The dental biofilm is considered to be the major biologic determinant common to the development of dental caries and the periodontal diseases.¹ Prior to discussing the role of oral cleanliness in prevention and control of these diseases, it is important to understand the nature of the dental biofilm and the ways with which it can be interfered.

Definition of the dental biofilm

A dental biofilm is defined as a consortium of microorganisms that is embedded in an extracellular polymeric matrix and sticks to dental surfaces.¹ Most clinicians still use the term dental plaque when referring to dental biofilms. However, the term dental biofilm is becoming more common among clinicians, probably because its role in oral diseases is an important area of study and the term is used in scientific literature. Moreover, dental biofilms are an archetypal example of a multispecies biofilm and share the same characteristics as other natural biofilms. Because of the etiologic role of dental biofilms in dental diseases such as caries, gingivitis, and periodontitis, dental professionals are trained in professional plaque control and to teach their patients to self-perform plaque control in order to prevent and manage biofilm-induced dental diseases. To appreciate the possible value of such interventions it is necessary to understand the biofilm composition and structure, keeping in mind that most dental biofilms live in harmony with the host and only result in dental disease when an ecological shift occurs within the biofilms. Furthermore, since teeth provide nonshedding surfaces with areas that are not easily accessible for mechanical plaque removal, other strategies may be relevant to prevent and control dental diseases.

Nature of the dental biofilm

Dental biofilms are well characterized and the development includes several stages, including pellicle formation, initial colonization with attachment of microorganisms, co-adhesion of microorganisms, succession, and growth of attached microorganisms and microcolonies into mature biofilms.² Modern molecular techniques have evolved dramatically in recent decades, and added important information to previous culture-based studies. Such techniques have resulted in identification of more than 700 taxa in the oral cavity,³ from a diverse group of microorganisms including bacteria, viruses, fungi, archaea, and protozoa.¹ In dental biofilm research there has been a focus on the bacteria, but lately understanding the fungus-bacteria interactions and how such cross-kingdom microbial communities influence the biofilm ecology has gained attention.^4,5 An association between microbial profile and oral health and disease has been suggested,⁶ but it is beyond the scope of this chapter to go into details of the diverse microbial composition of dental biofilm. Check QTreatment.

The structure of dental biofilms changes over time, from an initial colonization pattern dominated by adsorption of single bacteria and small multispecies aggregates of bacteria, which form complex multilayered microcolonies and biofilms after 24 to 48 hours.⁷ Molecular studies of mature biofilms from supra- and subgingival biofilms⁸ and occlusal surfaces⁹ using confocal microscopy and fluorescence in situ hybridization have demonstrated interesting structural features of such dental biofilms (Fig 6-1). For example, these studies have demonstrated in vivo biofilms comprise of a dense basal layer with a preferential localization of Actinomyces species (Figs 6-1b and c). In contrast, the outer layers seem to be more loosely structured (Figs 6-1a and c). Fungal species have recently been shown to be an important part of dental biofilm architecture, demonstrating hyphal networks with bacteria (Fig 6-1d).⁵ Co-localization of Candida species and bacteria such as streptococci suggest beneficial interactions that may affect the ecology of the biofilm.

The extracellular matrix is another important part of dental biofilms and consists of different types of biopolymers called extracellular polymeric substances (EPS), which include polysaccharides, extracellular DNA, proteins, and lipids. The EPS act as a scaffold that provides the mechanical stability of the biofilm by establishing a three-dimensional (3D) polymer network.¹⁰ Several functions of EPS have been ascertained involving adhesion, cohesion, nutrient source, and protective barrier against chemical and biologic substances. Despite the growing interest in exploring the role of the extracellular matrix and the development of new methods, there is still a need for deeper insight into the spatial structure and function of the matrix.¹¹ For example, it is not known how the EPS synthesis among oral bacteria can facilitate environmental and biologic niches. From studies of mixed-species oral biofilm models, it is understood that areas with low pH reside inside complexes of exopolysaccharides.¹² In addition, identification of acidogenic hot spots within 3D dental in situ biofilms support the existence of heterogenous pH landscapes in dental biofilms.¹³

Recognition that microorganisms grow in structural communities that differ phenotypically from their counterparts growing in planktonic phase, and understanding how they interact through communication and production of the biofilm matrix, is fundamental to prevent and treat biofilm-induced dental diseases. The finding that bacteria in biofilms can be up to 1,000 times more resistant to antimicrobials than the same bacteria growing in planktonic phase¹⁴ supports this view. Shifts in the balance of the resident microbiota driven by local environmental conditions, as described by the ecological plaque hypothesis,^1,15 can lead to demineralization in caries¹⁶ or loss of alveolar bone in periodontal disease.¹⁷

