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ORIGINAL ARTICLE
Year : 2021 | Volume
: 24
| Issue : 3 | Page : 313-320
Comparative evaluation of the cytotoxic effects of different oral antiseptics: A primary culture study
NZ Alpaslan Yayli1, S Keskin Tunc2, B Unalan Degirmenci3, A Dikilitas4, M Taspinar5
1 Department of Periodontology, Faculty of Dentistry, Van Yuzuncu Yil University, Van, Turkey
2 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Van Yuzuncu Yil University, Van, Turkey
3 Department of Prosthodontics, Faculty of Dentistry, Van Yuzuncu Yil University, Van, Turkey
4 Department of Periodontology, Faculty of Dentistry, Usak University, Usak, Turkey
5 Department of Medical Biology, Faculty of Medicine, Aksaray University, Aksaray, Turkey
Correspondence Address:
Dr. N Z Alpaslan Yayli
Department of Periodontology, Faculty of Dentistry, Van Yuzuncu Yil University, 65080, Van
Turkey
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/njcp.njcp_253_20
Abstract
Background: Dental plaque is a major oral health problem with severe consequences. Oral antiseptics provide important means for controlling dental plaque formation and are widely used by the public. However, some of these antiseptics have been shown to have side effects on oral tissues. Aim: In this study, we aimed to investigate the time and dose-dependent cytotoxic effects of various antiseptics on primary human gingival fibroblasts (HGF). Methods: HGF cells were obtained using primary culture techniques. The effects of various doses of 5 antiseptics containing Chlorhexidine-Gluconate (CHX), CHX with Benzydamine-Hydrochloride (Benzydamine-HCl), Povidone-Iodine (PVP-I), Benzydamine-HCl and Essential-Oil on HGFs were analyzed by using 2,3-bis (2-metoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide cell viability assay after 30, 60, and 180 s of exposure. Results: Cell viability analyses showed that cell death increased in an application time and dose-dependent manner. There was a statistically significant difference in the effects of each antiseptic on live-cell densities compared to the control group and each other (P < 0.001). Antiseptic containing 0.2% CHX showed the highest cytotoxicity on cells. The remaining viable cell density after administration of 0.2% CHX at a dose of 12.5% for 30 s is 35.19%. The high cytotoxic effect of 0.2% CHX was followed by 0.12% CHX with 0.15% Benzydamine-HCl, PVP-I and 0.15% Benzydamine-HCl groups. The lowest cytotoxic effect was observed for the Essential-Oil containing antiseptic solution. Conclusions: The results of this study show that these five antiseptic agents have variable effects on in vitro HGF proliferation. The doses and administration times of antiseptics should be controlled carefully during dental applications.
Keywords: Benzydamine hydrochloride, chlorhexidine, essential oil, cytotoxicity, in vitro
Introduction 
Dental plaque is the most important etiological factor for oral diseases such as dental caries and gingival diseases and has been reported to also affect systemic health including cardiovascular and nervous systems.[1] Plaque formation can be controlled by following dental hygiene practices and through mechanical treatments performed by dentists. However, in several cases such as the presence of deep periodontal pockets and furcation areas that are difficult to reach, after periodontal surgery and in cases where intermaxillary fixation is performed, mechanical approaches may not be sufficient to eliminate the pathogens.[1],[2] In such cases, the use of antimicrobial agents in addition to mechanical treatment has been suggested.[3] These antimicrobial agents include local or systemic antibiotics and antiseptic solutions with antibacterial effects.
