Year : 2008 | Volume
: 19 | Issue : 1 | Page : 29--35
Effect of three commercial mouth rinses on cultured human gingival fibroblast: An in vitro study
Flemingson, Pamela Emmadi, N Ambalavanan, T Ramakrishnan, R Vijayalakshmi
Department of Periodontics, Meenakshi Ammal Dental College and Hospital, Alapakkam Main Road, Maduravoyal, Chennai - 600 095, India
Department of Periodontics, Meenakshi Ammal Dental College and Hospital, Alapakkam Main Road, Maduravoyal, Chennai - 600 095
Aim: To examine the effect of three commercial mouth rinses (Hexidine 0.2%, Listerine Cool Mint, Betadine 1%) upon cultured human gingival fibroblast proliferation.
Materials and Methods: Human gingival fibroblasts were cultured and incubated in Dulbecco«SQ»s Minimum Eagle«SQ»s Medium containing Chlorhexidine, Listerine, Povidone-Iodine at varying concentrations (1%, 2%, 5%, 10%, 20% and 100% of the given solution) at 37°C for 1, 5 and 15 min. Control cells received an equal volume of Dulbecco«SQ»s Minimum Eagle«SQ»s Medium without adding mouth rinses, for similar duration of exposure at 37°C. Following incubation the media were removed, cells were washed twice with medium, supplemented with 10% Fetal Bovine Serum, and fibroblasts in the test and control group were allowed to recover in the same media for 24 h.
Results: In all the three groups, the proliferation inhibition was dependent on the concentration of solublized mouth rinses in the cell culture but independent of the duration of exposure to all three mouth rinses. The results showed that all three solutions were toxic to cultured human gingival fibroblasts, Chlorhexidine being the most cytotoxic. It was seen that at dilute concentrations (1% and 2% of given solutions) Listerine was more cytotoxic than Chlorhexidine and Povidone-Iodine.
Conclusion: These results suggest that Chlorhexidine, Listerine and Povidone-Iodine are capable of inducing a dose-dependent reduction in cellular proliferation of fibroblasts. The results presented are interesting, but to know the clinical significance, further studies are needed.
|How to cite this article:|
Flemingson, Emmadi P, Ambalavanan N, Ramakrishnan T, Vijayalakshmi R. Effect of three commercial mouth rinses on cultured human gingival fibroblast: An in vitro study.Indian J Dent Res 2008;19:29-35
|How to cite this URL:|
Flemingson, Emmadi P, Ambalavanan N, Ramakrishnan T, Vijayalakshmi R. Effect of three commercial mouth rinses on cultured human gingival fibroblast: An in vitro study. Indian J Dent Res [serial online] 2008 [cited 2021 Oct 18 ];19:29-35
Available from: https://www.ijdr.in/text.asp?2008/19/1/29/38929
Over the years, there have been numerous techniques and new technologies introduced for prevention and correction of periodontal disease. Less is the attention given towards the understanding of how periodontal structures become diseased and what happens to these structures during treatment. So it is important for one to get back to the basics in reevaluating what we are doing today.
During the past four decades, it has been generally accepted that dental plaque is the main etiologic agent of most forms of periodontal disease. It has been quite some time since the introduction of various chemical agents as anti-plaque agents. There are various chemical agents available commercially in the form of mouth rinses, which are known to have marked antiplaque activity. Among them, the most commonly used mouth rinses are Chlorhexidine, Listerine and Povidone-Iodine. The antibacterial activity of these agents has been extensively studied and their efficacy proved. Unfortunately, the toxic qualities of these agents do not seem to be reserved entirely for bacteria. The aim of the present study was to determine the detrimental effect of various commercially available mouth rinses such as Chlorhexidine Gluconate 0.2%, Listerine, Povidone-Iodine 1% upon cultured human gingival fibroblast proliferation.
Materials and Methods
The study consisted of eight male dental students in the age group of 20-30 years selected from Meenakshi Ammal Dental College and Hospital, Chennai.
Surgical procedure for harvesting connective tissue
Under adequate local anesthesia, a split-thickness flap was raised at the donor site using Bard Parker blade No. 15 and careful dissection was carried out to delineate the epithelium from the underlying connective tissue [Figure 1].
A wedge of gingival tissue from a non-periodontally involved maxillary molar was surgically excised from each of the subjects and transported in tubes containing Dulbecco's Minimum Eagle's Medium in an ice-container and an in vitro study was conducted in the Department of Biotechnology, Central Leather Research Institute, Chennai [Figure 2]. A small portion (full thickness) of the tissue from the adjacent side was taken for histological confirmation.
