this process is initiated by the development of a mineralized or denuded area on the root surface . internal bleaching , orthodontic tooth movement , traumatic injuries , and periodontal treatment often leave sequels in the periodontal ligament ( pdl ) and consequently cause root resorption . regarding the increasing prevalence of resorptive defects , choosing an optimal material for repair is very important . this material should be well tolerated by periradicular tissues and must be able to induce biological cell responses and contribute to tissue regeneration . a variety of materials have been used to fill root lesions . the resorbed area may be restored by the application of glass ionomers , composite resins , amalgam , or mineral trioxide aggregate ( mta ) . a recent study has indicated that mta is highly effective for filling root ends , repairing root perforations , and restoring damaged root structure . mta is a relatively new sealing material recommended for treatment of iatrogenic root perforations and several other defects due to caries , resorption , or trauma . this material received the food and drug administration approval in 1998 and mainly comprised portland cement , bismuth oxide , and gypsum ( ca ) . cell culture studies using different human and animal cells have shown optimal cell survival , proliferation , and attachment , as well as their fast growth on the mta surface . the biocompatibility of mta is believed to be due to the release of hydroxyl ions and subsequent formation of calcium hydroxide that occur in the hydration process . the gray - mta and tooth - colored mta or white wmta marketed as proroot mta ( dentsply tulsa dental ) were among the first mta products introduced to the dental market . mta - angelus ( angelus indstria de produtos odontolgicos s / a ) was introduced in 2001 but later underwent a color change to white and marketed as mta branco ( mtab ) ( angelus indstria de produtos odontolgicos s / a ) . mta - angelus and mtab exhibit no cytotoxicity or genotoxicity on various cell lines , with effects similar to those of mta on cell cultures . calcium - enriched mixture ( cem ) is a new endodontic cement with clinical applications similar to those of mta as well as similar ph , working time , dimensional changes , optimal flowability , and low film thickness . furthermore , it has shown favorable results in management of combined external / internal root resorption . materials used for treatment of cervical root resorptions are in close contact with the gingiva . the most ideal healing outcome after preparation and filling of a cervical root resorption area would be reformation of a normal attachment apparatus with healthy bone , pdl , and cementum . the purpose of this study was to investigate the cytotoxicity of a new formulation of mta named as nanohybrid mta , in comparison with cem cement and mta - angelus , using in vitro cell cultures of human gingival fibroblast ( hgf ) . the tested materials were white mta - angelus ( angelus , londrina , brazil ) ; cem ( bioniquedent , tehran , iran ) ; and nanohybrid mta containing three different nanoparticles ( based on the inventor 's claim ) ( tehran , iran ) . materials were prepared according to the manufacturers and inventor 's instructions and were placed in round teflon rings with a diameter of 1 cm and a height of 2 mm . in the first group ( a ) , materials were allowed to set for 24 h in a humid atmosphere . in the second group ( b ) , fresh materials comprised the third group ( c ) [ table 1 ] . for each material , the tested materials and subgroups used in the study for extract preparation , all specimens ( either at fresh or set state ) at the same time were placed into the wells of 24-well plates and immersed in 1 ml of dulbecco 's modified eagle 's medium ( dmem ) and incubated for 24 h. afterward , the extractions were filtered by 0.22-m pore size membranes ( orange scientific ; braine - l'alleud , belgium ) . human gingival fibroblasts ( hgf1-pi1 ; ncbi - c165 , pasteur institute cell bank , tehran , iran ) were grown as monolayer cultures at 37c ( 5% co2 , 95% humidity ) . the culture medium was dmem ( gibco , usa ) , supplemented with 10% fetal bovine serum ( fbs ) ( gibco , usa ) , 100 g / ml streptomycin , and 100 iu / ml penicillin . adherent cells at a logarithmic growth phase were detached by trypsin / ethylenediaminetetraacetic acid ( gibco , usa ) mixture . next , 5000 cells / well were placed on 96-well plates ( orange scientific ; braine - lalleud , belgium ) in complete medium and incubated for 24 h to obtain exponential cell growth . the culture medium was then replaced with 100 l of the tested materials original extracts ( supplemented with 10% fbs ) or control media ( positive control group consisted of distilled water and the negative control ( nc ) group consisted of complete