International Dental & Medical Journal of Advanced Research ● Vol. 3 ● 2017 1

International Dental & Medical Journal of Advanced Research (2017), 3, 1–6

O R I G I N A L A R T I C L E

Determination of cytotoxicity of dentine bonding agents, hydroxyethyl methacrylate and bisphenol alphaUpasana Jayaram Reddy

Department of Conservative Dentistry and Endodontics, A. J Institute of Dental Sciences, Mangalore, Karnataka, India

AbstractAim: The purpose of the study was to analyze cytotoxicity caused by dental adhesives on human gingival fibroblast (HGF) cell lines, compare the response of cultured HGFs to substances leached from a conventional total etch and self-etching adhesive system along with their individual components.Methodology: Healthy gingival tissue biopsies (explants) were used to obtain isolates of HGFs, which were cultivated in flasks till subconfluent monolayers obtained. Serial dilutions for each compound tested were made and elutes at all concentrations tested for were obtained at 48 h for each material which were then added at different concentrations to the HGF cultures, and cytotoxicity was analyzed by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results: Within the limitations of the in-vitro study, we conclude: All of the materials tested showed moderately or slightly cytotoxicity at the lower serial dilutions except bisphenol alpha and One Coat-7.0, which proved to be strongly cytotoxic among almost all the dilutions. The lowest cytotoxicity was obtained from Clearfil SE Bond at 0.3125 mg/mL and the highest from One Coat-7.0 at 5.9 mg/mL, among the three dentin bonding systems tested for cytotoxicity.Conclusions: Indicated that SE B had the lowest mean % of cell viability value and OC had the highest mean % of cell viability.

Introduction

Several dentin bonding systems have been developed over the last few decades with the aim to improve the bond between resin and tooth structures for more retentive restorations.

Bowen had proposed bisphenol alpha glycidyl methacrylate (BisGMA) resins to be bonded to enamel after etching, but he soon realized that resins would bond only to a clean substrate and not bond to dentin equally well. Clinically, bonding resins do not bond to dentin unless it is etched by a certain concentration of phosphoric acid and an intermediary dentin bonding system was required to bring together organic and inorganic components together.

Even today with all the deficiencies that Bis-GMA resins have it is still the most commonly used resin in restorative dentistry along with urethane dimethacrylate (UDMA) as primary components of bonding systems. Although the concept of ethical treatment of the patients extended back to Hippocrates (460-377 BC) the idea that new dental products have to be tested for safety before use is recent.[1] The urgent need of the hour was to confirm whether these Bis-GMA based resins are clinically safe.

Cytotoxicity tests are considered a sensitive and standardized method to determine the toxicity of a material containing significant amounts of biologically harmful leachable compounds. Culture medium of mammalian cells is the preferred method for the extraction of substance that can be released from a material because it is a physiological solution capable of extracting a wide range of chemical structures, not only those soluble in water.[2]

3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

The MTT assay is a sensitive, most widely used method to determine the mitochondrial enzyme activity and the cell function. It is highly predictable in measuring the cell viability, growth, and proliferation by using tetrazolium dye that is reduced by metabolically active cells having intact mitochondrial dehydrogenase enzymes.

The resinous monomers used in dentistry are formed by different organic molecules, such as bisphenol alpha GMA (Bis-

Keywords Biocompatibility, bisphenol alpha, cytotoxicity, hydroxyethyl methacrylate, self-etching primers, total etching primers

Correspondence Upasana Jayaram Reddy, Sheena House, Opposite Forum Fiza Mall, Pandeshwar, Mangalore - 575 001, Karnataka, India. Phone: +91-9845827259. Email: upasana.reddy.88@gmail.com

Received 27 November 2016; Accepted 02 January 2017

doi: 10.15713/ins.idmjar.57

2 International Dental & Medical Journal of Advanced Research ● Vol. 3 ● 2017

Cytotoxicity determination of bonding agents and components Reddy

GMA), triethylene glycol dimethacrylate (TEGDMA), UDMA, and hydroxyethyl methacrylate (HEMA), which work together as copolymeric chains. These monomers comprise the main organic basis of the majority of composite resins and dental adhesives.[3]

However, since various studies have been performed showing toxicity of most of the above-mentioned monomers, we have tested toxicity levels of HEMA and the chief precursor of Bis-GMA which is bisphenol alpha (BPA).

