Journal of Conservative Dentistry
Home About us Editorial Board Instructions Submission Subscribe Advertise Contact e-Alerts Login 
Users Online: 1145
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size
 


 
Table of Contents   
ORIGINAL ARTICLE  
Year : 2016  |  Volume : 19  |  Issue : 6  |  Page : 522-526
Cytotoxicity of two available mineral trioxide aggregate cements and a new formulation on human gingival fibroblasts


1 Department of Dental Biomaterials, School of Dentistry; Department of Tissue Engineering, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Periodontics, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Click here for correspondence address and email

Date of Submission20-Jul-2016
Date of Decision22-Sep-2016
Date of Acceptance14-Oct-2016
Date of Web Publication14-Nov-2016
 

   Abstract 

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.

Keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay; Angelus mineral trioxide aggregate; calcium-enriched mixture; calcium-enriched mixture cement; cytotoxicity; mineral trioxide aggregate

How to cite this article:
Torshabi M, Amid R, Kadkhodazadeh M, Shahrbabaki SE, Tabatabaei FS. Cytotoxicity of two available mineral trioxide aggregate cements and a new formulation on human gingival fibroblasts. J Conserv Dent 2016;19:522-6

How to cite this URL:
Torshabi M, Amid R, Kadkhodazadeh M, Shahrbabaki SE, Tabatabaei FS. Cytotoxicity of two available mineral trioxide aggregate cements and a new formulation on human gingival fibroblasts. J Conserv Dent [serial online] 2016 [cited 2017 Mar 26];19:522-6. Available from: http://www.jcd.org.in/text.asp?2016/19/6/522/194033

   Introduction Top


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.[1],[2],[3]

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 Top


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

Click here to view


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 Top


The results are presented in [Figure 1] and [Figure 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

Click here to view
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

Click here to view


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 Top


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.[7],[8],[9],[10],[11],[12],[13],[14],[15] 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 Top


  • 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.

 
   References Top

1.
Tronstad L. Root resorption – Etiology, terminology and clinical manifestations. Endod Dent Traumatol 1988;4:241-52.  Back to cited text no. 1
    
2.
Heithersay GS. Invasive cervical resorption. Endod Topics 2004;7:73-92.  Back to cited text no. 2
    
3.
Bergmans L, Van Cleynenbreugel J, Verbeken E, Wevers M, Van Meerbeek B, Lambrechts P. Cervical external root resorption in vital teeth. J Clin Periodontol 2002;29:580-5.  Back to cited text no. 3
    
4.
Dazey S, Senia ES. An in vitro comparison of the sealing ability of materials placed in lateral root perforations. J Endod 1990;16:19-23.  Back to cited text no. 4
    
5.
Heithersay GS. Management of tooth resorption. Aust Dent J 2007;52 1 Suppl:S105-21.  Back to cited text no. 5
    
6.
Roberts HW, Toth JM, Berzins DW, Charlton DG. Mineral trioxide aggregate material use in endodontic treatment: A review of the literature. Dent Mater 2008;24:149-64.  Back to cited text no. 6
    
7.
Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new root-end filling material. J Endod 1995;21:349-53.  Back to cited text no. 7
    
8.
Rao A, Rao A, Shenoy R. Mineral trioxide aggregate – A review. J Clin Pediatr Dent 2009;34:1-7.  Back to cited text no. 8
    
9.
Oliveira MG, Xavier CB, Demarco FF, Pinheiro AL, Costa AT, Pozza DH. Comparative chemical study of MTA and Portland cements. Braz Dent J 2007;18:3-7.  Back to cited text no. 9
    
10.
Perinpanayagam H. Cellular response to mineral trioxide aggregate root-end filling materials. J Can Dent Assoc 2009;75:369-72.  Back to cited text no. 10
    
11.
Torabinejad M, Parirokh M. Mineral trioxide aggregate: A comprehensive literature review – Part II: Leakage and biocompatibility investigations. J Endod 2010;36:190-202.  Back to cited text no. 11
    
