During the late stage of dental plaque formation, extracellular polymer synthesis is accomplished with a significant portion of the biofilm composed of extracellular matrix, but extracellular polymer formation most likely occurs at all stages of plaque formation.^7–16 Voids and channels encompassing the entire thickness of the biofilm form along the extracellular matrix and allow for the passage of nutrients. The biofilm acts as a selective permeable membrane and restricts ingress of antimicrobial agents, extracellular enzymes and noxious agents. The close proximity of organisms allows for symbiotic existence with gene transfer of protective factors between bacteria, extracellular matrix synthesis to support the entire community, production of by-products used for metabolism by other organisms and secretion of inhibitors of antimicrobial agents.

The properties of dental biofilms are listed in Table 2.^8–12 These properties confer increased resistance to antimicrobial agents, and the resistance of dental plaque bacteria to antibiotic and antimicrobial agents (for example, chlorhexidine) may be increased from 10-fold to more than 1,000-fold.^7–16 The standards for bactericidal dosages usually are determined on the basis of planktonic (liquid phase) growth. Such dosages do not apply to organisms growing on a biofilm, especially older, established biofilms. This fact is important when considering antimicrobial therapy for biofilms and puts into perspective the resistance to caries-preventive agents that some patients seen in clinical practice have. With maturation of the dental biofilm, bacteria become detached and colonize adjacent tooth surfaces or neighboring teeth.

S. mutans is considered the primary pathogen in dental caries development.^1–7 This cariogenic organism is acquired early in life but does not colonize predentate infants.^19–21 Soon after eruption of the first primary tooth, S. mutans colonization begins in 20 percent of 12- to 16-month-old children. In an inner-city population, S. mutans colonization has been seen in 25 percent of 12-month-olds and in 60 percent of 15-month-olds. The furrows in the tongue may be an ecological niche for this cariogenic organism even before eruption of the first primary tooth. Early acquisition of S. mutans is a major risk factor for early childhood caries and predicts future caries experience. When S. mutans colonization occurred by 2 years of age, the mean decayed missing filled surfaces of primary teeth score was 10.6 at 4 years of age, compared with a score of 3.4 for children in whom S. mutans colonized at 4 years of age. Almost 90 percent of children who were colonized by S. mutans by 2 years of age had dental caries, compared with only 25 percent of children not colonized by S. mutans by 2 years of age.

Early acquisition of Streptococcus mutans is a major risk factor for early childhood caries and predicts future caries experience.

As indicated previously, dental caries is a transmittable infectious disease. Initial infection may occur early in life for some children and result in an increased risk of developing caries and an increased number of tooth surfaces affected.^1–7,19,20 The major source of transmission in infants is their mothers.^19–21 Vertical transmission from mother to infant has been well-characterized by various techniques (bacteriocin, plasmid and chromosomal DNA typing). Successful colonization of the infant is related to inoculum dose, inoculation frequency and minimum infective dose. Transmission of S. mutans occurs in about 60 percent of infants when maternal salivary concentrations are 10⁵ colony-forming units (CFUs) per milliliter of saliva or greater, compared with only 6 percent transmission when the maternal concentrations were 10³ CFUs/mL of saliva or less. Horizontal transmission from family members, friends, day-care personnel and other children attending day care also is a means for colonization of infants. This information led the New York Department of Health¹⁹ to issue guidelines in 2005 to decrease maternal transmission of S. mutans to infants by reducing the maternal S. mutans reservoir through eliminating active caries and encouraging fluoride and chlorhexidine use, to discourage saliva-sharing activities (food tasting before feeding infants, toothbrush sharing), to encourage twice-daily toothbrushing in dentate infants, to avoid cariogenic feeding practices, and to encourage professional oral health examinations of infants before their first birthdays.