Oral antiseptics are generally utilized to reduce the microbial load in the oral cavity and to control bad breath. Due to their antimicrobial activity, they are also recommended to be used for the removal of supragingival plaque and treatment of gingivitis, prior to various oral and periodontal surgical procedures and during the postoperative maintenance phase. They usually contain chlorhexidine gluconate, ethanol, essential oils, and detergents as standard active ingredients.[4],[5]
In addition to having antibacterial activity, a safe antiseptic solution should not damage the tissues even during long-term treatments, not trigger any allergic conditions and not be absorbed through the oral mucosa.[6]In vitro cytotoxicity assays and cell culture techniques are widely utilized for analyzing the biocompatibility of different materials due to their advantages of being economical and repeatability of measurements as biological safety standards.[7] The use of cytotoxicity assays together with primary culture techniques is more informative compared to immortalized cell lines because primary cells do not have the genomic alterations that are present in almost all of the immortal cell lines that are mostly derived from either tumor tissues or are altered by viruses. Thus, primary cell lines can better represent the biological conditions that can be detected in vivo, such as stress response.[8]
Antiseptic agents undergo toxicity tests before entering the market, but various side effects that are clinically observable can be detected after prolonged use.[9],[10] It also appears that patients respond differently to different antiseptics. Some of the ingredients used in these agents may cause tissue irritation,[5] and various studies reported conflicting results regarding the effects of antiseptics on wound healing.[11] The association between antiseptic mouthwash and the risk of oral cancer development was also discussed in the literature.[12] Recently, the theory of stem cell division in cancer development has been proposed, which asserts that the duration and frequency of tissue contact with a cytotoxic agent may affect the risk of oral cancer development.[13],[14] These findings clearly show that the toxic effects of these oral antiseptic agents, which are frequently administered by both patients themselves and physicians in routine use, need to be carefully reassessed.
Studies on cytotoxicity assessments of oral antiseptics have generally focused on preparations containing chlorhexidine gluconate,[6],[15],[16],[17] and there are contradicting opinions regarding the toxic effects of chemicals containing povidone-iodine and essential oils.[5],[11],[17],[18],[19] In addition, sufficient data on the cytotoxicity of antiseptics containing benzydamine hydrochloride is still not available. Thus, the aim of this study was to investigate the cytotoxic effects of five different antiseptics containing different active ingredients (Chlorhexidine Gluconate, Benzydamine Hydrochloride, Povidone Iodine, and Essential Oil), which are frequently used in dental practice, on primary human gingival fibroblast (HGF) cells, with respect to application duration and dose.
Materials and Methods Solutions used
Growth medium consisted of Dulbecco's Modified Eagle's Medium (DMEM) High Glucose, 10% Fetal Bovine Serum (FBS), 1% L-Glutamine, and 1% Penicillin-Streptomycin. DMEM, L-Glutamine, Penicillin-Streptomycin, and Trypsin-EDTA were purchased from Capricorn Scientific®, Ebsdorfergrund, Germany. FBS, Phosphate Buffered Saline (PBS), 2,3-bis (2-metoxy-4-nitro-5-sulfophenyl)-5-
[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), 0.2% Chlorhexidine Gluconate (CHX), 0.12% CHX + 0.15% Benzydamine Hydrochloride (Benzydamine HCl), Povidone Iodine (PVP-I), only 0.15% Benzydamine HCl and Essential Oil were purchased from Biological Industries® (USA), Sigma® (Germany), Biotium® (USA), Klorhex® Mouthwash (Drogsan®, Turkey), Kloroben® Mouthwash (Drogsan®, Turkey), Batticon®Solution (Adeka®, Turkey), Tanflex® Mouthwash (Abdi Ibrahim®, Turkey), and Listerine® Cool Mint Oral Care Product (Johnson and Johnson®, Turkey), respectively.
Primary culture
This experimental controlled study was approved by the Ethics Committee of the Faculty of Medicine at Van Yuzuncu Yil University, Turkey (YYU-02-21.09.2017). HGF cells were obtained from a systemically healthy 25-year-old female donor who applied to Van Yuzuncu Yil University, Faculty of Dentistry, Department of Orthodontics with a complaint of malocclusion, had an indication of tooth extraction for orthodontic treatment and no gingivitis or gingival hyperplasia in her gums. Informed consent was obtained from the individual for all procedures. After the tooth was extracted, the gingival tissues were carefully collected from the neck of the tooth and then divided into small pieces in the cell culture laboratory and washed several times with growth medium. After washing, tissue pieces were transferred into a 25 cm2 flask containing 1 mL of medium and cultured for 24 h at 37°C, in a humidified chamber with 5% CO2. After surface attachment, the tissues were washed every 2–3 days with 1 mL of growth medium. Following the 8th-10th days of incubation, cells washed with PBS were grown under normal growth conditions. When the cells reached 70% confluency, they were trypsinized with 0.25% Trypsin-EDTA and passaged. All cytotoxicity tests were performed on cells at passage 3.
