Evaluation in vitro of cytotoxicity of dentin desensitizers on human gingival fibroblasts

Cite as: Vergara D, Villegas C, Pavicic M, Fritz M & Ehrenfeld I. Evaluation in vitro of cytotoxicity of dentin desensitizers on human gingival fibroblasts. J Oral Res 2015; 4(1):12-18. Abstract: The purpose of this study is to compare the cytotoxic effect of three materials, which have been used for treating dental hypersensitivity. Material and method: In vitro study. Clinpro (3M Co, St. Paul, MN. USA), Seal & Protect (Dentsply, DeTrey GmbH. Germany) and UltraEZ (Ultradent Products, Inc., S. South Jordan UT. USA) were used at concentrations of 0.1, 0.05, 0.01 and 0.001g/ml on human gingival fibroblasts. Furthermore, Clinpro and Seal & Protect were applied to this cell culture as polymerized disks. Toxicity was assessed at 24 and 48 hours by the use of the cell viability assay (MTT). Statistical analysis for cell viability was performed using two-way ANOVA and Tukey’s post hoc test. Statistical significance was set at 5%. Results: Seal & Protect and Clinpro were found to be highly toxic at 24 and 48 hours, reaching 70% toxicity at concentrations over 0.01g/ml. Seal & Protect and Clinpro polymerized disks were toxic at 24 and 48 hours. UltraEZ showed an increased between 46% and 67% in cell viability at 24 hours and between 8% and 45% at 48 hours. Statistical analysis showed differences between these three desensitizers when comparing concentration and control group (p<0.05). Discussion: UltraEZ did not have a cytotoxic effect and may be considered a compatible and safe material, whereas polymerized and non-polymerized Clinpro and Seal & Protect should be used with caution.


INTRODUCTION.
Dentine hypersensitivity is defined as a short and sharp pain arising after the exposure of dentin in response to external stimuli (osmotic, thermal, chemical or tactile), which cannot be ascribed to any other disease or dental defect 1,2 .This condition affects between 4.1 % and 74% of the population, being more frequent in women between 20 and 49 years old 3 .
Dentine hypersensitivity has been attributed to the stimuli, which act on exposed dentinal tubules 4 .The most effective treatment to eliminate its symptoms is sealing of exposed dentinal tubules 5,6 .This seal has been achieved through the use of desensitizing agents such as toothpastes 7 , fluoridated varnishes 8 and resin or glass ionomer-based agents with high rates of success at the beginning which decrease in time 9,10 .
Resinous dentine desensitizers are composed of glutaraldehyde, 2-hydroxyethyl methacrylate (HEMA), bisp-henol A-glycidyl methacrylate (BIS-GMA), photo-initiators and acetone, which have proven to be toxic in human gingival fibroblasts (HGF) 11,12 .When applying a desensitizing agent to the affected tooth in a cervical area, the contact of these compounds with the gum is inevitable.However, there are no studies indicating the effect of these agents on the periodontium in clinical practice.That is why the cytotoxic effect of desensitizing agents on human gingi-val fibroblasts have been studied, finding a high cytotoxicity and causing reactions in the gingival tissue due to the lack of protection of the gingiva during the application of the agent 13 .The main purpose of this study is to compare the cytotoxic effect of three dentinal desensitizers, Clinpro (3M ESPE), Seal & Protect (Dentsply) and UltraEZ (Ultradent) on cell cultures of human gingival fibroblasts.

Human Gingival Fibroblasts Culture
For this in vitro experimental study, a primary cell culture was performed using healthy gingival tissue associated to an unerupted third molar which was removed for orthodontic reasons from three young patients between 18 and 25 years of age, without use of medications and systemic diseases.Patients accepted the informed consent previously approved by the Ethics Committee of the Faculty of Medicine of the Universidad Austral de Chile (Authorization Number 2101.2014).A layer of gingival epithelial tissue was eliminated from the gingival connective tissue.This connective tissue was divided into 1-2mm fragments with a surgical scalpel.
Unless otherwise specified, the materials used for the preparation of culture medium were from Gibco® (Life Technologies Inc., Grand Island, NY, USA.).The fragments of connective tissue were cultured in 60 mm culture plates in a culture medium supplemented with 10% fetal bovine serum (FBS), 20% penicillin and 70% Dubelco's modified Eagle's medium (DMEM) (HyClone Laboratories Inc., Utah, USA) during three days at 37°C, 95% humidity with 5% CO 2 until a confluent cell monolayer was formed.Once the cells reached the desired confluence, they were recovered through trypsinization method using 0.25% trypsin and 1mm ethylenediaminetetraacetic acid (EDTA) solution.Then, they were placed into 75cm 2 culture flasks and cultured in complete culture medium (86.3% DMEM, 10% SBF, 2mm L-glutamine, 100U/ml penicillin and 100μg/ml streptomycin) at 37°C and 95% humidity and 5% CO 2 until the new formation of confluent cell monolayers.The medium was changed every 24 hours.Two lines of human gingival fibroblasts were obtained from different passages.The cells were stored in liquid nitrogen with 90% FBS and 10% dimethyl sulfoxide (DMSO) (Sigma Aldrich, St Louis, MO, USA.) until the toxicity test.
Materials and study groups.
Three desensitizing agents (Table 1) were used to create fourteen intervention groups and two control groups.Twelve groups corresponded to a solution of each desensitizer in complete culture medium (86.3% of DMEM, 10% FBS, 2mm L-glutamine, 100U/ml penicillin and 100μg/ ml streptomycin), getting four different concentrations per agent (0.1, 0.05, 0.01 and 0.001g/ml).Two other groups corresponded to Clinpro and Seal & Protect polymerized disks.These disks were prepared in 2x3mm metallic matrices and polymerized for 40 seconds with curing light Gnatus Optilight Max (1200mW/cm 2 ) (Gnatus, SP, Brazil).All experimental groups were cultured in the same complete culture medium previously used.A cell group in a culture medium without stimuli was used as negative control group and another with 10% of DMSO as positive control group.Results are presented according to the percentage of cell viability obtained from the negative control.S.D.: Standard Deviation.
a: Indicate significant differences between the experimental groups and negative control (p<0.05).b: Indicates no significant differences between the experimental groups and positive control (p>0.05)

