In Vitro Evaluation and Comparison of the Effect of Two Commercially Available Mouthwashes on Fibroblast Survival and Migration

Introduction

Periodontal disease is characterized by the destruction of the periodontal ligament and alveolar bone with subsequent clinical attachment loss caused by the response of susceptible host tissue to the microbial biofilm present within the oral cavity.¹ The optimal result was observed in the non-surgical elimination of plaque from periodontal pockets in combination with chemical prophylaxis.² Therefore, various antimicrobial mouth rinses are being used to prevent and treat periodontal disease.

Chlorhexidine (CHX) has been widely used in dentistry in mouthwash form. Indicated mainly for the treatment and maintenance of periodontal diseases and postoperative care, and are often referred to as the gold standard of anti-plaque agents.³ It is a bisbiguanide antiseptic, its use at a 0.12% concentration is indicated and approved between dental visits as part of a professional program in the management of gingivitis, in addition to scaling and oral hygiene instructions.⁴ Depending on its concentration, it has both bacteriostatic and bactericidal mechanisms of action. At lower concentrations, it alters the bacterial osmotic balance, releasing cytoplasmic components and thus promoting its bacteriostatic action. At high concentrations, CHX promotes the precipitation/coagulation of cytoplasmic proteins, which promotes its bactericidal action.³ It demonstrates a higher substantivity on the negatively charged surfaces within the oral cavity. Although the efficacy of CHX as an antiseptic is proven, its effect on tissues, particularly during the healing process, has been questioned.

Essential oil-containing (EO) mouthwash is the most prescribed over-the-counter mouthwash after CHX.⁴ It is a combination of phenol-related essential oils, including thymol (0.060%), eucalyptol (0.091%), menthol (0.042%), and methyl-salicylate (0.064%) in a 26.9% hydroalcoholic vehicle. EO is a first-generation antiseptic and does not have as much substantivity as CHX. Its mode of action is through bacterial membrane damage, as well as inhibiting bacterial enzyme action. EO exert antimicrobial activity against various oral pathogens, reduce plaque biofilm accumulation and present anti-inflammatory and prostaglandin synthetase inhibitor activity which can occur at concentrations lower than that needed for antibacterial activity.⁴

Fibroblasts are the predominant cell types in connective tissues of the periodontium. They play an important role in tissue repair through their proliferation, synthesizing extracellular matrix (ECM), and control of collagen deposition at the wound site. They play a vital role in the maintenance of healthy periodontium, synthesize, and maintain a heterogeneous group of connective matrices throughout the periodontium, and exhibit motility and contractility functions, which help shape structural tissue organization during regeneration and development. Fibroblast migration is essential for normal wound healing.^2,5 Wound healing is a dynamic process involving the coordinated action of both resident and migratory cell populations within the extracellular matrix (ECM) and cytokines. Synthesis of ECM by fibroblasts at the wound site is an intricate process that depends on the contents of the matrix within the wound, such as type I, III, and V collagen, fibronectin, glycosaminoglycans, and various cytokines/growth factors produced by inflammatory cells, keratinocytes, and fibroblasts themselves.²

Since there are very few studies that evaluate the effects of essential oil-containing mouthwashes on fibroblast cell survival and migration, the purpose of this study was to evaluate and compare in vitro, the effects of two mouthwashes that are commercially available, on the survival and migratory capacity of human gingival fibroblasts, to assess potential differences in wound healing.

Materials and Methods

Source of data

Human gingival fibroblasts were obtained from the repository of Maratha Mandal’s Nathajirao Halgekar Institute of Dental Science and Research Centre, Belagavi, India. Samples were analysed at the Central Research Laboratory at Maratha Mandal’s N.G.H Institute of Dental Science and Research Centre, Belagavi.

Method of collection of data

The present study focused on two commercially available mouthwashes, chlorhexidine 0.12% (CHX) and essential oils containing mouthwash (EO). The viability of fibroblasts following the use of six different concentrations of CHX and EO mouthwash treatment (100%, 50%, 25%, 12.5%, 6.25%, 3.125%) was assessed using MTT Assay, followed by assessment of cell migration by performing the Cell migration assay, by using those two concentrations of CHX and EO, in which lesser reduction in viability of fibroblasts was observed.