XTT cytotoxicity test
Prior to cytotoxicity testing, 8 × 103 cells were seeded into each well of the 96-well plates and incubated overnight at 37°C, in a humidified chamber containing 5% CO2. The antiseptics to be applied were filtered and prepared in 6 different doses as 0.5%, 2.5%, 12.5%, 25%, 50%, and 100% in growth medium. Each of the prepared antiseptic doses was administered to the cells for 3 different durations, 30 s, 60 s, and 180 s. Only medium was added to the control groups, where no antiseptic was applied. Antiseptic solution types, their application durations and doses in the experimental groups are shown in [Table 1]. At the end of the indicated periods, the media containing antiseptics were removed and each well was washed with PBS. Afterward, growth medium was added into each well and the cells were incubated overnight in a humidified chamber at 37°C with 5% CO2. At the end of the incubation, XTT assay was performed according to the manufacturer's instructions.
Table 1: Antiseptic solution types, their application durations and doses in the experimental groups35 μL of XTT solution was added into each well of the plate, and incubation was performed for 5 h at 37° C in a humidified chamber containing 5% CO2. At the end of the experiment, absorbance values were measured at 450 nm with a microplate reader. All tests were repeated at least 3 times. The degree of cytotoxicity was determined according to the classification made by Sletten & Dahl (in case of a drug application, when cell viability is less than 30%, between 30% and 60% and more than 60% with respect to controls, it is severely, moderately or mildly cytotoxic, respectively).[15],[20]
Statistical analysis
For the statistical analysis, NCSS (Number Cruncher Statistical System) 2007 Statistical Software (NCSS LLC, Kaysville, Utah, USA) program was used. While evaluating the data, GLMM (General Linear Mixed Model) analysis was performed in order to investigate the effects of antiseptic type, dose, and application duration variables on live-cell density measurements for the comparison of quantitative data as well as descriptive statistical methods (mean, standard deviation, median, frequency, and ratio). Post-hoc evaluations were performed with the corrected Bonferroni test. The results were expressed in a 95% confidence interval and P < 0.05 significance level.
Results The live-cell density measurements following dose and duration dependent administration of antiseptics are shown in [Figure 1],[Figure 2],[Figure 3]. After 30 s of incubation, the highest cell viability was observed in the control group where no antiseptic solution was applied, which was 2.11 ± 0.01. The absorbance value obtained when 0.2% CHX was applied at a dose of 2.5% was 1.88 ± 0.05, and when it was applied at 12.5%, it was 0.74 ± 0.01. Following the application of only 0.15% Benzydamine HCl at 100% dose, the remaining live cell density was 1.85 ± 0.04, while the application of 0.12% CHX + 0.15% Benzydamine HCl at 12.5% dose resulted in the remaining viable cell density with an absorbance of 1.74 ± 0.01 and 0.54 ± 0.02 after application of 100% dose. The absorbance value measured after the application of 100% Essential Oil was 1.95 ± 0.06, and the measured absorbance value after the application of PVP-I at 100% dose was 1.80 ± 0.04. The remaining viable cell densities after 30 seconds of antiseptic administration at all doses are shown in [Figure 1].
Figure 1: Viable cell density following 30 sec application of the antiseptic solutions on HGF cells at different doses
Figure 2: Viable cell density following 60 sec application of the antiseptic solutions on HGF cells at different doses
Figure 3: Viable cell density following 180 sec application of the antiseptic solutions on HGF cells at different dosesViable cell density following 60 s application of the antiseptic solutions on HGF cells at different doses is shown in [Figure 2]. The absorbance value measured after application of 0.2% CHX at 25% dose was 0.47 ± 0.03, and after applying only 0.15% Benzydamine HCl at 100% dose, it was 1.85 ± 0.05. Following the application of 0.12% CHX + 0.15% Benzydamine HCl at a dose of 50%, absorbance values measured for analyzing live cell density was 0.95 ± 0.15. When Essential Oil was applied at 100% dose, this absorbance value was 1.87 ± 0.01 and when PVP-I was applied at 100% dose, it was 1.79 ± 0.05.