How to interfere with the dental biofilm

The common goal for the dental practitioner is to help the patient maintain good oral health and thereby avoid imbalance in dental biofilms. This has traditionally been endeavored by teaching patients good oral hygiene measures. From clinical observations it is known that if clinically biofilm-free conditions are obtained, caries lesion progression can be arrested, but there is no evidence of the threshold of biofilm quantity.¹⁸ However, the age of the biofilm may play a role. For example, dental biofilms need to be well established, exceeding 2 days, to be able to cause pH drops below the critical pH resulting in demineralization of enamel.¹⁹ Increased biofilm accumulation has also been shown to increase inflammation, the risk of gingivitis and halitosis, and suppression of beneficial bacteria.¹ Therefore, the strategy could be to aim at preventing biofilms from maturing. When the biofilm becomes thicker, a shift in the biofilm composition can take place, caused, for example, by reductions in oxygen and nutrients from saliva that favor specific micro-organisms.

The purpose of mechanical plaque removal is prevention of initial colonization of oral microorganisms, mechanical disruption of the biofilm, and possible removal of the biofilm. There is limited information on the effect of tooth brushing on biofilm. In order to investigate the effectiveness of different plaque removal methods, in vitro studies have been used to investigate the efficacy of various types of toothbrushes. One in vitro study showed that 16-hour-old biofilms adhered more tenaciously than initial biofilms,²⁰ which may be explained by the fact that adhering bacteria need time to anchor themselves to the surface and grow. The same study also demonstrated that the bacteria in the inner part of the biofilms were the most difficult to remove and required direct contact with the toothbrush bristles. Another study showed that complete removal of the biofilm cannot be achieved and biofilm will always be left behind.²¹ A recent study investigated the impact of oral hygiene discontinuation on supragingival microbiomes.²² This study demonstrated that the microbial composition of supragingival biofilm samples collected after 4, 7, and 10 days of oral hygiene cessation differed significantly from the baseline samples. Even though the data were collected from healthy individuals and not correlated with change in clinical endpoints, the data suggest that cessation of tooth brushing may change the composition of the microbiota and thereby alter the ecology of the biofilm in favor of disease.²²

Evidence on the combined effect of mechanical and chemical plaque removal is also lacking, and one study of 4-day biofilm grown intraorally on titanium disks showed incomplete biofilm removal.²³ The use of antibacterial agents is intended to achieve the same goal as mechanical plaque removal, including inhibition of bacterial adhesion and colonization, as well as disturbance of mature biofilms. However, certain agents such as chlorhexidine and essential oils could potentially also inhibit bacterial growth and metabolism and modify biofilm biochemistry and ecology. The fact that part of the dental biofilm is difficult to remove can be explained by the biofilm structure described above, with compact inner layers of biofilm.^8,9 Future research should provide deeper knowledge of the various components of the biofilm matrix, and a possible approach could be to develop inhibitors to disrupt the stability of the 3D matrix.¹¹

On the other hand, as described above oral biofilms in dynamic balance with the host may not constitute a health problem. In fact, the resident microflora may prevent colonization of exogenous microorganisms that could drive the ecological balance towards disease development. Black pigmented dental biofilms are examples of biofilms that are difficult to remove but have a suggested positive association with lower caries experience.²⁴ In appreciation of ecological principles, an alternative approach to eliminate the biofilm is to try to control or even manipulate the biofilm to maintain a healthy balance with the host; for example, by introducing replacement strategies including pre- and probiotics (see Chapter 11). It has been argued that the practitioner should not only focus on elimination of the biofilm but rather maintain good oral health through a harmonious coexistence between the oral resident microbiota and the host.²⁵

Clinical effect of biofilm control on dental caries

By definition, dental caries is a multifactorial, biofilm-mediated disease that develops in the sites favoring accumulation and maturation of the microbial deposits.²⁶ Thus, it is apparent that regular removal of the biofilm should act as a prerequisite for the control of caries development. However, many other determinants (see Chapter 17) might take a role in the caries process, by interacting with the metabolic processes in the biofilm, or by affecting the fluctuations of minerals at the interface with the tooth surface. Such a complex interplay of different variables determines the likelihood for caries lesion progression as well as the rate at which this occurs at any plaque-covered site.²⁷ Therefore, the impact of oral cleanliness alone is difficult to evaluate.