Evidence of cytotoxicity.
Cytotoxicity of the desensitizer was assessed by cell viability using dimethyl-thiazolyl-diphenyl tetrazoliumbromide (MTT) assay, which is converted to insoluble formazan by the action of dehydrogenases enzymes.
HGFs were seeded in 24-well plates at a concentration of 20,000 cells/well, with complete culture medium for 24 hours at 37°C to allow proliferation and cell adhesion.Then, the culture medium was replaced by a new complete culture medium containing the desensitizing concentration and the polymerized disk.During this procedure, the medium was protected from light to prevent polymerization of resin-based agents.The observation and control period for each desensitizer was repeated in triplicate.Each group was observed at 24 and 48 hours when the culture medium was removed and 100μl of MTT (Sigma Aldrich, St. Louis, MO, USA.) were added at a concentration of 0.55mg/ml per well for 4 hours.After completing the period of time, this medium was removed and each plate was washed with phosphate buffered saline (PBS), pH 7.4 (NaCl 124mmol/l, Na 2 HPO 4 10mmol/l and KH-Darmstadt, Germany) in a proportion of 2:10.After 5-10 minutes incubation, optical density (O.D.) was read at a wavelength of 570nm using a spectrophotometer (Bio-Mate TM 3S Waltham, MA USA, Thermo Fisher Scientific Inc.).The percentage of viability was obtained according to the International Organization for Standardization (ISO) for biological evaluation of medical products, MTT cytotoxicity assay.

Statistical analysis
Cell viability was assessed for the different materials, concentrations and times using two-way ANOVA, followed by Tukey tests to establish differences between the groups (GraphPad InStat®, Version 6.0.5.San Diego, CA, USA).The level of statistical significance was set at 5%.

RESULTS.
Clinpro and Seal & Protect dental desensitizers significantly decreased cell viability of human gingival fibroblasts at 24 and 48 hours reaching a toxicity greater than 70% at concentrations higher than 0.01g/ml.Clinpro and Seal & Protect, in its polymerized state, decrease cell viability at 24 hours to 75% and 21%, respectively, while at 48 hours, both desensitizer decreased to 11%.
In contrast, UltraEZ was capable of increasing cell via-disks of the other two desensitizers was not possible.It is not possible to compare UltraEZ with polymerized discs of the other desensitizing agents as it is a gel without the possibility of light or hardening.
UltraEZ is a desensitizer based on two components, potassium nitrate and sodium fluoride.Studies on the cytotoxicity of sodium fluoride at concentrations of 0.0095% and 0.07% have indicated that cell viability decreases to 11% when assessed at 12 hours 13 .Despite having 0.25% sodium fluoride, UltraEZ did not generate cytotoxicity in this study.Duraphat ® , a material which presents 5% sodium fluoride and is indicated for the treatment of dentinal hypersensitivity and dental caries prevention was evaluated in a study by Hoang-Dao et al. 14 along with two other materials, Isodan ® and Shellac F. The toxicity test using MTT assay revealed that Duraphat ® was the least toxic material followed by Shellac F. and Isodan ® , evaluating the same concentrations used in this work for each desensitizer.This shows that cell viability increases when the concentration of the materials is reduced and methacrylate components act as toxic in these cell groups.The increase in cell viability when applying UltraEZ on HGF cannot be explained on the basis of the components listed by its manufacturer.
Camps et al. 15 compared cell viability of resin-based desensitizing agents on L-929 mouse fibroblast cell lines using MTT assay by interpositioning a dentin disk as a barrier between fibroblasts and the desensitizer, concluding that all of the materials used in this study had a low cytotoxicity.In that study, Seal & Protect showed a cell viability of 88% at 48 hours.These data are in contrast with the results of Sengün et al. 16 , who conducted a study of cytotoxicity in human gingival fibroblasts directly exposed to the desensitizers at three concentrations: 0.1, 0.3, and 0.5μl/ml, assessing cell viability through MTT assay at 48 hours, in addition to a count of viable cells at 24 and 48 hours, reporting that all the desensitizers were cytotoxic.In the study by Sengün, Seal & Protect showed a range of cell viability between 40% and 85% at the different concentrations evaluated.The count of viable cells for Seal & Protect was 50% at 24 hours and bility compared to the negative control group (p=0.021).The results for each desensitizer are detailed in the following table (Table 2).
The positive control of 10% DMSO achieved a 3% cell viability at 24 hours, and a 5% at 48 hours.
Statistical analysis shows statistically significant differences for the three desensitizers when comparing concentration v/s negative control group (p<0.05).At concentrations above 0.01g/ml, Clinpro and Seal &Protect did not show statistically significant differences compared with the positive control group at 24 hours (p>0.05).