BPA was synthesized close to 100 years ago and recently data begun to emerge indicating BPA exposures, even those in the range generally experienced by any population, having adverse effects on human health. They are components of resin-based dental sealants and composites that are increasingly used in both preventive and restorative oral health care.[2]

However these components alone, as well as in combination with composite resins are capable to reach concentrations to damage pulp after diffusing through dentin.[14,15] As polymerization is incomplete free monomers can be detected by different analytic methods.[16,20]

In dentistry, more than 98% of the restorations are of polymers and monomers. This study was to focus on the toxicity levels of different unpolymerized monomers as single components or in their commercially available form which is used by practitioners on a day to day basis.

Cytotoxicity study using MTT assay

The list of armamentarium and materials used in the study [Table 1].

Methodology

Three commercially available dentin bonding systems were investigated and two individual components used in bonding agents were also tested namely BPA and HEMA. Of them two are self-etching adhesives and the third is a total etch adhesive. The primer and bond of each were taken as separate groups for the self-etch systems. The experiment was conducted in the following steps.

Table 1: Composition of different dentine bonding agents used in the studyProduct/manufacturer CompositionClearfil Se Bond ‑ Bond (Kuraray Noritake Dental Inc., Okayama, Japan) MDP

Bis‑GMAHEMAHydrophobic aliphatic methacrylateColloidal silicadl‑camphorquinone

Clearfil Se Bond ‑ Primer (Kuraray Noritake Dental Inc., Okayama, Japan) MDPHEMAHydrophobic aliphatic methacrylatedl‑camphorquinoneWater

Clearfil Protect Bond Bond (Kuraray Noritake Dental Inc., Okayama, Japan) MDPBis‑GMAHEMAHydrophobic aliphatic dimethacrylatedl‑CamphorquininoN, N‑diethanol p‑toluidineSilanated colloidal silicaSodium fluoride

Clearfil Protect Bond‑Primer (Kuraray Noritake Dental Inc., Okayama, Japan) MDP2‑HEMA12‑Methacryloyloxydo decylpyridinium bromideHydrophilic aliphatic dimethacrylateWater

One Coat 7.0 (Coltene/Whaledent AG, Altstatten, Switzerland) HEMAEthanol

2‑HEMA‑HEMA (Sigma‑Aldrich Chemicals Pvt. Limited, Bengaluru, India) 2‑HEMASynonyms: 1,2‑Ethanediol mono (2‑methylpropenoate)Glycol methacrylateHEMA

BPA (Sigma‑Aldrich Chemicals Pvt. Limited, Bengaluru, India) BPASynonyms: 2,2‑Bis (4‑hydroxyphenyl) propane 4,4′‑Isopropylidenediphenol

MDP: 10‑Methacryloyloxydecyl dihydrogen phosphate, HEMA: Hyrdoxyethyl methacrylate, BPA: Bisphenol alpha, Bis‑GMA: Bisphenol alpha glycidyl methacrylate

International Dental & Medical Journal of Advanced Research ● Vol. 3 ● 2017 3

Reddy Cytotoxicity determination of bonding agents and components

Growth and maintenance of cell cultures

Tissue pieces were minced to approximately 0.3 cm of the gingival tissue biopsy which was taken from the site of extraction of patients who required orthodontic extraction of teeth, and thus human gingival fibroblasts (HGFs) were isolated from them. These cells are frequently used in biological assessment of resin based materials because they are in close proximity with restorative dental materials in the oral cavity, more clinically relevant and are sensitive cells easy to isolate.[17-19,22] These free monomers alter cell functions in the oral cavity.[29]

Dulbecco’s Modified Eagle’s Medium (DMEM) containing 25 cm3 tissue flasks were used for explant cultivation, supplemented with 5% fetal bovine serum, penicillin, at 37°C, and 5% CO2. Cells were counted on a Nuebaer’s Chamber and seeded, followed with an incubation for 24 h in 5% CO2 at 37°C.

Sample preparation and elution

Serial dilutions for each compound tested were made in Eppendorf and the testing components were placed in cell culture medium DMEM and subjected to the cytotoxicity assay.

Addition of elute to cultured fibroblasts

Cells were randomly divided and seeded into 96 well plates. There were seven experimental groups and two control groups.