	DENTAL BIOFILM PRESERVES ENAMEL INTEGRITY THROUGH POSTERUPTION MATURATION

TOP ABSTRACT DENTAL BIOFILM: ACQUIRED ENAMEL... DENTAL BIOFILM PRESERVES ENAMEL... DYNAMIC PROCESS OF CARIES... MODULATION OF THE DENTAL... CONCLUSIONS REFERENCES

 
Enamel is composed of HAP (92–94 percent), water (2–3 percent), carbonate (2 percent), trace elements (sodium, magnesium, potassium, chloride, zinc, which total about 1 percent), lipids (< 1 percent) and fluoride (0.01–0.05 percent).^21,22 The mineral component (HAP) forming enamel, dentin and cementum is not in a pure form but, instead, contains considerable amounts of carbonate, sodium, magnesium and chloride, as well as a small quantity of fluoride.

At the time of tooth eruption, HAP is susceptible to acid dissolution. The enamel of newly erupted teeth is permeable; soluble fluids are able to penetrate up to 200 µm into subsurface enamel. As noted previously, the newly erupted tooth is covered rapidly by acquired enamel pellicle. Fortunately, saliva is supersaturated with calcium and phosphate, and fluoride is preferentially sequestered by pellicle and plaque.^7,21–26 In this environment, the enamel undergoes posteruption maturation via the replacement of more acid-soluble carbonate-rich substituted HAP with more acid-resistant HAP and fluoridated HAP.^21,22 With the availability of calcium, phosphate and fluoride from saliva and the dental biofilm, fluoride becomes incorporated into HAP by means of absorption of fluoride onto pre-existing HAP crystals, fluoride exchange for hydroxyl groups in HAP and new crystal growth of fluoridated HAP from fluoridated mineral phases. With posteruption maturation, the porosity and permeability of the enamel is reduced substantially. Some of the salivary proteins (statherin, PRPs) in the pellicle also may bind to HAP crystals in the surface and subsurface enamel and provide additional protection against organic acids produced by cariogenic organisms.

Caries susceptibility can be reduced substantially by treatment of recently erupted teeth with topical fluoride solutions.²¹ Caries formation in newly erupted teeth exposed to topical fluoride is approximately one-half that of teeth that have not been exposed to fluoride.

	DYNAMIC PROCESS OF CARIES FORMATION ALONG THE BIOFILM-ENAMEL SURFACE INTERFACE

TOP ABSTRACT DENTAL BIOFILM: ACQUIRED ENAMEL... DENTAL BIOFILM PRESERVES ENAMEL... DYNAMIC PROCESS OF CARIES... MODULATION OF THE DENTAL... CONCLUSIONS REFERENCES

 
The process of dental caries formation begins when dietary sucrose is made available to acidogenic bacteria in the dental biofilm.^21,22,26 The pH rapidly decreases from a resting pH of 7.0 to a pH of less than 5.0 within the biofilm fluid and along the interface between the biofilm and enamel surface, and the critical pH (about pH 5.5) at which enamel undergoes dissolution is reached.

The process of demineralization and remineralization is a dynamic process, with periods of demineralization interspersed with remineralization. The effects of demineralization can be reversed if there is adequate time between acidogenic challenges to allow for remineralization to occur.

Although dental plaque is saturated with calcium and phosphate, the rapid increase in hydrogen ions (for example, acid) of more than 100-fold, and potentially as much as 1,000-fold (pH 4.0), provides a strong driving force to diffuse hydrogen ions into the fluid in the pores surrounding HAP crystals in sound surface and subsurface enamel. This process results in demineralization of the subsurface enamel by means of calcium and phosphate’s moving toward the enamel surface, which results in egress of calcium and phosphate from the subsurface enamel into the overlying biofilm.

The transport of calcium and phosphate from the subsurface enamel is due to the driving force caused by the high concentration of soluble calcium and phosphate within dental plaque, which is higher than soluble calcium and phosphate in the subsurface enamel. This driving force is created by an increase in hydrogen ions of at least 100-fold to potentially 1,000-fold in plaque secondary to active fermentation of fermentable dietary carbohydrates. Because of the high calcium and phosphate content in the overlying pellicle and dental plaque, a certain amount of the calcium and phosphate reprecipitates in the superficial layers of the affected surface enamel and helps retain an intact enamel surface layer.