12.
Camilleri J. Characterization of hydration products of mineral trioxide aggregate. Int Endod J 2008;41:408-17.  Back to cited text no. 12
    
13.
Parirokh M, Torabinejad M. Mineral trioxide aggregate: A comprehensive literature review – Part I: Chemical, physical, and antibacterial properties. J Endod 2010;36:16-27.  Back to cited text no. 13
    
14.
Asgary S, Shahabi S, Jafarzadeh T, Amini S, Kheirieh S. The properties of a new endodontic material. J Endod 2008;34:990-3.  Back to cited text no. 14
    
15.
Asgary S, Ahmadyar M. One-visit endodontic retreatment of combined external/internal root resorption using a calcium-enriched mixture. Gen Dent 2011;60:e244-8.  Back to cited text no. 15
    
16.
Hakki SS, Hakki EE, Nohutcu RM. Regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by basic fibroblast growth factor and dexamethasone in periodontal ligament cells. J Periodontal Res 2009;44:794-802.  Back to cited text no. 16
    
17.
Hunt TK, Knighton DR, Thakral KK, Goodson WH 3rd, Andrews WS. Studies on inflammation and wound healing: Angiogenesis and collagen synthesis stimulated in vivo by resident and activated wound macrophages. Surgery 1984;96:48-54.  Back to cited text no. 17
    
18.
International Organisation for Standardisation. International Standard ISO 10993–12 Biological Evaluation of Medical Devices – Part 12: Sample Preparation and Reference Materials. Geneva: International Organisation for Standardisation; 2002.  Back to cited text no. 18
    
19.
Koh ET, McDonald F, Pitt Ford TR, Torabinejad M. Cellular response to mineral trioxide aggregate. J Endod 1998;24:543-7.  Back to cited text no. 19
    
20.
Williams DF. On the mechanisms of biocompatibility. Biomaterials 2008;29:2941-53.  Back to cited text no. 20
    
21.
Salehimehr G, Nobahar SH, Hosseini-Zijoud SM, Yari S. Comparison of physical and chemical properties of Angelus MTA and new endodontic restorative material. J Appl Pharm Sci 2014;4:105-9.  Back to cited text no. 21
    
22.
Sakaguchi R, Powers J. Craig's Restorative Dental Materials. 13th ed. St. Louis: Mosby Elsevier; 2012.  Back to cited text no. 22
    
23.
Ciasca M, Aminoshariae A, Jin G, Montagnese T, Mickel A. A comparison of the cytotoxicity and proinflammatory cytokine production of EndoSequence root repair material and ProRoot mineral trioxide aggregate in human osteoblast cell culture using reverse-transcriptase polymerase chain reaction. J Endod 2012;38:486-9.  Back to cited text no. 23
    
24.
Keiser K, Johnson CC, Tipton DA. Cytotoxicity of mineral trioxide aggregate using human periodontal ligament fibroblasts. J Endod 2000;26:288-91.  Back to cited text no. 24
    
25.
Jaberiansari Z, Naderi S, Tabatabaei FS. Cytotoxic effects of various mineral trioxide aggregate formulations, calcium-enriched mixture and a new cement on human pulp stem cells. Iran Endod J 2014;9:271-6.  Back to cited text no. 25
    
26.
Ghoddusi J, Tavakkol Afshari J, Donyavi Z, Brook A, Disfani R, Esmaeelzadeh M. Cytotoxic effect of a new endodontic cement and mineral trioxide aggregate on L929 line culture. Iran Endod J 2008;3:17-23.  Back to cited text no. 26
    
27.
Camilleri J, Montesin FE, Di Silvio L, Pitt Ford TR. The chemical constitution and biocompatibility of accelerated Portland cement for endodontic use. Int Endod J 2005;38:834-42.  Back to cited text no. 27
    

Top
Correspondence Address:
Fahimeh S Tabatabaei
Department of Dental Biomaterials, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.194033

Rights and Permissions


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]



 

Top
 
 
 
  Search
 
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed636    
    Printed28    
    Emailed0    
    PDF Downloaded85    
    Comments [Add]    

Recommend this journal