DISCUSSION.
Assessing toxicity using MTT assay allows comparing cell viability of gingival fibroblasts exposed to different concentrations of desensitizers used in dental practice.Clinpro and Seal & Protect showed a highly cytotoxic behavior on human gingival fibroblasts, indicating that the higher the desensitizer concentration used is, the less cell viability.
At concentrations of 0.05 and 0.01g/ml, cell viability of human gingival fibroblasts treated with Clinpro and Seal & Protect was very low, less than 10% at 24 and 48 hours.Therefore, the highest concentration used for these desensitizers did not show statistically significant differences compared with the positive control (p=0.295).
The values of cell viability of Seal & Protect increase between 24 and 48 hours, while the values of Clinpro continued to decline at 48 hours.Polymerising the material to reduce the release of their components into the culture medium succeeded in reducing cytotoxic effects.However, the decrease in cell viability can be compared at the lowest concentration of each desensitizing used (0.001g/ml) at 24 hours, but it progressed over time and it was about 0.01g/ml at 48 hours.Clinpro presented a higher cell viability in measurement twice than polymerized Seal & Protect.On the contrary, UltraEZ was capable of promoting cell viability between 46% and 67% at 24 hours and between 8% and 45% at 48 hours.Since UltraEZ is a desensitizing gel and cannot use curing light or be set, the comparison with the polymerized 10% at 48 hours, demonstrating a high cytotoxicity at all concentrations evaluated and compared with the results obtained in this research.Seal & Protect has a many components.From among them, the cytotoxicity di and trimethacrylate resins, dipentaerythritol penta acrylate monophosphate 17,18 and triclosan 19 have been studied.Also, its composition is similar to adhesive systems which reported a high cytotoxicity 20 .It has been shown that the combined use of different types of monomers has a synergistic effect in cellular toxicity of human gingival fibroblasts 21 .
The cytotoxicity of Clinpro has not been reported yet.Since it is composed of acrylate copolymers, itaconic acid and 2-hydroxyethyl methacrylate (HEMA), it is considered as a resin-modified glass-ionomer 22 .Studies on resinmodified glass-ionomer cements, have shown to release HEMA in solution for being photopolymerized and even hyperpolymerized 23 .
HEMA is a monomer present in a large number of dental biomaterials 24 .This monomer affects proliferation, apoptosis and cell cycle 25,26 .Aditionally, it has been studied the inflammatory response in gingival fibroblasts, causing an increase in the levels of reactive oxygen species, cyclooxygenase-2, tumor necrosis factor-alpha gene expression and prostaglandin E2 release 27 .
While the clinical behavior of desensitizer on the gingiva cannot be ensured because there are no studies evaluating cellular toxicity in humans, such factors as the clearance from the gingival sulcus and the saliva, which dilutes the concentrations and reduces the lenght of time for contact of these materials with the periodontium, could mitigate the cytotoxic effects of the desensitizers and not generate evident lesions at the clinical level.
It is necessary to carry out studies in three-dimensional oral mucosa cell cultures or in animal to identify toxicity and inflammatory changes through histopathological sections, in order to better quantify the periodontal tissue response to the exposure of these biomaterials.In the absence of this kind of studies, the clinician should be careful when handling these materials in cervical areas, as being in contact with the gum, could generate responses in the periodontium, causing migration of periodontal attachment, exposing dental tissue and producing dentinal hypersensitivity again.
The results of this study indicate that UltraEZ has no cytotoxic effect, but it increases cell viability.However, Seal & Protect and Clinpro, both in their polymerised and non-polymerised form, were highly toxic at the concentrations evaluated in this study.

ACKNOWLEDGMENTS:
This research is based on the thesis of Diego Vergara and Claudio Villegas, which was carried out as a requirement for obtaining the degree of Bachelor in Dentistry at Universidad Austral de Chile in June 2014.Thanks for the financial support from the School of Dentistry and the Research and Development Management of the Universidad Austral de Chile.

Table 2 .
Cytotoxicity of dentin desensitizers on human gingival fibroblasts assessed at 24 and 48 hours.