Following were the experimental and control groups:

GROUP Ⅰ - Fibroblasts without any treatment (Control-DMEM media)

GROUP Ⅱ - Fibroblasts treated with chlorhexidine (Peridex-0.12%)

GROUP Ⅲ - Fibroblasts treated with essential oil containing mouthwash (Listerine cool mint)

Evaluation of cell survival using MTT

assay MTT assay was carried out to evaluate the viability of fibroblasts following CHX and EO-containing mouthwash treatment.

The procedure of MTT assay

The human gingival fibroblast cells were allowed to grow in a Dulbecco's Modified Eagle's Medium (DMEM) medium consisting of 10% Fetal Bovine Serum (FBS) and 1% Antibiotic–Antimycotic 100 × solution as a supplement. Approximately 5×10³ cells were seeded in a 96-well microtiter plate and maintained at 37°C in 95% humidity and 5% CO₂ overnight. The cells were then treated with different concentrations of CHX and EO for 60 s and further incubated for another 72 h. The wells were washed twice with phosphate buffered saline (PBS). 20 µL of the MTT reagent solution 5 mg/ mL of PBS was supplemented in each well and the plate was incubated for 4 h in the dark. When the formazan crystals were formed, they were dissolved by the addition of 100 µL dimethyl sulfoxide (DMSO) and by using a microplate reader, absorbance was recorded at 570 nm (Bio-Rad, California, USA).⁶

Evaluation of cell migration (Cell migration assay)

The concentrations of the mouthwashes that showed less reduction in the viability of fibroblasts in the MTT assay were selected and used to evaluate cell migration using the cell migration assay.

The procedure of cell migration assay (Figure 1)

Around 5×10³ cells were seeded in a 24-well microtiter plate and maintained at 37°C in 95% humidity and 5% CO₂ overnight. The cells were treated with CHX and EO for 60 s. Cells were then rinsed thrice with DMEM and allowed to recover for 24 hours in DMEM containing 10% FBS. By using the tip of the P200 pipette, a 250-micron scratch was made within each well that resembled an incision line. Non-adherent cells were removed and fresh DMEM was added. Cells were allowed to fill in the gap for three days, stained with acridine orange, and photographed with an inverted microscope. For each sample, three measurements were taken perpendicular to the length of the scratch. Later, the mean and standard deviation of three measurements were acquired for each condition.⁴

Statistical analysis

• Intergroup comparison (2 groups) was done using a t-test.
• Intergroup comparison (>2 groups) was done using one-way ANOVA, followed by pairwise comparison using a post hoc test.

For all the statistical tests, P <0.05 was statistically significant, keeping α error at 5% and β error at 20%, thus giving power to the study as 80%.

* = statistically significant difference (P <0.05)

** = statistically highly significant difference (P <0.01)

# = non-significant difference (P >0.05) … for all tables

Results

Following the assessment of cell viability using six different concentrations of CHX and EO mouthwash treatment (100%, 50%, 25%, 12.5%, 6.25%, 3.125%) through the process of MTT assay, assessment of cell migration was done by using those two concentrations of CHX and EO in which lesser reduction in viability of fibroblasts was observed. Therefore 25% and 6.25% concentrations of CHX and EO were chosen to assess cell migration.

The mean viability of cells after treatment with six different concentrations of CHX and EO mouthwashes is tabulated in Table 1. As the concentration of CHX and EO in Group 2 and Group 3 decreased, the viability of fibroblasts increased.

Figure 1a shows the mean viability of fibroblasts after treatment with six different concentrations of CHX mouthwashes. As the concentration of CHX decreased, the mean viability of fibroblasts increased. CHX at 100% concentration showed mean cell survival of 21.19% which was the lowest, while the highest % of mean cell survival of 113.90% was observed with 3.125% of CHX.