medium ) . louis , mo , usa ) assay was used to determine the influence of different materials on the viability and proliferation of hgf . after 24 and 72 h of incubation of cells in the presence of test extracts , the medium was removed from each well , the cells were washed with phosphate - buffered saline , and 100 l of the mtt solution ( 5 mg / ml ) was added to each well ; cells were incubated for an additional 3 h. the resulting formazan crystals were dissolved by dimethyl sulfoxide solvent ( sigma - aldrich ) . the optical density ( od ) of the plates was read using a spectrophotometer ( anthos 2020 , austria ) , at a test wavelength of 570 nm and a reference wavelength of 620 nm . the viability of the treated cells was computed as a percentage of the mean nc value . cytotoxicity responses were rated as severe ( < 30% ) , moderate ( 30%60% ) , slight ( 60%90% ) , or noncytotoxic ( > 90% ) . statistical analysis was performed using graphpad prism version 6.01 ( graphpad prism software , inc . results were subjected to one - way anova followed by tukey 's post hoc test for pairwise comparisons . statistical significance was set at p < 0.05 . the tested materials were white mta - angelus ( angelus , londrina , brazil ) ; cem ( bioniquedent , tehran , iran ) ; and nanohybrid mta containing three different nanoparticles ( based on the inventor 's claim ) ( tehran , iran ) . materials were prepared according to the manufacturers and inventor 's instructions and were placed in round teflon rings with a diameter of 1 cm and a height of 2 mm . in the first group ( a ) , materials were allowed to set for 24 h in a humid atmosphere . in the second group ( b ) , fresh materials comprised the third group ( c ) [ table 1 ] . for each material , the tested materials and subgroups used in the study for extract preparation , all specimens ( either at fresh or set state ) at the same time were placed into the wells of 24-well plates and immersed in 1 ml of dulbecco 's modified eagle 's medium ( dmem ) and incubated for 24 h. afterward , the extractions were filtered by 0.22-m pore size membranes ( orange scientific ; braine - l'alleud , belgium ) . human gingival fibroblasts ( hgf1-pi1 ; ncbi - c165 , pasteur institute cell bank , tehran , iran ) were grown as monolayer cultures at 37c ( 5% co2 , 95% humidity ) . the culture medium was dmem ( gibco , usa ) , supplemented with 10% fetal bovine serum ( fbs ) ( gibco , usa ) , 100 g / ml streptomycin , and 100 iu / ml penicillin . adherent cells at a logarithmic growth phase were detached by trypsin / ethylenediaminetetraacetic acid ( gibco , usa ) mixture . next , 5000 cells / well were placed on 96-well plates ( orange scientific ; braine - lalleud , belgium ) in complete medium and incubated for 24 h to obtain exponential cell growth . the culture medium was then replaced with 100 l of the tested materials original extracts ( supplemented with 10% fbs ) or control media ( positive control group consisted of distilled water and the negative control ( nc ) group consisted of complete medium ) . louis , mo , usa ) assay was used to determine the influence of different materials on the viability and proliferation of hgf . after 24 and 72 h of incubation of cells in the presence of test extracts , the medium was removed from each well , the cells were washed with phosphate - buffered saline , and 100 l of the mtt solution ( 5 mg / ml ) was added to each well ; cells were incubated for an additional 3 h. the resulting formazan crystals were dissolved by dimethyl sulfoxide solvent ( sigma - aldrich ) . the optical density ( od ) of the plates was read using a spectrophotometer ( anthos 2020 , austria ) , at a test wavelength of 570 nm and a reference wavelength of 620 nm . the viability of the treated cells was computed as a percentage of the mean nc value . cytotoxicity responses were rated as severe ( < 30% ) , moderate ( 30%60% ) , slight ( 60%90% ) , or noncytotoxic ( > 90% ) . statistical analysis was performed using graphpad prism version 6.01 ( graphpad prism software , inc . results were subjected to one - way anova followed by tukey 's post hoc test for pairwise comparisons . statistical significance was set at p < 0.05 . the results are presented in figures 1 and 2 . relative cell viability obtained from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using human gingival fibroblasts exposed to different materials for 24 h ( n = 6 ) . complete medium and distilled water were used as negative and positive controls , respectively relative cell viability obtained from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using human gingival fibroblasts exposed to different materials for 72 h ( n = 6 ) . complete medium and distilled water were used as negative and positive controls , respectively a significant decrease in cell proliferation was seen in all