavity side the dentine in the cavity side was cleaned with sterile water and dried with gently blown air . three self - etching bonding agents as well as the president and vitrebond controls were applied to the dentine according to the manufacturers recommendations . the enzymatic activity of target cells was analyzed using the mtt ( 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyl tetrazolium bromide ) assay after 24 h of test material exposure . the mesh was removed from the stainless - steel holder of the perfusion chamber and immediately inserted into 0.5 ml of freshly prepared mtt solution ( 1 well/1 mesh ) in a 48-well tissue culture plate . the plates were incubated for 2 h. mitochondrial dehydrogenase in living cells converts the yellow water - soluble tetrazolium salt 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5 diphenyltetrazolium bromide into dark blue formazan crystals that are stored in the cytoplasm of the cells . then , the mtt solution was removed , and the mesh was washed twice with 0.5 ml of phosphate - buffered saline solution . dimethyl sulfoxide ( dmso ; 250 l ) was added to each well to dissolve the formazan . the plate was agitated on a shaker for 30 minutes to enhance the dissolution of the formazan . a 200-l aliquot was drawn from each well and transferred to a 96-well tissue culture plate . the mean optical density of the negative control group was set to represent 100% viability . the results for the experimental groups and the positive control were expressed as the percentages of the negative control . the statistical analysis was performed by applying the non - parametric mann - whitney test ( p<.05 ) . the percentages of viable cells compared to the controls for g bond , clearfil se bond x and clearfil s bond are shown in figure 4 . the average percent cell viability after exposure to g bond , clearfil se bond x , clearfil s bond and vitrebond was 113.03 , 111.83 , 90.98 and 59.90 , respectively . vitrebond was more toxic than g bond , clearfil se bond x and clearfil s bond ( p<.01 ) , whereas the experimental group did not have any toxicity compared with the negative control group ( p>.05 ) . in a previous study , the cytotoxicity levels of self - etching dentine - bonding agents ( g bond , clearfil se bond x and clearfil s bond ) were evaluated using the agar overlay technique . this analysis showed that the g bond and clearfil s bond were both moderately cytotoxic , whereas the clearfil se bond x appeared to be more toxic than the other 2 bonding agents . the amount of cells destroyed by each of the three dentine bonding agents was not significantly different.3 because this was only a preliminary evaluation of the cytotoxicity of the three dentine - bonding agents , it was not possible to determine the level of toxicity quantitatively . hence , a qualitative study of a dentine barrier model simulating clinical practice was needed to obtain reliable data . this study revealed that the percent of viable cells for g bond , clearfil s bond and clearfil se bond x was 113.03 , 90.98 and 111.83 , respectively . in this study , three dentine bonding agents were studied under two systems . g - bond and clearfil s bond were processed in one step , and a two - step system was used for the self - etching primer bonding system of clearfil se bond x. in the all - in - one system , the etching , priming and bonding steps are combined together as one step for resin - bonding . in contrast , in the two - step self - etching primer bonding system , the etching and primer steps are combined together as a single step that is separate from applying the resin bonding agent prior to restoration of the tooth with a resin composite . in the clearfil s bond experiment , this value was lower than the other groups , but the difference was not statistically significant . the difference may be due to the presence of hydrophobic dimethacrylate in the composition . hydrophilic monomers such as hema or tegdma have been shown to be cytotoxic , but to a lesser degree than a more hydrophobic monomer.17 however , self - etching adhesive systems were less cytotoxic than the total - etching system.18 the preparation of materials for experiments significantly has been reported to alter the apparent cytotoxicity of the materials.19 thus , every effort was made to simulate in vivo conditions in the laboratory . however , it is not possible to create an environment that totally replicates clinical conditions . cell toxicity assays have been performed both in cell lines and primary cells , but immortalized cell lines are more stable than primary cells.20 no differences have been observed in the growth of bovine pulp - derived cell lines transfected with simian virus that were seeded on dentine discs and those seeded on tissue culture plates.21 scanning electron microscopy ( sem ) also showed biocompatibility between the dentine and cells . thus , bovine teeth are an appropriate choice for use with the bovine pulp cell line in this experiment . it has been demonstrated that 0.5 mm of dentine can reduce material toxicity to 75% and that 1 mm of dentine can reduce toxicity by up to 90% of the control value ( i.e. , the value obtained when dentine was not present).22 most cytotoxicity research uses 500 m for the standard thickness of the dentine barrier slice in the pulp chamber model.23 the permeability of the dentine varies in different regions of the tooth . therefore , the region of dentine that is selected is important when evaluating restorative materials in terms of bond strength and cytotoxicity . maroli et al24 showed , using scanning electron micrographs , that there are more tubular openings in the cervical third of approximal sections than in occlusal and middle . the cervical area of the bovine tooth was used as the dentine barrier in this experiment . with respect to transdentine permeability characteristics , bovine dentine at the level of the cemento - enamel junction seems to be a suitable alternative for coronal human dentine.25 the use of perfused dentine barrier models is a widely accepted method of evaluating the biological properties and the toxicity of dental restorative materials that will come into contact with dentine . however if contamination occurs during the experiment , the experiment will be ineffective because the complete growth of the cell culture in the mesh requires 14 days . moreover , researchers must have adequate experience with and knowledge of 3-d cell culture techniques . each step requires meticulous technique , especially the installation of the perfusion system , which is a 3-way connection with a flow rate similar to blood flow in the actual pulp . another advantage of this study was the ability to reduce the quantity and frequency of animals used in experiments . an experiment with animals is needed when the results from of in vitro studies are ambiguous . in accordance with the first priority of iso 7405 , it is recommended to avoid the use of animals for experiments if the study can possibly be conducted in vitro . all three self - etching dentine - bonding agents were non - toxic compared with the negative control group ( p>.05 ) . these agents could be used clinically in cases where there is more than .5 mm of remaining dentine above the pulp .
objective : the aim of this study was to evaluate the cytotoxicity of three dentine bonding agents ( g - bond , clearfil s3 bond and clearfil se bond x ) in cell - culture perfusion.methods:in this experiment , 8104 tcpc sv40 cells ( bovine - pulp - derived cells transfected with simian virus 40 large t - antigen ) in mem - alpha media , 20%fcs were seeded on mesh in a 6-well plate and incubated at 37 c with 5% co2 . after 2 days 
 , the mesh inserts were transferred to a 24-well plate and incubated in mem - alpha media , 20%fcs with 50 g / ml of ascorbic acid at 37 c with 5% co2 for 14 days . 
 the test materials were applied to the dentine discs of dentine barrier models . 
 three - dimensional cell cultures in mesh and perfusion conditions were generated in this experiment . 
 each material , as well as the negative control ( president ) and the positive control ( vitrebond ) , was tested in 5 models with the tests repeated in triplicate . 
 the mtt assay was used to determine cell viability after the diffusion of leachable toxicity from the tested materials through the dentine discs.results:the cell survival rate with g - bond and clearfil s3 bond was 113.03 and 90.98 percent , respectively , whereas that with clearfil se bond x was 111.83 percent . 
 all three dentine bonding agents had no toxicity compared with the negative control group ( p>.05).conclusions : all three self - etching dentine - bonding agents are nontoxic . 
 pulp damage caused by these three bonding agents is unlikely to occur in the clinic .