Introduction
Dental caries, periodontal disease, and oral mucosal lesions are major public health problems worldwide, which are caused by oral biofilm. Systemic diseases, such as cardiovascular disease and complications during pregnancy, have been reported to relate with oral microflora [
1,
2]. In particular, aspiration pneumonia has been caused by oral bacteria for elderly people and immunocompromised patients [
3,
4]. Oral health is closely related with general health and quality of life (QOL) [
5]. These facts indicate that it is important to suppress dental plaque formation and development to maintain QOL.
In the process of dental plaque formation, acquired enamel pellicle forms on hard tissue such as tooth surface [
6], and then, early colonizers were primarily composed of Gram-positive species such as
Streptococcus adhere to the surfaces. Secondary colonizers attach primary bacteria already anchored to teeth or tissues, which is important for the development of dental plaque [
7,
8]. Fusobacteria play a central role in physical bridges to mediate the co-aggregation of bacterial cells and promote the anaerobic microenvironment [
9].
Fusobacterium nucleatum is the predominant species in the healthy oral cavity and markedly increases in the oral cavity with periodontal disease. The inhibition of biofilm formation by periodontopathic bacteria including
F. nucleatum, therefore, is regarded as an effective strategy for preventing periodontal diseases.
It has been shown that the biological properties of a coating of biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymers, which have a phospholipid polar group that mimics a biomembrane, are completely harmless to humans, reducing protein adsorption and bacterial adhesion and inhibiting cell attachment [
10‐
12]. We previously reported that a coating of non-aqueous MPC-polymer on coverslips decreased bacterial adhesion, suppressed biofilm formation, and attributed these effects to the “superhydrophilicity” of MPC-polymer-coated surfaces [
13]. MPC-polymer application markedly inhibited both the adherence and biofilm formation of
Streptococcus mutans on saliva-coated hydroxyapatite and streptococcal adherence to oral epithelial cells and reduced the adherence of
F. nucleatum to streptococcal biofilms in vitro [
14]. In the small-scale clinical trial, mouth rinsing with MPC-polymer inhibited the increase of oral bacterial numbers, especially
S. mutans in vivo, indicating that MPC-polymer coating in oral cavity can be useful for preventing oral infections including dental caries by preventing microbial adherence to oral surfaces [
14]. Clinical effect of MPC-polymer on preventing microbial adherence to oral surfaces needs to verify more details because the previous study was a small-scale trial and parallel study design. The aim of this study is to clarify whether MPC-polymer can suppress bacterial adherence in oral cavity by a crossover clinical trial. We also investigated the number of
F. nucleatum, which is the key bacterium forming dental plaque, using clinical samples.
Discussion
It is well known that microorganisms often survive within biofilms, which results in environmental problems and various infectious diseases [
24]. During the biofilm formation and its maturation, co-aggregation and co-adhesion of bacteria are critical [
9]. Inhibition of bacterial adherence, therefore, has been considered an effective strategy for prevention of infectious diseases. To destroy the biofilm in the oral cavity, many studies have been conducted on the effective strategies in dental hygiene practice. As a mechanical method, tooth brushing and tongue scraping are used to remove microorganisms. Mouthrinses and toothpastes containing antibacterial compounds, such as chlorhexidine, cetylpyridinium chloride (CPC), and triclosan, are commonly used to prevent the growth and the biofilm formation of bacteria. Currently used disinfectants, however, induce adverse effects, such as extrinsic brown staining of teeth and restorations, toxic to mucous membranes, burning sensation, and mouth irritation [
25]. Taking into account these disadvantages of the disinfectants, a novel strategy to inhibit the bacterial adherence and dental plaque formation is required.