The role of fluoride

Presence of fluoride in the oral cavity, due to its well-recognized therapeutic effect, masks the effect of mechanical plaque removal. The majority of clinical studies that focused on plaque removal and showed inhibition of the caries process were performed using fluoridated dentifrices. A series of Cochrane systematic reviews provide solid evidence collected from a number of clinical trials, that regular tooth brushing with fluoride-containing toothpaste is associated with a clear reduction of caries increments.^28,29 Moreover, supervised procedures of oral care among children lead to greater benefits as compared to self-performance. Although these benefits could be attributed to the improved delivery of fluoride to the oral cavity on a regular basis, the biologic effect of tooth cleaning should not be rejected. It is interesting to note that the remarkable decline of dental caries that occurred in the last decades of the 20th century in many Western European countries, was not associated with any particular preventive strategy; the emphasis on oral hygiene education, along with the regular use of fluoride toothpaste, proved to be as successful a method as education combined with additional fluoride delivery, other than toothpaste.³⁰

The effect of tooth brushing separately from that of fluoride was investigated in the 1970s.^31,32 Koch and Lindhe³¹ reported the results of a 3-year clinical trial in 9- to 11-year-old children involved in a preventive program based on daily supervised tooth brushing with nonfluoride dentifrice (“placebo”), or on other preventive regimes such as tooth brushing with NaF toothpaste, fortnightly mouth rinsing with 0.5% NaF solution, and fortnightly mouth rinsing with distilled water. They found that supervised daily brushing without fluoride significantly contributed to oral cleanliness, and reduced the plaque levels as well as gingival inflammation, but was of little value for control of dental caries. Those children who brushed their teeth under supervision with NaF dentifrice experienced about 50% lower caries increment than those in the placebo brushing group. It is important to note, however, that the number of new caries lesions that developed over the study period was significantly lower in children using NaF toothpaste than in those rinsing their mouth with 0.5% NaF solution (Fig 6-2). The other interesting observation was that smooth surfaces (the buccal surfaces, in particular) benefited from brushing most of all, most likely due to the easy access for cleaning as well as for the delivery of fluoride. The study by Horowitz et al³² came to a similar conclusion, that the supervised use of the nonfluoride toothpaste had only marginal effect on the caries rates over the 2-year period; however, certain limitations of the study design, such as interrupted interventions during the summer periods, limit the value of their findings.

The effect of supervised versus individually performed tooth cleaning

The second important point is that oral cleanliness is a behavior-dependent factor.³³ The usual methods to control accumulation of the dental biofilm on the dental surfaces are tooth brushing and interdental flossing. The quality of the biofilm removal makes a difference: a number of studies have shown that although fluoride is present, the caries experience is lower in the individuals with good oral hygiene.^34–36 Professionally performed or supervised tooth cleaning has been shown to be effective in caries reduction by a number of research groups.^36–38 In particular, an emphasis of the mechanical plaque control on the erupting surfaces has been shown to have an effect.^39,40 However, in all of these studies fluoride in different formulations was involved. Interestingly, in a study focused on the plaque removal from proximal surfaces,⁴¹ a caries-inhibiting effect of the regular supervised interdental flossing procedure in children was demonstrated in comparison with the self-performed unsupervised flossing. However, the use of fluoridated toothpaste under unsupervised conditions of interdental flossing did not have a superior effect to that of nonfluoridated toothpaste.⁴¹

Individually performed tooth brushing combines many variables, such as frequency and duration of brushing, the brushing method, and the toothbrush used, and these may affect the efficacy of the plaque removal.⁴² Thus, infrequent brushers demonstrated a higher incidence of caries lesions than frequent brushers, particularly in the primary dentition.⁴³ A considerable variety in the choice of toothbrushes (manual or powered; varying in bristle shape, bristle size, and number of filaments) as well as in the tooth brushing methods implies that these may play a role in the oral cleanliness. However, no single tooth brushing technique or toothbrush design has been demonstrated to be superior. The most important principle is to access all available tooth surfaces, and to spend sufficient time for plaque removal, regardless of the basic method of tooth brushing that is used.⁴⁴ Unfortunately, not all tooth surfaces are equally accessible to cleaning; interproximal spaces or deep occlusal fissures are relatively inaccessible to the toothbrush filaments. The use of dental floss is a widely accepted measure for plaque removal from the interproximal space; however, there is very limited evidence regarding the anti-caries effect of unsupervised interdental cleaning, in permanent or in primary dentition.^45,46 Although a few early studies demonstrated a significant reduction of proximal caries in primary teeth as a result of supervised flossing,^41,47 the professional-quality flossing is a hardly achievable goal. Moreover, the topical fluoride exposure combined with tooth brushing may override the effectiveness of flossing.⁴⁸

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