Comparative Evaluation of Efficacy of 0.2% Chlorhexidine and 1% Chitosan Mouthwash on Microbial Growth in Patients Using Dental Prosthesis: A Clinical Study

Introduction

The mouth is one of the most heavily colonized areas in our body with various types of microorganisms. Few microorganisms eventually adhere to the oral cavity forming dental plaque. Dental plaque is the major causative factor for two common oral disease conditions, dental caries and periodontal diseases.

Plaque is responsible for the initiation and progression of periodontal diseases and they are correlated with the amount of plaque accumulation and development of gingivitis.¹ Dental prostheses may influence the periodontal conditions, the risk of caries, and the amount of stress on natural teeth.^2,3 Various researchers have shown that there is an increase in the mobility of abutment teeth, gingival inflammation, and formation of periodontal pockets after dental prosthesis fitting.^4,5 According to some authors, removable partial denture is associated with increased plaque accumulation, not only on tooth surfaces in direct contact with the denture, but also on the teeth in the opposing arch, and in certain cases, even on the buccal surfaces of teeth.⁶ These alterations lead to poor oral hygiene.

The gingival health is adversely affected by removable partial dentures, with it being most severe when the removable prosthesis is in close contact with the gingival margin. The differences in gingival inflammation between the dental prosthetic plate and dental bar design suggest that covering more gingival tissue promotes development of gingivitis, which may subsequently predispose the area to periodontal disease.⁷

One of the ways to prevent bacterial growth is rinsing with an antibacterial mouthwash. Chlorhexidine (CHX) is the gold standard mouthwash that can be used to prevent and reduce bacterial growth and inflammation.⁸ However, the use of chlorhexidine mouthwash daily may exhibit certain side effects, such as oral mucosa irritation, taste perception alteration, burning sensation, and tooth staining.⁹ Alternatively, many studies have reported Chitosan (CHT) as a biomaterial that possesses antibacterial properties.

Chitosan is the deacetylated (to varying degrees) form of chitin. It is said to be the second most abundant natural biopolymer on earth next to cellulose, is widely distributed in nature, and occurs in arthropods, crustaceans, fungi, and yeast.¹⁰ However, studies on the antibacterial activity of chitosan as a mouthwash in clinical scenario remain minimal due to the water-insoluble property of the material. Many studies proved that 1% microcrystalline chitosan solution can reduce the bacterial total plate count to 99.05%.¹⁰ Therefore, this study attempted to evaluate the antibacterial effect of 1% chitosan solution in comparison to 0.2% chlorhexidine mouthwash on the bacterial levels of the oral cavity with a dental prosthesis.

Materials and Methods

The chlorhexidine mouthwash used in this study was the commercially available 0.2% chlorhexidine gluconate mouthwash (Hexidine, ICPA Health Product Ltd, India). Even the chitosan solution used in this study was the commercially available solution with a composition 1% chitosan and 0.25% acetic acid (BGfresh, DH Medical Technology Co. Ltd, China). Both the rinsing solutions were placed in 40 identical 100 mL bottles and were randomly labeled 1 to 40. This clinical trial was conducted in the Department of Prosthodontics, BIDSH, Patna. The study included 40 prosthesis-wearing patients in the age group of 35 to 55 years. All the patients were explained about the protocol of the study and informed consent was taken. The study was conducted in compliance with the Helsinki Declaration of 1975.

Patients with non-plaque induced gingivitis or periodontitis, those on antibiotic therapy, those using mouthwash for the past three months, patients with systemic diseases, smokers, pregnant and lactating mothers were not involved in the study. The present study included 40 prosthesis-wearing patients with plaque-induced gingivitis in the age group 35 to 55 years. They were randomly divided into two groups of 20 each.

The first group was advised to rinse with 15 mL 0.2% CHX mouthwash, and the second group with 15 mL 1% chitosan mouthwash twice daily for 30 seconds after 30 min of their regular tooth brushing for 15 days. Any history of allergy to chitosan and chlorhexidine was recorded prior to the commencement of the study. All patients underwent oral prophylaxis after sample collection on day 0. The plaque samples were acquired at baseline and on the 15th day. The plaque samples were striated, cultured on blood agar plates and incubated at 40°C for 48 hours for microbial growth. The Colony Forming Unit (CFU) is a unit used in microbial counting to estimate the number of viable microbes. Microbial colonies were counted using an automatic colony counter machine.

Statistical analysis

Statistical analysis was performed using MS Excel 2007 version. For each parameter used in the study, the average and standard deviation were calculated. To determine the significant difference between different concentrations and different time intervals, unpaired t test was used. The student's t test was applied for evaluation of the mean microbial count within the groups. An independent t test was used to assess the changes from day 0 to day 15. The same test was used to compare the two disinfectants. The statistical significance level was set at P <0.05.

Results

Comparison within the groups using the "paired t-test" showed a significant curtailment in both the groups (P <0.001). Independent t-test was performed for the proximity of microbial colony count between the groups on day 15. Intergroup comparison of microbial count within the groups was done using independent t-test (P <0.05).