Here, we demonstrated the inhibitory effects of mouthwash with 5% MPC-polymer coating in the oral cavity on the number of oral bacteria by a randomized, crossover clinical study. Importantly, MPC-polymer has been approved by the Food and Drug Administration (FDA) and applied for various purposes, such as contact lenses and cosmetics. We previously have shown that (i) MPC-polymer coating to plastic coverslips reduces retention of human pathogenic microorganisms including
Staphylococcus aureus,
S. mutans,
Pseudomonas aeruginosa, and
Candida albicans in vitro [
13], and (ii) MPC-polymer application markedly inhibited both the adherence and biofilm formation of
S. mutans on saliva-coated hydroxyapatite and oral epithelial cells [
14]. These findings show that superhydrophilicity of MPC-polymer-coated surfaces, but not disinfectant action, may inhibit the adherence of pathogenic bacteria to teeth and/or oral mucosa. In addition, MPC-polymer coating has a role for reducing the protein adsorption. However, the salivary proteins included in the acquired enamel pellicle, such as histatin and statherin, have protective effects of demineralization, prevention of acidic dissolution of the teeth, and antibacterial/antifungal effects [
26‐
28]. In the future, therefore, the development of MPC-polymer with the ability of absorbance of selective salivary proteins are desired.
Fusobacteria play a central role as physical bridges to mediate the co-aggregation of bacterial cells and promote an anaerobic microenvironment.
F. nucleatum is a predominant and key bacterium in the dental plaque formation and closely associates with other periodontal pathogens [
9]. Production of hydrogen sulfide by
F. nucleatum is known to be associated with halitosis [
29]. MPC-polymer coating inhibits the adherence of
F. nucleatum to streptococcal biofilms in vitro [
14]. Here, we showed that 5% MPC-polymer gargle significantly suppressed the increase of
F. nucleatum to 50% (Fig.
4b). These findings suggest that mouthwash with MPC-polymer coating may suppress the adherence of
F. nucleatum to the oral cavity and may inhibit the maturation of dental plaque.
F. nucleatum has been reported to be involved in the development of colon cancer via activation of oncogenic signaling, recruitment of tumor-infiltrating immune cells, and interference of the host immunity [
30‐
32]. The control of
F. nucleatum is important to reduce the risk of colon cancer as well as oral infectious diseases.
To count the bacterial number in the oral cavity, we employed the gargling method. Although it is almost impossible to count the actual bacterial number in the oral cavity, we can estimate the actual number from a sample that is appropriately taken. It has been reported that the bacterial number obtained by the gargling method well reflects the number of bacteria that inhabit in the oral cavity [
18]. The bacteria adhered to oral mucosa, tooth surfaces, and tongue, therefore, can be collected by the gargling and the bacterial number in the gargling sample may be well correlated to the whole number of bacteria inhabiting in the oral cavity. To assess the number of oral bacteria in gargling sample, we used a device adopted for rapid oral bacteria quantification system (electronic bacterial counter). Although the electronic bacterial counter has a limit of the detection of bacterial counts (less than 10
5 cells/ml), the number of microorganisms in the saliva usually exceeds 10
5 cells/ml [
20]. The result by an electronic counting method did not show a highly significant difference in comparison with the result by a culture method (Fig.
3a, b). A difference in bacterial viability may be one of the reasons for which there was low significant difference. Moreover, an electronic bacterial counter is lower sensitivity than the culture method. However, the data evaluated by electronic bacterial counter was well correlated with the data evaluated by a culture method (
r2 = 0.7814). As the electronic bacterial counter is simple and easy-to-use operation, this device is useful for counting oral bacteria. Therefore, we can easily check the number of oral bacteria in gargling sample from elderly people at home and compromised hosts in a hospital. We will examine the effect of MPC-polymer coating for elderly people and compromised hosts in the future.
In conclusion, we suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adhesion. We believe that MPC-polymer is a potent compound for the control of oral microflora to prevent oral infection including dental caries, periodontal diseases, and halitosis. In this study, we focused on the oral bacterial adherence after MPC-polymer treatment. To know the initial microorganism recovery after the intervention, we will evaluate the clinical oral health status including the status of tongue coating.
As MPC-polymer treatment protected human oral keratinocytes from the damage induced by CPC [
33], MPC-polymer can be used for the purpose on the protection from the damage of oral mucosa in combination with other commercially available disinfectants.