Figure 1b depicts the mean viability of fibroblasts following treatment with six different concentrations of EO mouthwashes. As the concentration of EO decreased, the mean viability of fibroblasts increased. EO mouthwash at 100% concentration showed mean cell survival of 20.78% which was the least, while the highest % of mean cell survival of 117.81% was observed with 3.125% of EO mouthwash.

Table 2 and Figure 2 represent the mean scratch width of the control group, CHX-treated group, and EO-treated group in nanometres.

Table 3 and Figure 3a represent the intergroup comparison of cell survival between all three groups (control group, CHX treated, and EO treated groups) using one way ANOVA test.

No statistically significant difference was seen for the values between all three groups (P >0.05).

Table 4 and Figure 3b represent the intergroup comparison of cell migration between all three groups using one way ANOVA test. A statistically highly significant difference was seen for the values between all three groups (P <0.01).

Discussion

In several inaccessible areas like deep periodontal pockets after periodontal surgery, mechanical approaches may not be sufficient to eliminate the pathogens.^7,8 The goal of periodontal treatment is to halt the bacteria/host‑induced inflammation and provide a healthy periodontium. Nonsurgical/ surgical therapy has been the mainstay of periodontal disease management, and plaque control is considered the cornerstone in preventing this chronic disease.⁹ In such cases, the use of antimicrobial agents in addition to mechanical treatment has been suggested._7,10

These antimicrobial agents include local or systemic antibiotics and antiseptic solutions with antibacterial effects. Due to their antimicrobial activity, they are recommended to be used for the elimination 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 and essential oils.^7,11,12

Chlorhexidine gluconate efficiently eliminates Gram-positive and Gram-negative bacteria, their vegetative forms, yeasts, fungi, protozoa as well as viruses. It modulates the metabolism of Candida, a fungi, and promotes reduction of their pathogenicity.¹³

Essential oil containing mouthwash contains a fixed combination of four essential oils as active ingredients (thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042) and kills microorganisms by disrupting their cell walls and inhibiting enzymatic activity.¹⁴

Postoperative pain and sensitivity are the main reasons why chemical plaque control is the most preferred method of oral hygiene maintenance following periodontal surgery. Both CHX and EO-containing mouthwashes are proven to reduce oral bacterial survival and inhibit gingival inflammation and plaque accumulation.¹⁵

However, there are concerns that these products are harmful to oral cells including gingival fibroblasts.¹⁶

Fibroblasts are essential to the wound-healing process.¹⁷

They are considered the main architect-builders of periodontal tissue. The effect of mouthwashes on growth, survival, migration, and proliferation, should be thoroughly assessed when they are used as an addition to mechanical periodontal therapy.⁹

Very few studies in the literature have assessed the migration capacity of human gingival fibroblasts following CHX and EO treatment. Hence in the present study, the effects of two types of mouthwashes on survival and migration of gingival fibroblast were determined and compared between the groups.

Chlorhexidine is found to be toxic to mammalian cells at lower concentrations than that used in the oral cavity.¹⁸ Several authors associate chlorhexidine with cytotoxic effects on gingival epithelial cells, on cells from the periodontal ligament, on keratinocytes, macrophages, osteoblasts, and osteoclasts.^19-25 It was also reported that chlorhexidine causes erythrocytes and neutrophil lysis, inhibits the protein synthesis in fibroblasts.^26-28 CHX is toxic, even in low concentrations, for different cell types including fibroblasts in culture.^29,30

Our findings on CHX support those of previous in vitro studies. Similar to our findings, Marzena Wyganowska-Swiatkowska et al. (2016), Abdel-Rahman Youssef et al. (2020), Sangeetha Kolathuparambil Sukumaran (2021) and many others in their studies proved the in vitro cytotoxicity of CHX on human gingival fibroblasts.^2,31,32,9

The findings of our study agree with that of Jeffery J. Pucher et al. (l993), who used three concentrations of CHX, i.e., 0.002%, 0.005% for 1 hour, and 0.12% for 30 seconds, and found that the lowest % of viable cells were seen in 0.12% CHX treated group, even though they were treated just for 30 seconds, and the maximum% of viable cells were found in 0.02% CHX treated cells. This implies that with the rise in the dosage of CHX, the % of viable cells decreased, as observed in our study.²⁷

