Cytotoxicity of two available mineral trioxide aggregate cements and a new formulation on human gingival fibroblasts.

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.

INTRODUCTION

External root resorption is a progressive process that causes destructive loss of tooth structure. This process is initiated by the development of a mineralized or denuded area on the root surface.[1] Internal bleaching, orthodontic tooth movement, traumatic injuries, and periodontal treatment often leave sequels in the periodontal ligament (PDL) and consequently cause root resorption.[123]

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.[4] 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).[5] A recent study has indicated that MTA is highly effective for filling root ends, repairing root perforations, and restoring damaged root structure.[6]

MTA is a relatively new sealing material recommended for treatment of iatrogenic root perforations and several other defects due to caries, resorption, or trauma.[7] This material received the Food and Drug Administration approval in 1998[8] and mainly comprised Portland cement, bismuth oxide, and gypsum (Ca). It includes fine hydrophilic particles that harden in the presence of moisture or blood.[9] 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.[10] MTA also showed favorable healing characteristics.[11] 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.[12]

MTA products of different manufacturers may be found in dental markets worldwide. 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.[13] MTA-Angelus (Angelus Indústria de Produtos Odontológicos S/A) was introduced in 2001 but later underwent a color change to white and marketed as MTA Branco (MTAB) (Angelus Indústria de Produtos Odontológicos 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.[11] 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.[14] Furthermore, it has shown favorable results in management of combined external/internal root resorption.[15]

Materials used for treatment of cervical root resorptions are in close contact with the gingiva.[16] 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. As the wound site progresses, the fibroblasts become the predominant cell type present.[17] 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).

MATERIALS AND METHODS

Material preparation

Sample preparation and extraction were carried out according to ISO 10993-12 standard.[18] 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.[18] In the first Group (A), materials were allowed to set for 24 h in a humid atmosphere. In the second Group (B), the discs were removed from the Teflon rings after 30 min of setting. Fresh materials comprised the third Group (C) [Table 1]. For each material, three discs (n = 3) were prepared for each time point.

Table 1

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-μm pore size membranes (Orange Scientific; Braine-l'Alleud, Belgium).

Cell culture

Human gingival fibroblasts (HGF1-PI1; NCBI-C165, Pasteur Institute Cell Bank, Tehran, Iran) were grown as monolayer cultures at 37°C (5% CO2, 95% humidity). The culture medium was DMEM (Gibco, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 μg/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-l’Alleud, Belgium) in complete medium and incubated for 24 h to obtain exponential cell growth. The culture medium was then replaced with 100 μL 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). Six replicates were assessed per extract or control.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) (Sigma-Aldrich, St. 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 μL 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 mean OD of the NC wells was set to represent 100% viability. 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%).

Data analysis

Statistical analysis was performed using GraphPad Prism version 6.01 (GraphPad Prism software, Inc. La Jolla, CA, USA). 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.

RESULTS

The results are presented in Figures 1 and 2.

Figure 1

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

Figure 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 72 h (n = 6). Complete medium and distilled water were used as negative and positive controls, respectively

After 24 h of exposure

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).

After 72 h of exposure

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.

The effect of time on the viability of cells exposed to different cements

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).

The effect of state of the cements on the viability of cells

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). There was no significant difference between MTA and CEM. After 72 h of exposure, the sequence of toxicity was the same as previous with no significant difference between MTA and CEM.

As expected, full setting (A) decreased the material's cytotoxicity. 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).

DISCUSSION

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.[19] The term biocompatibility refers to the capability of materials in dealing well with the host response.[20] The cytotoxic potential of materials is commonly evaluated to determine their biocompatibility before the conduction of clinical studies. Materials showing cell toxicity in vitro are probably highly toxic in vivo. 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. MTA- Angelus has shorter setting time and better workability than conventional MTA. The setting time of CEM cement is also lower than conventional MTA. Furthermore, studies showed that the antibacterial effect of CEM cement is more than MTA.[789101112131415] 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.[21]

The cytotoxicity of dental materials can be assessed by several techniques. The MTT assay is a commonly used technique to assess the metabolic activity of cells.[22]

Cell type is an important factor to consider in an experimental design for MTT assay. Laboratory cell lines such as osteoblasts,[23] PDL fibroblasts,[24] human dental pulp stem cells,[25] and mouse L929 fibroblasts[26] 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).[26] However, Camilleri et al. indicated that fresh MTA was more biocompatible than its set state.[27]

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.[24]

In the current study, nanohybrid MTA showed significant cytotoxicity when cells were exposed to fresh elutions of this cement. The toxicity increased over the tested time periods. Set nanohybrid MTA (Groups I and II) had higher cytotoxicity after 72 h compared to 24 h. In our previous study,[25] 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.

CONCLUSION

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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.