Viable cell density following 180 s application of the antiseptic solutions on HGF cells at different doses is shown in [Figure 3]. When applications at 100% dose were considered, the absorbance values measured for 0.2% CHX, only 0.15% Benzydamine HCl, 0.12% CHX + 0.15% Benzydamine HCl, Essential Oil, and PVP-I were 0.36 ± 0.03, 1.83 ± 0.02, 0.52 ± 0.06, 1.85 ± 0.03, and 1.72 ± 0.02, respectively.
GLMM analysis was carried out to examine the effects of antiseptic, dose, and time of administration variables on live cell density. While antiseptic, dose, and time were added to the model as independent variables, live-cell density variable was added as a dependent variable. In the model, the effects of antiseptic, dose and time variables alone were found statistically significant (P < 0.01). When the interactions between variables were investigated, only antiseptic type-dose interactions were significant (P < 0.001), while antiseptic type-time, dose-time, and antiseptic type-dose-time interactions were statistically insignificant (P = 0.115, P = 0.659, P = 0.996, respectively).
When the live-cell densities (absorbance values) of each antiseptic compared to the control group and each other were examined independent of application duration and dose, the difference between all antiseptic types was found to be statistically significant (P < 0.001). The highest viable cell density was observed in the control group, to which no antiseptic treatment was applied. This was followed by antiseptics containing Essential Oil, only 0.15% Benzydamine HCl, PVP-I and 0.12% CHX + 0.15% Benzydamine HCl as the active ingredient, while the lowest live-cell density was observed in the samples on which 0.2% CHX was applied.
Regardless of antiseptic type and application duration, there was a significant difference in dose-dependent cell death (absorbance values) in each group (P < 0.001). The highest viable cell density was observed in the 0.5% dose-treated groups; this was followed by 2.5% dose, 12.5% dose, 25% dose and 50% dose, and the lowest viable cell densities were determined in groups where antiseptics were administered at 100% dose.
Evaluation of the importance of application durations between treatment groups (regardless of antiseptic type and dose) revealed that the highest live-cell density (absorbance values) was obtained in measurements taken after 30 s of contact, followed by 60 s, and 180 s and there was a statistically significant difference among each duration period (P = 0.029 between 30 and 60 s, P = 0.001 between 30 and 180 s and P = 0.001 between 60 and180 s).
When the antiseptic type–dose interaction was examined independent of time, a statistically significant difference was found between the control groups and antiseptics in terms of cell viability in all the data evaluated when the antiseptic was applied at a dose of 12.5% or more. When 2.5% dose was administered, there was no statistically significant difference in the number of live HGF cells between groups treated with 0.2% CHX and 0.12% CHX + 0.15% Benzydamine HCl and between groups receiving only 0.15% Benzydamine HCl and PVP-I (p > 0.05). For the 0.5% dose applications, the only statistically significant difference in terms of live cell density was between 0.2% CHX group and the other groups (P < 0.001) [Table 2].
Table 2: Viable cell densities with respect to antiseptic type and dose interaction, independent of the application duration variableThe study results showed that the antiseptic solution containing 0.2% CHX as the active ingredient is the antiseptic with the highest cytotoxicity to HGF cells at all durations and all doses. The remaining viable cell density after administration of this antiseptic at a dose of 12.5% for 30 s is 35.19%. While the second most cytotoxic antiseptic solution was 0.12% CHX + 0.15% Benzydamine HCl, antiseptic solution containing essential oil as the active ingredient was the agent with the lowest cytotoxicity.
Discussion This is an experimental, controlled, in vitro study evaluating the cytotoxic effects of various antiseptics containing different active components on HGF cells. To the best of our knowledge, no other study has evaluated the cytotoxic effects of all of the above-mentioned antiseptics on primary HGF cell culture comparatively with respect to dose and application duration.