Similar results were also reported in the study by Emma P et al. (2007) where 1%, 2%, 5%, 10%, 20%, and 100% CHX mouthwash were used for 1, 5, and 15 min, and a direct correlation between the concentrations of CHX and the fibroblast cell death was observed.⁵

The comparison and analysis of the results from studies on the effects of CHX on periodontal tissues is complicated and practically impossible due to different research methodologies applied by different authors. In particular, the duration of cell exposure, the CHX concentrations and the media used varied.³¹

Essential oil decreases the chances of infection with Herpes viruses HHV-1 or HHV-2 and the influenza virus by around 100%.³³ Essential oils also inhibit the aggregation and multiplication of bacteria.³⁴ EO in this study also showed a reduction in cell viability, which supports the findings of previous study by Matheus Melo Pithon et al.³⁵ As there is an increase in the concentration of EO, the viability of cells decreases. This finding was similar to previous reports by Emmadi et al. (2007) and many others.^5,2

In the present study, the comparison between the test groups and control group was done and no statistically significant difference (P >0.05) was seen in the viability of gingival fibroblasts compared to the negative control group, which was contrary to the reports of Jonatas Rafael de Oliveira et al. (2018), who observed a statistically significant difference between the control group and the CHX and EO treated groups.³⁶

In our study, the comparison of viability of cells was done between CHX and EO treated groups, and no significant difference between the groups (P >0.05) was noted. This finding was not in agreement with the findings reported by Emmadi P et al. (2007) who observed that 1%, 2%, and 5% concentrations of EO showed more reduction in cell viability than CHX, whereas 10%, 20%, and 100% EO showed a lower reduction in viability than CHX. Thus, at lower concentrations, EO was more cytotoxic than CHX, whereas at higher concentrations, CHX was more cytotoxic than EO.⁵ Nevertheless, contradicting results were also reported by Shaimaa Ali Hamouda Ali El Basuony et al. (2018) and Nazli Zeynep Alpaslan Yayli et al. (2021) who reported that CHX demonstrated more cytotoxic effect when compared to EO, unlike the results of our study where no statistically significant difference was observed between CHX and EO treated groups.^16,7

For success in periodontal treatment and to obtain expected regeneration, it is necessary that the site contains viable and healthy cells of the periodontium and these cells must adhere to the root surfaces of the teeth. When mouthwash is used as an adjunct to mechanical therapy or as a subgingival irrigant, it should not produce any harmful effect on the fibroblasts.

Hence to evaluate the effect of CHX and EO mouth-washes on the migration of human gingival fibroblasts (HGFs), the cell migration assay was carried out in the present study, and our findings are in accordance with the study conducted by Ugur Mercan et al. (2016),  where they observed inhibition of cell migration in a cell migration assay model following CHX treatment,¹⁷ and Peng Zhou1et al. (2021) also observed inhibition of cell migration in a cell migration assay model following the use of 10% CHX and 10% EO.³⁷

In a study by Ioannis Tsourounakis (2012), similar to our study, cell viability and migration capacity were assessed following CHX and EO exposure to fibroblasts, and the results of cell viability were contrary to ours with CHX showing a more toxic effect when compared to EO, unlike results of our study where no significant difference in cell viability was observed between CHX and EO group. However the results of cell migration were in agreement with results of our study with CHX inhibiting cell migration more than EO.⁴

Conclusion

Both the chlorhexidine (CHX) and essential oil (EO) containing commercially available mouthwashes showed a reduction in viability of human gingival fibroblasts after one minute exposure, and with an increase in the concentration of both the mouthwashes, the fibroblast viability decreased. Although comparable results were observed when cell viability was assessed, when the migration capacity was assessed, CHX showed a significantly higher scratch width compared to EO indicating its higher toxicity. Hence EO can be considered a safe alternative to CHX.

Conflicts of interest

The authors declare that they have no conflict of interests

Acknowledgments

We would like to thank Dr. Vijay MK for supporting us with the laboratory analyses.