After a cariogenic acid challenge, the resting pH of the dental plaque is restored, and the driving force of the increased hydrogen ion concentration no longer is a factor.^21,22,26 This process allows for remineralization of the affected enamel to begin. The hydrogen ion concentration equalizes in the dental biofilm and in the fluid in the pores surrounding the HAP crystals of the demineralized subsurface enamel. Calcium and phosphate ions are transported passively into the subsurface demineralized enamel from the saliva and biofilm. The primary driving force in remineralization is supplied by the supersaturated calcium and phosphate concentrations in saliva and plaque as compared with the less saturated fluid in the sub-surface enamel pores. The process of demineralization and remineralization is a dynamic process, with periods of demineralization interspersed with remineralization. The effects of demineralization can be reversed if there is adequate time between acidogenic challenges to allow for remineralization to occur. The figure²⁷ shows that caries is a dynamic process. If adequate measures are instituted, the balance can be tipped toward remineralization, and clinically detectable caries can be avoided.

View larger version (52K): [in this window] [in a new window]   Figure. The caries balance: a schematic diagram of the balance between pathological and protective factors in the caries process. (Reproduced with permission of Blackwell Publishing from Featherstone.27 )

The presence of fluoride within the biofilm is important in limiting demineralization. In the presence of a small quantity of fluoride (0.03 to 0.08 parts per million) within biofilms, saliva and artificial calcifying fluids, remineralization is favored over demineralization.^{2,21,22,26,28,29} Fluoride acts as a catalyst and increases the rate of HAP and fluoridated HAP formation. Only a minute fluoride concentration is needed to initiate and maintain the remineralization process. Formation of fluoridated HAP creates a mineral that resists acid to a similar extent as fluorapatite.^21,22,26 Fluoride from saliva or exogenous sources such as fluoride rinses, gels, varnishes and toothpastes is taken up preferentially by biofilms, lessens the effects of an acidogenic challenge and facilitates remineralization when the resting pH returns to 7.0. However, similar to increased concentrations of calcium and phosphate in biofilms, saliva and artificial calcifying fluids, excessive levels of fluoride lead to rapid mineral precipitation on the enamel surface and obturation of the surface enamel pores that communicate with the underlying demineralized lesion. This process further limits remineralization of the subsurface demineralized enamel. There is an optimal level of calcium, phosphate and fluoride in dental biofilm, saliva and artificial calcifying fluids that allows for remineralization of the enamel lesion to its full depth.

Remineralization of subsurface enamel lesions requires a considerable amount of exposure time.^{21,22,26,28,29} The calcium content of enamel is about 30 moles per liter. The calcium content in saliva is about 1 mmol/L and about 2 mmol/L in an optimal calcifying solution. To replenish the lost mineral in demineralized enamel with calcium from saliva or a calcifying solution, it would be necessary to supply 10,000 volumes of saliva or a calcifying solution to re-form one volume of remineralized enamel.²² This implies that it would take considerable time to replace completely the mineral phases lost in enamel owing to demineralization.

Calcium, phosphate and fluoride derived from saliva and sequestered in the dental biofilm are required for effective remineralization and maintenance of the enamel surface integrity (Figure). Although it seems like a daunting task, remineralization occurs on a daily basis after an acidogenic challenge. The ability of saliva alone to remineralize enamel was recognized four decades ago when it was shown that white-spot lesions followed for more than six years reverted to sound enamel in 32 percent of cases, were arrested and remained stable in 43 percent of cases, and progressed to cavitation in only 25 percent of cases.³⁰

Many of the white-spot lesions occurring around brackets worn by patients who are undergoing orthodontic treatment will revert to sound enamel after orthodontic treatment with or without exposure to daily topical fluoride rinses.^31,32 Orthodontists also are aware that rinses and gels containing high fluoride concentrations do not allow white-spot lesions adjacent to brackets to revert back to the normal translucent luster of sound enamel. Rather, treatment of white-spot lesions with high-concentration topical fluoride results in unsightly white opacification of enamel lesions owing to occlusion of surface porosities communicating with the subsurface white-spot lesion. This clinical observation is understandable, considering information presented about surface porosity occlusion with high concentrations of fluoride. This observation also highlights the need to use low-fluoride rinses, gels and tooth-pastes for optimal remineralization of white-spot lesions; to avoid unesthetic enamel opacification; and to restore normal enamel luster.