Generally, in vitro cytotoxicity tests are performed on fibroblasts and epithelial cells.[6],[15],[16] Various chemical and environment factors may affect the proliferation of the epithelial cells, which in turn causes the epithelium to be thinner or become more prone to ulcer. In these conditions, epithelium with increased permeability becomes more sensitive to toxic agents.[21] Thus, the connective tissue microenvironment in the deeper layers may be disrupted and connective tissue cells, especially fibroblasts, may be harmed. Similarly, the connective tissue harboring fibroblast cells remains exposed to the oral environment until re-epithelialization occurs at the wound site formed after various periodontal treatments and oral surgical procedures. For these reasons, fibroblast cells were used to evaluate the cytotoxic effects of antiseptics in this study. The viability of HGF cells exposed to antiseptics was evaluated by XTT assay, which is a colorimetric, fast and alternative method, with high reliability.[22]
Antiseptics may have cytotoxic effects depending on their active ingredients.[5],[15],[16] Therefore, different active ingredients that are used in antiseptic dental solutions were selected for the present study. The contact durations of the antiseptics were determined by considering the manufacturers' application instructions. Accordingly, the shortest application time was selected as 30 s and the cytotoxic effect was also evaluated after longer periods of application due to the possibility of the antiseptic being kept in the mouth for a longer period.[16] The toxic effects of antiseptics were also examined by using various doses based on the information that is available in the literature,[13],[17],[18] to determine the doses which show strong toxicity in vitro and to provide data for routine use.
Our results showed that the antiseptic type, application duration and dose variables had significant effects on cytotoxicity independently, but antiseptic-dose interaction was significant only in multiple interactions. This result is consistent with the studies in the literature.[17],[18] When the antiseptic types were compared, it was determined that the antiseptic solution containing 0.2% CHX was the most cytotoxic antiseptic against HGF cells compared to the control group, which was not exposed to any antiseptic, after all application durations and at all doses. It is striking that the cytotoxic effect was observed even at much lower doses than the doses usually applied in clinical use. The high cytotoxic effect of 0.2% CHX was followed by 0.12% CHX + 0.15% Benzydamine HCl, PVP-I and only 0.15% Benzydamine HCl groups. The lowest cytotoxic effect was observed for the Essential Oil containing antiseptic solution.
With its wide spectrum antibacterial activity, CHX is considered the gold standard in dentistry among all antiseptics.[23] This antiseptic adheres to hard and soft tissues for a long time and is slowly released at an effective dose, which helps to maintain its antimicrobial effect for longer durations.[24] According to the results of the present study, exposure to 0.2% CHX at a dose of 12.5% and above causes severe cell death in HGF cells. This indicates the importance of the type of the antiseptic ingredient and its dose in cell death. These results are consistent with several studies in the literature.[5],[16],[17] Some studies clinically confirm the toxic effects of CHX at the cellular level.[10] The determination of severe cytotoxicity in the groups containing the combination of 0.12% CHX and 0.15% Benzydamine HCl suggests that these results may be mostly related to the CHX active substance, because the cytotoxic effect of the other groups containing only 0.15% Benzydamine HCl were classified as mild according to Sletten and Dahl.[15],[20]
Benzydamine HCl is a non-steroidal anti-inflammatory anesthetic with antimicrobial and antifungal properties.[25] Since there are limited data on the cytotoxicity of Benzydamine HCl in the literature, our study results could not be compared. Nevertheless, the data obtained in our study can be used for comparison for subsequent studies. The fact that Benzydamine HCl is an antibacterial agent with low cytotoxic effects on fibroblasts suggests that it can be used safely to achieve faster wound healing for conditions such as post-operative period.
In this study, it was observed that the cytotoxic effect of the antiseptic solution containing PVP-I as the active ingredient was lower than the CHX containing antiseptics and was included in the group causing the mild cytotoxicity[15],[20] compared to the control group, at the highest dose and the longest contact time. PVP-I is a water-soluble complex, made up of polyvinylpyrrolidone and iodine that exhibits antibacterial characteristics of iodine while stains and irritations caused by iodine are minimized.[26] Although PVP-I has been used in the treatment of wounds for a long time, there are conflicting results in the literature regarding its effects on wound healing.[11],[27] This may be due to the type of the cell line used, the passage stages, the technique used and the time of application.