groups exposed to all cements compared to the nc group ( p < 0.05 ) . the most cytotoxic material in this time point was the subgroup c of angelus mta with 42% cell viability . as shown in figure 1 , the least cytotoxic material in this time point was the group a of cem which showed 15% reduction compared to the nc group and this reduction was statistically significant ( p < 0.05 ) . in the second mtt assay ( 72 h ) , a significant decrease in cell proliferation was noted in all groups ( p < 0.05 ) . however , in subgroup a of cem cement and mta , there was no significant difference compared to the nc group ( p < 0.05 ) . experimental mta was still moderately cytotoxic at 72 h. the most cytotoxic material in this time point was the subgroup b of angelus mta and the least cytotoxic material in this time point was the group a of this material . all samples of experimental mta ( groups a , b , and c ) had slight cytotoxic effects after 24 h of incubation and moderate cytotoxic effects after 72 h of incubation . mta and cem at subgroup a caused a time - dependent increase in cell viability , while in other groups , a significant time - dependent decrease in cell viability was seen ( p < 0.05 ) . in the fresh samples ( subgroup c ) , the sequence of toxicity was mta > cem and experimental ( p < 0.05 ) at 24 h. however , after 72 h of exposure , there was no significant difference between these groups ( p > 0.05 ) ( all fresh samples showed moderate cytotoxicity in 72 h ) . in the 30 min set samples ( subgroup b ) , the sequence of toxicity was mta > cem > experimental ( p < 0.05 ) at 24 h. there was no significant difference between these groups after 72 h of exposure . in the 24 h set samples ( subgroup a ) , the sequence of toxicity was experimental > mta and cem ( p < 0.05 ) . after 72 h of exposure , the sequence of toxicity was the same as previous with no significant difference between mta and cem . however , compared with extracts of the other tested materials at the same setting ( 24 h ) , experimental mta presented a higher toxicity to the hgfs at both time intervals ( p < 0.05 ) . a significant decrease in cell proliferation was seen in all groups exposed to all cements compared to the nc group ( p < 0.05 ) . the most cytotoxic material in this time point was the subgroup c of angelus mta with 42% cell viability . as shown in figure 1 , the least cytotoxic material in this time point was the group a of cem which showed 15% reduction compared to the nc group and this reduction was statistically significant ( p < 0.05 ) . in the second mtt assay ( 72 h ) , a significant decrease in cell proliferation was noted in all groups ( p < 0.05 ) . however , in subgroup a of cem cement and mta , there was no significant difference compared to the nc group ( p < 0.05 ) . experimental mta was still moderately cytotoxic at 72 h. the most cytotoxic material in this time point was the subgroup b of angelus mta and the least cytotoxic material in this time point was the group a of this material . all samples of experimental mta ( groups a , b , and c ) had slight cytotoxic effects after 24 h of incubation and moderate cytotoxic effects after 72 h of incubation . mta and cem at subgroup a caused a time - dependent increase in cell viability , while in other groups , a significant time - dependent decrease in cell viability was seen ( p < 0.05 ) . in the fresh samples ( subgroup c ) , the sequence of toxicity was mta > cem and experimental ( p < 0.05 ) at 24 h. however , after 72 h of exposure , there was no significant difference between these groups ( p > 0.05 ) ( all fresh samples showed moderate cytotoxicity in 72 h ) . in the 30 min set samples ( subgroup b ) , the sequence of toxicity was mta > cem > experimental ( p < 0.05 ) at 24 h. there was no significant difference between these groups after 72 h of exposure . in the 24 h set samples ( subgroup a ) , the sequence of toxicity was experimental > mta and cem ( p < 0.05 ) . after 72 h of exposure , the sequence of toxicity was the same as previous with no significant difference between mta and cem . however , compared with extracts of the other tested materials at the same setting ( 24 h ) , experimental mta presented a higher toxicity to the hgfs at both time intervals ( p < 0.05 ) . materials often applied for restoration of cervical resorption defects may directly contact the gingival tissues . thus , aside from other requirements , these materials must be biocompatible to the gingival tissue . the term biocompatibility refers to the capability of materials in dealing well with the host response . the cytotoxic potential of materials is commonly evaluated to determine their biocompatibility before the conduction of clinical studies . we aimed to investigate the cytotoxicity of a new formulation of mta named as nanohybrid mta , in comparison with cem cement