After incubation for 48 h, the medium was removed and refilled with 100 µl in every well with test solution. The plates were incubated for another 24 h followed by evaluation of cytotoxicity.

MTT assay and spectrophotometric analysis

As first stated by Mosmann in 1983, the MTT assay[30] outlines the capability of mitochondrial dehydrogenase enzymes in cells to convert the yellow water-soluble tetrazolium salt into dark purple formazan crystals. The number of surviving cells is thus, directly proportional to the level of the formazan product formed.

About 100 µl MTT dye was added to each well-containing cells treated with various extracts of bonding agents and the control wells. Optical density was determined by dissolving the MTT formazan intercellular reaction product with dimethyl sulfoxide and the spectrophotometric absorbance was measured at 630 nm using an enzyme-linked immunosorbent assay microplate reader (Lisa Plus, Aspen Diagnostics, India). Mean was calculated with three readings from every well.

The following formula was used to estimate the percentage of cell viability:

Absorbance of sample × 100% of cell viability =Absorbance of control

Results

The results of the percentage of cell survival for the single components HEMA and BPA revealed different patterns. With HEMA, the cell death increased as the concentration of the material was increased.

Whereas BPA demonstrated a different pattern, where there was almost no cell death at the highest concentration (20 mg/mL) tested, followed by a sudden increase in cell death at subsequent concentrations (i.e., 10, 5, 2.5, 1.25, 0.625 mg/mL) until 0.0156 mg/mL where the percentage of cell viability was again high indicating low cell death.

Scoring criteria are given by Dahl et al.[4] was used to rate cytotoxicity. The percentage of cell viability for each component of bonding agent was recorded, and the results were tabulated and subjected to statistical analysis.

The mean value Graph 1 showing the cell viability of HEMA and BPA test groups at different concentrations on HGF cell lines.

The mean value Graph 2 showing the cell viability of all the concentrations of the five compounds tested for cytotoxicity.

Discussion

Several dentine bonding adhesives have been introduced into restorative dentistry and are formed by different organic molecules such as GMA, Bis-GMA, TEGDMA, UDMA dipentaerythritol pentaacrylate monophosphate, and HEMA which function as copolymeric chains.[5]

Graph 1: The mean value showing the cell viability of hydroxyethyl methacrylate and bisphenol alpha test groups at different concentrations on human gingival fibroblast cell lines

Graph 2: % Cell viability

4 International Dental & Medical Journal of Advanced Research ● Vol. 3 ● 2017

Cytotoxicity determination of bonding agents and components Reddy

Most of the composites and sealants used in dentistry are based on Bis-GMA.[6] Reports revealed that in-situ polymerization is not complete and that free monomers can be detected by different analytical methods.[6]

The cytotoxicity of dentine bonding agents depends generally on dentine permeability, adhesive composition and time elapsed after their placement.

The cytotoxicity of dentin primers and bonding agents was determined on HGFs cell line by MTT assay in the current study (Figures 1 and 2).

The results of the percentage of cell survival for the single components HEMA and BPA, which were tested for cytotoxicity by MTT assay revealed different patterns. With HEMA, the cell death increased as the concentration of the material was

increased, whereas BPA there was almost no cell death at the highest concentration (20 mg/mL) tested, followed by a sudden increase in cell death at subsequent concentrations until 0.0156 mg/mL where the percentage of cell viability was again high indicating low cell death.

A search of literature revealed that as of today very few have tested the cytotoxicity of BPA, found in composites and sealants for two reasons:• As a by-product of other ingredients in dental composites

and sealants that have degraded• As a trace material left over from the manufacture of other

ingredients used in dental composites and sealants.[7]

“Dental sealant exposure to BPA occurs primarily with the use of sealants containing BPA dimethacrylate. This exposure is

Figure 1: Human gingival fibroblasts as seen under compound microscope during cell culture at different concentrations on hema

Figure 2: Human gingival fibroblasts as seen under compound microscope during cell culture at different concentrations on bisphenol‑a

International Dental & Medical Journal of Advanced Research ● Vol. 3 ● 2017 5

Reddy Cytotoxicity determination of bonding agents and components

considered an infrequent event with little relevance to estimating general population exposures.”