Low calcium, low phosphate and low fluoride concentrations provide an environment for ideal remineralization.

Low calcium, low phosphate and low fluoride concentrations provide an environment for ideal remineralization. In addition, the use of low-fluoride–concentration rinses, gels and tooth-pastes in infants and young children is important in reducing the likelihood of fluorosis’ occurring in their developing permanent dentitions, while attempting to reduce the number of carious lesions.

	MODULATION OF THE DENTAL BIOFILM TO MAINTAIN ENAMEL INTEGRITY

TOP ABSTRACT DENTAL BIOFILM: ACQUIRED ENAMEL... DENTAL BIOFILM PRESERVES ENAMEL... DYNAMIC PROCESS OF CARIES... MODULATION OF THE DENTAL... CONCLUSIONS REFERENCES

 
Several agents are capable of delivering calcium, phosphate and fluoride to saliva and dental biofilms and assisting in the remineralization of caries.^{1–7,22,26,28,29,33–37} The availability of calcium, phosphate and fluoride ions is important in people who are prone to caries.

Amorphous calcium phosphate (ACP) has been integrated into over-the-counter chewing gums and gels to deliver calcium to dental biofilms and the enamel surface.^26,33–37 The milk protein casein phosphopeptide (CPP) stabilizes calcium and phosphate ions in ACP solutions and can bind as many as 25 calcium ions, 15 phosphate ions and five fluoride ions per molecule. CPP-ACP is taken up by dental biofilms and localizes to the enamel surface as nanoparticles. Calcium, phosphate and fluoride from CPP-ACP are released during acidogenic challenges, help maintain the supersaturated state of these ions in the biofilm and promote remineralization over demineralization. Results of laboratory and in vivo studies have demonstrated significant uptake of CPP-ACP by biofilms and deposition of minerals into artificial caries-like enamel lesions.^33–36 The clinical significance of these data continues to be studied.

Deposition of minerals into enamel lesions after use of CPP-ACP–containing chewing gums and solutions is detectable.^26,33,34 Also, when CPP-ACP is integrated into glass ionomer restoration materials, significant reductions in secondary caries adjacent to these restorations have been seen in laboratory investigations.^26,33,34

Dental biofilms and enamel surfaces also may benefit from exposure to commercially available calcifying agents with fluoride.^{28,29,33–37} A carbopol-based remineralizing gel has shown considerable promise in laboratory studies.^28,29 This gel contains bioavailable calcium, phosphate and fluoride and affects carieslike lesion formation and progression in permanent and primary tooth enamel.

Sugar alcohols have a significant effect on cariogenic bacteria in dental biofilms, are deemed to be noncariogenic and do not promote dental caries.^38–40 They include xylitol, sorbitol, mannitol, maltitol, lactitol and erythritol. Sugar alcohols, in particular xylitol—found in chewing gum, candies and lozenges—have several beneficial effects on the prevention of dental caries. These include reduction in the amount of dental plaque and the plaque index; reduction in mutans streptococci in plaque and saliva; reduction in extracellular and intracellular polysaccharide matrix production; reduction in the binding of mutans streptococci to the acquired enamel pellicle, leading to less adherent plaque; and decrease in acid production in plaque. Incorporating both fluoridated dentifrices and xylitol into a prevention program for children at risk of developing caries is beneficial.

Vertical transmission of S. mutans from mother to child is affected by the daily use of xylitol-containing chewing gum.^38–40 In pregnant women and new mothers, chewing xylitol-containing chewing gum had the greatest effect on reducing vertical transmission when compared with using chlorhexidine rinse and fluoridated toothpastes. For 2-year-old children, the transmission rates were 10 percent with xylitol-containing chewing gum, 29 percent with chlorhexidine rinse and 49 percent with fluoridated toothpaste.^38,39 When the children were 6 years old, the colonization rate was 52 percent with xylitol-containing chewing gum, 86 percent with chlorhexidine rinse and 84 percent with fluoridated toothpaste. An additional advantage was that the mothers using xylitol-containing chewing gum had the lowest caries increment.