and mta - angelus . nanohybrid mta is a mixture of three different nanoparticles ( based on the inventor 's claim ) , cem cement is composed of calcium oxide , sulfur trioxide , phosphorus pentoxide , silicon dioxide , and some minor components , while according to its manufacturer , mta- angelus is composed of 80% portland cement and 20% bismuth oxide . nanohybrid mta is a new experimental cement , but there are so many studies on cem cement and mta- angelus . furthermore , studies showed that the antibacterial effect of cem cement is more than mta . one study comparing cem cement with angelus mta showed that cem cement has better chemical and physical properties than angelus mta , but angelus mta is preferred in terms of setting time . the mtt assay is a commonly used technique to assess the metabolic activity of cells . cell type is an important factor to consider in an experimental design for mtt assay . laboratory cell lines such as osteoblasts , pdl fibroblasts , human dental pulp stem cells , and mouse l929 fibroblasts are often used in such investigations . cultured hgfs were used in the current study to better simulate the oral clinical environment . despite several advantages , mta has some major drawbacks such as prolonged setting time and extended maturation phase . both set and fresh mta were evaluated in the current study to determine whether it was the setting process that caused cytotoxicity or the by - products released by the set materials . our results revealed significant differences among the three states of the same material ( p < 0.05 ) , except for the nanohybrid mta group , which interestingly showed no significant difference between the set state and fresh formula ( p < 0.05 ) . another study also found higher cytotoxicity of fresh mta when compared with the set form of the same material ; significant differences were also noted among the time intervals ( 24 , 48 , and 72 h ) . however , camilleri et al . indicated that fresh mta was more biocompatible than its set state . the current study showed that the proliferation of hgfs exposed to completely set cem and mta resembled that of the controls after up to 72 h of incubation , suggesting low cytotoxicity of these materials . the results of other studies on the cytotoxicity of completely set mta were similar to ours . in the current study , nanohybrid mta showed significant cytotoxicity when cells were exposed to fresh elutions of this cement . set nanohybrid mta ( groups i and ii ) had higher cytotoxicity after 72 h compared to 24 h. in our previous study , nanohybrid mta had cytotoxic effects on dental pulp stem cells after 24 , 48 , and 72 h , which is in agreement with the results of the current study . disintegration of nanohybrid mta during extraction or its composition may explain its significant cytotoxicity in all states . set cem and set mta - angelus exerted similar , favorable effects on the mitochondrial activity and viability of fibroblastswithin the limitations of this in vitro study , nanohybrid mta could not be recommended as a material of choice for cervical root resorption . set cem and set mta - angelus exerted similar , favorable effects on the mitochondrial activity and viability of fibroblasts within the limitations of this in vitro study , nanohybrid mta could not be recommended as a material of choice for cervical root resorption .
aim : the purpose of this study was to investigate the cytotoxicity of nanohybrid mineral trioxide aggregate ( mta ) in comparison with calcium - enriched mixture ( cem ) cement and mta - angelus , using human gingival fibroblasts ( hgfs).materials and methods : nine disc - shaped specimens of each material ( in 2 set stat : a , set for 24 h ; b , set for 30 min ; and c , fresh stat ) were prepared . 
 hgfs were exposed to tested materials extracts or control media . 
 cytotoxicity testing was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay in two time intervals.statistical analysis : results were evaluated by one - way anova and t - test . 
 statistical significance was set at p < 0.05.results:cem 
 cement demonstrated favorable cell viability values when completely set ( 24 h set mta = 24 h set cem ) at both time intervals . 
 interestingly , 24 h after incubation , cem in groups b and c demonstrated higher cell viability values than mta ( p < 0.05 ) . 
 however , after 72 h of incubation , these groups of cem and mta showed equal cell viability . 
 all samples of nanohybrid mta had slight cytotoxic effects after 24 h of incubation , and moderate cytotoxic effects after 72 h of incubation.conclusion:set cem and set mta - angelus exerted similar , favorable effects on cell viability . however , within the limitations of this in vitro study , the results suggest that nanohybrid mta could not be recommended as a material of choice for cervical root resorption .