All though earlier studies by Soderholm[8] suggest that the resins leached from materials are of no significance, however in 2012, the FDA reiterates that “recent studies provide reason for concern about the potential effects of BPA on the brain, behavior, and prostate gland of fetuses, infants, and children.” Which resulted in this study on BPA and HEMA as individual components in the individual composite resins and their effect at cell levels in the laboratory.

Almost all manufacturers add HEMA for additional bond strength to dentin.[9] The molecular weight of HEMA is 130.14 and is highly water soluble. High concentrations and early release of HEMA may release free unpolymerized HEMA and come in contact with cell cultures. In this study, cell viability was determined by the mitochondrial activity analysis carried out using MTT based cytotoxicity assay.

The studies by Bruno Neves et al. (2010)[10] demonstrated that the self-etching system was partially biocompatible since the primer was cytotoxic and observed that the adhesive resin was positioned in an intermediate level of biocompatibility.[10]

Based on the mean values, the results of the percentage of cell viability for the three dentin bonding systems used in the study showed that the cell viability was the highest for SE Bond followed by Protect Bond, SE Primer, Protect Primer and One Coat. Indicating that One Coat is the most cytotoxic and Clearfil SE Bond is the least cytotoxic among all the tested dentin bonding systems. Indicating that all the dentin bonding systems were cytotoxic especially the primers were most cytotoxic. However, One Coat 7 which contains both the primer and bonding agent in one bottle were found to be most cytotoxic in this study.

Most clinical researchers have observed the highest toxicity within the first 24 h after the placement. Hanks et al. (1991) and Krifka et al. (2012) have reported HEMA induced apoptosis in vitro at 24 h. In this study, the dentin bonding agents were kept in contact up to 48 h and recorded maximum toxicity at the end of 48 h.

Demirci et al. (2008)[11] reported that dentin primers and dentin bonding agents of Clearfil SE Bond resin and Clearfil Protect Bond resin decreased cell survival in a dose-related manner. This applies to all materials used to test and of the resin based material used for testing.[22-26] A total-etching bonding system is more cytotoxic than a self-etching bonding system.[12,13,28]

Conclusion

It was observed that all the bonding systems and the two components HEMA and BPA showed a drastic variation in cytotoxic response, clearly indicating them to have cytotoxic effects at concentrations corresponding to concentrations released into the oral cavity.

All materials were initially toxic at the highest concentration and the toxicity decreased over time except for BPA where the highest concentration showed no toxicity at all and as the

concentration decreased there were strongly cytotoxic and again at the lowest concentration they proved to be moderately cytotoxic. One Coat-7.0 proved to be strongly cytotoxic at all the concentrations tested for.

The clinical significance of our study is that most of the byproducts of BPA based resin materials is a matter of harm to patient, dentist as well as the assistant handling the material. There is no one ideal material available to the clinician,[21] thus continued research is mandatory in this direction to avoid any complications.

References

1. Anusavice KJ. Biocompatibility of dental materials. Phillips Science of Dental Materials. 11th  ed. Ch. 8. St. Louis, MO: Saunders; 2003.

2. de Moraes Porto IC. Polymer Biocompatibilty. INTECH 1A7Q2 Publications; 2012.

3. Arossi GA, Dihl RR, Lehmann M, Cunha KS, Reguly ML, de Andrade HH. In vitro genotoxicity of dental bonding agents. Mutagenesis 2009;24:169‑72.

4. Dahl JE, Frangou‑Polyzois MJ, Polyzois GL. In-vitro biocompatibility of denture relining materials. Gerodontology 2006;23:17‑22.

5. Kaya A, Undeger U, Aydin S, Omurlu H, Basaran N. Genotoxicity evaluation of dentine bonding agents by comet assay. Int Endod J 2011;44:807‑16.

6. Huang FM, Chang YC. Cytotoxicity of dentine bonding agents on human pulp cells in vitro. IEJ 2002;35:905‑9.

7. Council on Scientific Affairs Statement. Bisphenol A and Dental Materials. US: American Dental Association (ADA); 2014.

8. Soderholm KJ, Phil M. Bis‑GMA based resins in dentistry: Are they safe? JADA 1999;130:201‑9.

9. Chen RS, Lui CC. Cytotoxicity of three dentin bonding agents on human dental pulp cells. J Dent 2003;31:223‑9.

10. Cavalcanti BN, Barschneider LR, Marques MM. Cytotoxicity of substances leached from a conventional and a self‑etching adhesive system on human pulp fibroblasts. Braz Dent Sci 2010;13:10‑4.