The in vitro study was to determine the detrimental effect of various concentrations of chemical anti-plaque agents which are available as commercial mouth rinses such as Chlorhexidine gluconate (0.2%), Listerine and Povidone-Iodine(l%), upon cultured human gingival fibroblast proliferation. No commercial financial support was obtained for the study.
The method used for the culture of gingival fibroblasts was as reported by Kent et al.  The tissue was washed three times in phosphated buffered saline containing antibiotics of higher strength. The function of this balanced salt solution was to maintain pH and osmotic pressure in the medium and to provide adequate concentration of essential inorganic ions. Then the tissue biopsies were cut into small pieces using a sterile technique under a laminar flow hood [Figure 3] and put in a test tube containing 0.1% collagenase. It was left overnight at 37°C and later centrifuged at 2000 rpm for 5 min. The cells got sedimented and formed cell pellets. The medium was later decanted and pellet resuspended in culture medium containing Dulbecco's Minimum Eagle's medium supplemented with sodium pyruvate (O.lg/1), L-glutamine (1.16 g/l), streptomycin sulfate (100 mg/1), Penicillin (1,00,000 µ/1) and 10% Fetal Bovine Serum (Complete DMEM). The medium was tested for endotoxin with the Limulus Amoebocyte Lysate as described by the manufacturer.
Cells were grown at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air [Figure 4]. Satisfactory attachment of fibroblasts to the culture flasks was obtained in 24-48 h. The monolayers of cells, about 2 to 3 × 106 cells/cu mm were formed in two to three weeks. The media were changed and the sub-culturing was done after the cells reached confluence as seen under phase contrast microscope [Figure 5].
Sub-culture of fibroblast
Cells growing in a monolayer can be sub-cultured by physical means like shaking, magnetic stirring or by chemical methods like proteolytic enzymes (trypsin) and chelating agents (EDTA). Fibroblast sub-cultures were prepared from the primary cultures by removing the spent medium, washing the tissue fragments several times with sterile Phosphated Buffered Saline and dissociating the fibroblasts from the outgrowth by treatment with 0.5 to 0.8 ml of trypsin and Ethylene Diamine Tetra Acetic (EDTA) acid. The cells treated with trypsin and EDTA were incubated at 37°C for 2 min, after which cells were seen dissociating from the culture flasks. The cells were dislodged from the flasks by gentle tapping and the enzyme activity was stopped by adding 5 to 8 ml Dulbecco's Minimum Eagle's medium containing 10% Fetal Bovine Serum. The cells for the third, fourth and fifth passages were sub-cultured by a similar method.
Experimental design for exposure of human gingival fibroblast cultures to commercial mouth rinses (Chlorhexidine, Listerine, Povidone-Iodine)
The tissue was processed for fibroblast culture and sub-culture and the effect of anti-plaque agents on fibroblast proliferation was assessed. Considering the commercially available concentrations as 100%, the anti-plaque agents were diluted to 1%, 2%, 5%, 10%, 20% and 100%. The cultured fibroblasts were divided into four groups.
The control group was not exposed to any of the mouth rinses.
Group 1. Those exposed to Chlorhexidine gluconate (CHX group)
Group 2. Those exposed to Listerine (List group)
Group 3. Those exposed to Povidone-Iodine (P-I group)
Using 96-well culture plates, gingival fibroblasts were seeded at 10,000 cells/well in growth media containing Dulbecco's Minimum Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100 mg streptomycin/ml, 100 units penicillin/ml and 1.16 mg L-glutamine/ml and allowed to attach and grow at 37°C. After 24 h, cells were incubated in Minimum Eagle's Medium supplemented with 1%, 2%, 5%, 10% and 20% and 100% of the given commercial mouth rinses (Hexidine 0.2%, Listerine cool mint, Betadine 1%). The media containing the chemical agents were serum-free in order to avoid precipitation reaction. All culture samples were exposed to the given concentrations for 1, 5 or 15 min. After exposures of the cells to the mouth rinses at the given concentrations, the cells were rinsed twice with Minimum Eagle's Medium supplemented with 10% Fetal Bovine Serum and were allowed to recover in growth media for 24 h.
The cell proliferation was studied by spectrophotometric analysis.