According to the results of the present study, the antiseptic solution with the least cytotoxic effects compared to the control group is the antiseptic solution containing Essential Oil at all doses and all application durations. There are studies in the literature that are in contradiction with the findings of our study.[18],[28] Essential Oils are complicated mixtures of the volatile hydrophobic substances obtained from secondary plant metabolites. The antibacterial effects of Essential Oils are generally related to the changes in the permeability and integrity of the bacterial cell membrane.[29] Although these agents have anti-plaque and anti-gingivitis effects, they contain ethyl alcohol that causes acetaldehyde accumulation in oral tissues.[13] Our data indicate that the antiseptic solution containing Essential Oil, which we examined in our study, may be cytostatic rather than cytotoxic.
Nowadays studies have focused on whether there is any association between the development of oral cancer and daily antiseptic mouthwash use. Recently, López-Lázaro came up with the theory of stem cell division in cancer development,[13],[14] where he emphasizes that the orderly consumption of alcoholic beverages that include cytotoxic doses of ethanol will decrease the life of some cells which cover oral cavity, pharynx and esophagus and the stem cells in the deeper layers will be forced to divide more often. He also states that when stem cells are divided to replace the damaged cells, cancer causing mutations also occur which might cause the formation of cancerous regions in self-regenerating tissues.[14] Antiseptics and similar chemical agents may disrupt the microenvironment of the tissue, start the uncontrollable differentiation process and may result in the transformation of the fibroblast cells to cancerous cells. Due to all these reasons, it was important to evaluate the cytotoxic effects of oral antiseptics, which both individuals routinely use and frequently recommended as a part of the treatment applied by dentists.
One of the limitations of our study was that the antibacterial effects of the antiseptic solutions on oral pathogens have not been investigated. Therefore, options for a holistic assessment and determination of biocompatibility index are limited. However, it has been proven in the literature that each of the antiseptics included in the study, has sufficient antibacterial activity,[4],[25],[26],[29] while there is a lack of evidence of their cytotoxic effects on HGF cells. Another limitation was that different age groups and different genetic characteristics could not be evaluated because HGF cells were obtained from a single donor. Furthermore, since the study was performed in mono-layer cells, it is difficult to fully reflect the results of a complex system involving fibroblasts, multilayered epithelial cells and defense cells. In the oral cavity, the cells are not only interconnected, but are also affected by the internal environment of the oral cavity, and this cannot be simulated by in vitro cell culture.
This study focused on the determination of possible cytotoxic effects of these agents and the cytotoxicity levels were found to be higher than expected. To provide more accurate data, these tests should be performed in individual organoid models, since the genetic structure of the individuals may be one of the main reasons for the different response to these antiseptics in the clinic. Therefore, in the future, antiseptic use may be decided through individual organoid based treatment protocols.
Conclusion The results obtained in our study show that these five antiseptic agents have variable effects on in vitro HGF proliferation. Cell death increased in an application time and dose-dependent manner. The highest cytotoxic effects were seen in CHX containing groups. In the groups containing only Benzydamine HCl and Essential Oil, cell death was relatively lower and mild cytotoxic effects were observed. The dose and duration of administration of the antimicrobial agents should be carefully controlled for optimal results. Daily routine use of antiseptics with proven cytotoxic effects should be carefully discussed. In addition, clinicians should be aware of the potential adverse effects of antiseptics and warn their patients against the misuse of these products. These regimens should be considered when interventional surgical procedures are planned because the antiseptics come into contact with the connective tissue, which may lead to significant effects on wound healing. Furthermore, the possible risk of oral cancer development should not be overlooked as long-term and routine use of antiseptic may lead to the need for continuous renewal of fibroblasts. An antiseptic agent that is not harmful against body cells needs to be developed.
Acknowledgements
This study was funded by the authors and Van Yuzuncu Yil University, Turkey (Project No: THD-2018-6452). The authors would like to thank the Director of the Scientific Research Projects of Van Yuzuncu Yil University for their support.
Ethical approval
All procedures performed in studies involving human participant were in accordance with the ethical standards of the Committee of the Faculty of Medicine at Van Yuzuncu Yil University (YYU-02-21.09.2017) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent
Informed consent was obtained from the participant included in the study.
Financial support and sponsorship
This study was funded by the authors and Van Yuzuncu Yil University, Turkey (Project No: THD-2018-6452).
Conflicts of interest
There are no conflicts of interest.
References 
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