Table 1 shows the relationship between bacterial CFU count at baseline and 15 days after the use of mouthwash in Group A (CHX) and Group B (CHT). These mouthwashes were effective in reducing bacterial CFU counts.

Figure 1 shows the relationship between the two groups. The CFU counts were similar in both the groups, both at baseline and post-treatment. The bacterial count reduction in each group after the use of respective mouthwash was significant. However, the intergroup comparison of reduction in bacterial counts did not demonstrate significant difference between Group A and Group B (Table 2). 

Table 2 shows the relationship between the mean reduction in the bacterial counts of Group A and Group B. Both the mouthwashes were effective in reducing bacterial counts; however, Group B was slightly more effective in reducing the bacterial colonies at 15 days when compared to Group A.

Discussion

This study aimed to evaluate and compare the antimicrobial effect of two types of mouthwashes, 0.2% Chlorohexidine and 1% Chitosan in patients wearing dental prosthesis. Results of this study indicated that the mouth rinses evaluated demonstrated an effective antibacterial effect against bacteria in the oral cavity of patients wearing dental prosthesis. These conclusions were supported by the clinical parameters and microbiologic outcomes.

One of the adverse effects of the dental prosthesis is unfavorable oral hygiene that leads to gingivitis, enamel demineralization, caries, and few patients tend to develop periodontal problems. It is recommended to use mouthwashes following prosthodontic treatment to decrease plaque accumulation. Because of the greater risk of caries in dental prosthesis-wearing patients, additional measures along with use of fluoridated dentifrices become necessary. In the present study, highest reduction in the mean bacterial CFU (3.725x10² ) was noted with Group B mouthwash after use for 15 days in patients wearing dental prosthesis. However, the difference in the mean value of bacterial count reduction between Group A and Group B was not significant (P >0.05). This could be due to short experimental period and small sample size.

Chlorohexidine gluconate has been in use for more than three decades for both prevention and therapy of periodontal diseases owing to its bactericidal and bacteriostatic activities and findings of several in vitro and in vivo studies have proven the same.¹¹ 0.2% CHX has been accepted as the gold standard owing to its prolonged bacteriostatic action for 8-12 hours.¹² The results of the present study are in agreement with a previous study which investigated the efficacy of 0.2% CHX mouthwashes.¹³

During the last 20 years, chitosan received a great deal of attention for both its medical and pharmaceutical applications, with several reports on chitosan-based products used as antibiotics, antiparasitic, anaesthetics, and painkillers, growth promoters, and biosensors.¹⁴ Recently, authors have begun exploring the use of chitosan-based products in the oral cavity. The results of studies that analyzed the antibacterial activity clearly indicated that water-soluble chitosan is a promising antibacterial agent for use in mouthwashes. Chitosan has limited cytotoxicity, with no antimicrobial resistance while possessing bio-adhesiveness, anti-inflammatory, and antimicrobial properties, and thus can be considered as a good therapeutic option. The antibacterial activity of chitosan is influenced by its degree of deacetylation (DD), molecular weight (MW), and acidity (pH). The characteristics of effective chitosan are DD 85%, high MW, and pH 6. The antimicrobial action and bio-adhesiveness of chitosan appear to be a perfect match for usage in oral care products. However, most mouthwashes used these days are carefully formulated to obtain antimicrobial activity, among other properties. Chlorhexidine has been associated with certain side effects such as bitter taste, formation of extrinsic stains on the teeth and tongue, increased risk of caries due to fermentation and alcohol content, discoloration, altered taste perception, metallic taste, and cytotoxic effects on cells.¹⁵ All these disadvantages have led to the development of more natural and biocompatible agents like chitosan.

Overall, the results of the current study validate a stable and active chitosan-based mouthwash formulation for human usage. The chitosan mouthwash possesses lower toxicity and higher antimicrobial activity than the commercial mouthwash tested. Hence chitosan mouthwash is a safer, valid, and viable alternative to the already existing mouthwashes.¹⁶

Conclusion

This clinical trial revealed that chitosan has adequate antibacterial activity to reduce total bacteria count and its effectiveness did not significantly differ from chlorhexidine when tested in dental prosthesis-wearing patients. Both the mouthwashes were effective in maintaining low bacterial counts in the oral cavity of patients wearing dental prosthesis. The outcome of this study could allow CHT to be considered as a mouth rinse. The exact antibacterial mechanism of CHT is still clearly not defined. Further investigations are needed to evaluate the potential of CHT as an effective antiplaque mouth rinse and also to develop an ideal concentration of CHX in mouthwashes to avoid the unwanted side effects. Overall, these results prove that the development of chitosan mouthwash was successful and it can be a valid and viable alternative to the already existing mouthwashes. The antibacterial activity of chitosan is influenced by its DD, MW, and acidity (pH)

Conflict of Interest

None