The cell proliferation was measured spectrophotometrically at 570 nm by the (3-[4,5 Dimethyl Thiazol-2-yl]-2,5-Diphenyl-tetrazolium Bromide (MTT, Sigma, USA) assay,  which yields a purple forazan Complex owing to cleavage of tetrazolium ring by dehydrogenase enzymes solubilized by acidified isopropanol [Figure 6]. The intensity of the color measured was directly proportional to the cell number because only living cells cause this conversion. The procedure involves the addition of MTT stock solution (5 mg/ml) to each culture being assayed, equal to one-tenth of the original culture volume and incubated for 3 to 4 h. At the end of the incubation period the medium was removed and the converted dye was solubilized with acidic isopropanol (0.04-0.10 N hydrochloric acid in absolute isopropanol). Absorbance of the converted dye was measured at a wavelength of 570 nm with background substraction at 650 nm.
The collected data were tabulated and analyzed using descriptive and inferential statistics. To assess all the parameters, mean percentage was used. To compare between the timings and between concentrations, Analysis of Variance (ANOVA) was used and p-values were obtained [Table 1],[Table 2],[Table 3].
Inference for [Table 1],[Table 2],[Table 3]
In all these three groups, there was no statistically significant difference in the percentage of reduction of cells between timings. But there was a statistically highly significant difference between the percentage of cell reduction for various concentrations. The rate of inhibition increased with the increase in concentration.
The fibroblast is the predominant cell type in the soft connective tissues of the periodontium and consequently plays a central role in normal function and in pathologic alterations. Fibroblasts synthesize and maintain a diverse group of connective tissue matrices throughout the periodontium and exhibit motility and contractility, functions, which help shape structural organization of the tissue during regeneration and development.
Wound healing is a dynamic process involving the coordinated action of both resident and migratory cell populations within the extracellular matrix (ECM) and cytokines. The biologic control of ECM synthesis by fibroblasts at the wound site is a complex process dependent upon the matrix constituents within the wound (collagens Types I, III, V, fibronectin and glycosaminoglycans) and cytokines/growth factors produced by inflammatory cells, keratinocytes and fibroblasts themselves. Therefore, fibroblasts play a pivotal role in tissue repair, as, by their proliferation and ECM synthesis, they control collagen deposition at the wound site.
Chlorhexidine, a polybiguanide, Listerine, a combination of phenol-related essential oils and Povidone-Iodine, a complex containing polyvinyl pyrrolidone and Iodine in an aqueous solution have been reported as mouthwashes to be effective against a range of organisms.
The ability of Chlorhexidine to non-selectively kill oral microbiota makes it an excellent agent to indiscriminately affect mammalian cells.  In fact, the effects of Chlorhexidine on a variety of mammalian cells have shown this drug to be a toxic agent and at doses similar to or below those introduced into the oral cavity. For example, 215 µg/ml (237 µm) reduced sperm motility by 50%. It has been reported that 0.2% Chlorhexidine cause the disruption of the polymorphonuclear leukocyte cell membrane and fixation of the cytoplasmic contents.  In the presence of 0.01% Chlorhexidine, resting macrophages released lysosomal enzymes into the medium. Human skin epithelial cells exhibited growth inhibition and differential staining at concentrations as low as 0.05 mM. Furthermore, in erythrocytes, 1 mM Chlorhexidine produced 100% hemolysis and treatment with 2 µm to 200 µM of Chlorhexidine produced a dose-dependent inhibition of Na-K ATPase activity. It was demonstrated that exposure to 0.004% Chlorhexidine for 3 h would inhibit amino acid incorporation into protein in human newborn fibroblasts.  Furthermore, 0.2% Chlorhexidine for 30 sec caused significant cell death and inhibition of amino acid incorporation in human newborn fibroblasts. In the same study, it was shown that 0.01% Chlorhexidine caused significant cell death in human gingival fibroblasts and that incubation for 10 min of 0.004% Chlorhexidine prevented human gingival fibroblasts from synthesizing protein 4 h later.
In recent investigations using cells from the periodontium, Chlorhexidine has been shown to impair human fibroblast attachment to root surfaces and to affect cellular proliferation  as well as total protein production in vitro. 
Also Mariotti et al.  suggested that Chlorhexidine will induce a dose-dependent reduction in cellular proliferation and that concentrations of Chlorhexidine that have little effect on cellular proliferation can significantly reduce both collagen and noncollagen protein production of human gingival fibroblasts in vitro.
Wilken et al.  in their recent study have proved the in vitro cytotoxicity of Chlorhexidine on human gingival fibroblasts.
Fibroblasts used in these experiments were obtained from eight human gingival connective tissue biopsies from the students of Meenakshi Ammal Dental College in the age group of 20-30 years with clinically healthy periodontium.