11. Demirci M, Hiller KA, Bosl C, Gallerb K, Schmalzb G, Schweikl H. The induction of oxidative stress, cytotoxicity and genotoxicity by dental adhesives. Dent Mater 2008;24:362‑71.

12. Chiang SL, Jiang SS, Wang WJ, Chiang HC, Chen PH, Tu KH, et al. Characterization of arecoline‑induced effects on cytotoxicity in normal human gingival fibroblasts by global gene expression profiling. Toxicol Sci 2007;100:66‑74.

13. Vajrabhaya LO, Pasasuk A, Harnirattisai C. Cytotoxicity evaluation of single component dentin bonding agents. Oper Dent 2003;28:440‑4.

14. Schmalz G, Schuster U, Koch A, Schweikl H. Cytotoxicity of low pH dentin‑bonding agents in a dentin barrier test in vitro. JOE 2002;28:188‑92.

15. Pulgar R, Olea‑Serrano MF, Novillo‑Fertrell A, Rivas A, Pazos P, Pedraza V, et  al. Determination of bisphenol A and related aromatic compounds released from bis‑GMA‑based composites and sealants by high performance liquid chromatography. Environ Health Perspect 2000;108:21‑7.

16. Theilig C, Tegtmeier Y, Leyhausen G, Geurtsen W. Effects of Bis‑GMA and TEGDMA on proliferation, migration, and tenascin

6 International Dental & Medical Journal of Advanced Research ● Vol. 3 ● 2017

Cytotoxicity determination of bonding agents and components Reddy

expression of human fibroblasts and keratinocytes. J  Biome d Mater Res 2000;53:632‑9.

17. Bakopoulou A, Papadopoulos TF, Garefis P. Molecular toxicology of substances released from resin‑based. Dental restorative materials. Int J Mol Sci 2009;10:3861‑99.

18. Moharamzadeh K, Brook IM, Noort RV. Biocompatibility of resin‑based dental materials. Materials 2009;2:514‑48.

19. Franz A, Konig F, Lucas T, Watts DC, Schedle A. Cytotoxic effects of dental bonding substances as a function of degree of conversion. Dent Mater 2009;25:232‑9.

20. Ferrance JL. Resin composite‑state of the art. Dent Mater 2011;27:29‑38.

21. Karapinar‑Kazandag M, Bayrak OF, Yalvac ME, Ersev H, Tanalp J, Sahin F, et al. Cytotoxicity of 5 endodontic sealers on L929 cell line and human dental pulp cells. Int Endod J 2011;44:626‑34.

22. Ding SJ, Kao CT, Chen CL, Shie MY, Huang TH. Evaluation of human osteosarcoma cell line genotoxicity effects of mineral trioxide aggregate and calcium silicate cements. J  Endod 2010;36:1158‑62.

23. Gulden M, Jess A. Cytotoxicity potential of hydrogen peroxide in cell cultures. Free Radic Biol Med 2010;49:1298‑305.

How to cite this article: Reddy UJ. Determination of cytotoxicity of dentine bonding agents, hydroxyethyl methacrylate and bisphenol alpha. Int Dent Med J Adv Res 2017;3:1-5.

24. Pameijei CH, Zmener O. Resin materials for root canal obturations. DCNA 2010;54:325‑44.

25. Brzovic V, Miletic I, Zeljezic D, Mladinic M, Kasuba V, Ramic S, et al. In vitro genotoxicity of root canal sealers. IEJ 2009;42:253‑63.

26. Porto IC, Andrade AK, Guênes GM, Ribeiro AI, Braz R, Castro CM. In vitro potential cytotoxicity of an adhesive system to alveolar macrophages. Braz Dent J 2009;20:195‑200.

27. Vajrabhaya LO, Korsuwannawong S, Bosl C, Schmalz G. The cytotoxicity of self‑etching primer bonding agents in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:e86‑90.

28. Angiero F, Farronato G, Dessy E, Magistro S, Seramondi D, Farronato D, et  al. Evaluation of the cytotoxic and genotoxic effects of orthodontic bonding adhesives upon human gingival papillae through immunohistochemical expression of p53, p63 and p16. Anticancer Res 2009;29:3983‑8.

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © Reddy UJ. 2017