The gingival fibroblasts in the fourth passage were seeded at 10,000 cells/well using 96-well culture plate. After 24 h, cells were incubated in Minimum Eagle's Medium supplemented with 1%, 2%, 5%, 10%, 20% and 100% of the given commercial mouth rinses (Hexidine 0.2% Listerine cool mint, Betadine 1%) for 1,5 and 15 min. The cells were then rinsed twice with Minimum Eagle's Medium supplemented with 10% Fetasl Bovine Serum and were allowed to recover in growth medium for 24 h after which cell proliferation was studied by spectrophotometric analysis.
Differences between times of exposure of mouth rinses and concentrations as observed for cellular proliferation was determined using Analysis of variance (ANOVA).
In this study it was found that gingival fibroblast proliferation was inhibited by exposure to Chlorhexidine which was similar to the results of earlier studies. ,,,,
As depicted in graphs 1-6, it was noted that there was no significant change in the effect of Chlorhexidine on gingival fibroblast between time intervals (1, 5, 15 min) at concentrations of 1%, 2%, 5%, 10%, 20% and 100% (P > 0.05). This result was in accordance with a previous study  which stated that the effect of Chlorhexidine on gingival fibroblasts occurs rapidly, since a 1-min exposure to Chlorhexidine was as effective as 15-min exposure in retarding gingival fibroblast proliferation. These data are similar to another in vitro study, which demonstrated that a time of short exposure to Chlorhexidine was equally effective in impairing human gingival fibroblast function as longer exposure times. 
As depicted in Graphs 1-6, it was also noted in this study that between different time intervals (1, 5, 15 min) exposure of gingival fibroblasts to Listerine and Povidone-Iodine had no statistically significant difference (P > 0.05).
It was observed in this study that when gingival fibroblasts were exposed to different concentrations of Chlorhexidine, the fibroblast cell death increased as the concentrations of Chlorhexidine increased. This was reflected in the results, which showed a statistically significant difference (P , which stated that Chlorhexidine reduced gingival fibroblast proliferation in a dose-dependent manner.
As depicted in Graphs 1-6, there was a statistically significant difference (P , which stated that concentration as low as 0.004% Chlorhexidine, resulted in impaired cellular function and/or cell death.
It was also found in this study that Povidone-Iodine at commercially available concentrations is cytotoxic to fibroblast cells. This result was in agreement with a previous study,  which found that Povidone-Iodine at concentration of 5.0% to 0.05% was lethal to canine embryonic fibroblasts in vitro. The findings were in accordance with studies conducted by Lineaweaver et al. and Barnhardt et al., , who proved the cytotoxic effect of Povidone-Iodine on human gingival fibroblasts.
As depicted in Graphs 1-6, it was seen that in dilute concentrations as low as 1% and 2% of the three commercially available mouth rinses, proliferation inhibition was more with Listerine when compared to Chlorhexidine and Povidone-Iodine. But when the fibroblast cells were exposed to a higher concentration (5%, 10%, 20% and 100%) of the three commercially available mouth rinses, Chlorhexidine was found to be more cytotoxic as compared to Listerine and Povidone-Iodine.
Do all these findings and data portend that the use of Chlorhexidine and the other anti-plaque agents as an oral rinse to reduce dental plaque is dangerous?
There have been extensive clinical studies using Chlorhexidine, Listerine and Povidone-Iodine showing significant effect on preventing dental plaque accumulation and gingival inflammation. , The results obtained in this study demonstrate the detrimental effect of these three mouth rinses on gingival fibroblast proliferation, which could interfere with wound healing.
The results obtained with Chlorhexidine are comparable with the earlier studies. ,,,,, Hence, introduction of Chlorhexidine, Listerine and Povidone-Iodine to periodontal connective tissue cells during initial therapy, after surgical procedures, during regeneration of the periodontium, may have important consequences on healing.
In conclusion, this study demonstrates that these anti-plaque agents that are used as mouth rinses have adverse effects on human gingival fibroblasts proliferation in vitro. In vitro studies have their own limitations in that they actually do not mimic the clinical situation. Owing to the fact that mouth rinses are mainly effective against supragingival plaque and that they are able to penetrate only up to 4% into the subgingival area,  the limitations of in vitro studies can be taken to our advantage. An exception would be if the solutions were used for subgingival irrigation, either by the clinician or the patient.
The results of this in vitro study question the extensive usage of mouth rinses.
The results of this study show that all three given mouth rinses caused fibroblast proliferation inhibition. Hence it may be concluded that these mouth rinses when exposed to gingival fibroblasts are harmful and can have important consequences in wound healing. The results presented are interesting, but to know the clinical significance, further studies are needed.
However, further studies involving larger sample size will give a better understanding about the inhibition of fibroblasts proliferation, which would have an impact on healing of oral wounds.
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