Vertical transmission of Streptococcus mutans from mother to child is affected by the daily use of xylitol-containing chewing gum.

Other antimicrobial agents have been used to control pathogenic organisms in dental biofilms.^{2,3,6,8–11,41} Povidone-iodine is a mucosal antiseptic that has been used in medicine and periodically has been used as a topical agent to control early childhood caries. It has had some success in reducing caries in infants at high risk of developing early childhood caries, and is water-soluble and nonirritating. However, iodine hypersensitivity, history of thyroid disease or dysfunction, and pregnancy are contraindications to povidone-iodine use. Delmopinol hydrochloride is a surface-active agent with low antimicrobial potency.¹¹ It inhibits extra-cellular polysaccharide (glucan) synthesis by S. mutans, and it purportedly reduces bacterial acid production and disaggregates existing plaque in vitro. Triclosan is a broad-spectrum biocide found in many antibacterial detergents, soaps and surface decontaminants. Triclosan is incorporated into fluoridated dentifrices as a way to reduce supragingival plaque and gingivitis,^8,9,37 and triclosan-containing dentifrices enhance the anti-caries potential of fluoride in toothpastes. Chlorhexidine is an antimicrobial agent used for controlling plaque in periodontal disease.^2,3,6,41 It has been advocated for caries prevention and remineralization of early childhood caries in infants and young children. Chlorhexidine is available in solution form and as a varnish.

Fluoride-releasing dental materials used to restore carious teeth and to prevent caries in pits and fissures are another exogenous source for long-term low fluoride delivery.^42,43 Typically, these dental materials release a large amount of fluoride while undergoing setting reactions and then release relatively constant low levels of fluoride for prolonged periods. Results of laboratory studies show decreased secondary caries formation adjacent to a wide variety of fluoride-releasing materials.^21,26,28 The fluoride content of tooth structure within about 8 millimeters of the restoration increased significantly. The saliva and dental biofilm fluoride contents also were elevated and affected the tooth structure susceptible to a cariogenic challenge and the metabolism of cariogenic bacteria owing to crucial bacterial enzymes involved in acid formation. These materials can be recharged with fluoride during daily application of fluoridated toothpastes or with the occasional use of topical fluoride rinse or gel.

The final point to be considered is the microbial composition of the dental biofilm. Several methods may be used to alter the cariogenicity of the biofilm. Pro-biotic and molecular genetic techniques have been used to replace cariogenic organisms such as mutans streptococci and Lactobacillus species with strains of bacteria that are not cariogenic.^8,44–46

Several mutated strains of S. mutans that lack the machinery to efficiently metabolize fermentable carbohydrates to organic acids have been developed. One example is S. mutans with a glucosyltransferase C (gtfC) gene mutation.^8,44 The pathogenicity of both S. mutans and S. sobrinus is related to their acidogenic potential and ability to form water-insoluble extracellular and enzymatically undegradable polysaccharides from sucrose.

These extracellular polysaccharides (glucans) promote adhesion and colonization of cariogenic organisms and mediate protection against antimicrobial agents and resistance to toxic compounds. Synthesis of these glucans is via glucosyl-transferase B, glucosyltransferase C and glucosyl-transferase D genes. The introduction of a mutated gtfC gene that affects the ability of S. mutans to produce extracellular glucans has resulted in a decrease in extracellular matrix component of mixed oral biofilms from 51 to 33 percent of the biofilm volume.⁴⁴

The biofilm also had a 16-fold increase in its diffusion coefficient, indicating that the biofilm would be more permeable, as well as less able to protect against antimicrobial and toxic agents and decreasing microbial adhesion. Similarly, organisms with mutations in genes that encode adhesins that allow binding to salivary glycoproteins and engage in coaggregation are factors in disrupting the formation and colonization of dental biofilms by pathogenic organisms.^8,45

The potential for genetic engineering offers an opportunity for displacing and replacing cariogenic bacteria with noncariogenic bacteria while maintaining normal oral homeostasis.

	CONCLUSIONS

TOP ABSTRACT DENTAL BIOFILM: ACQUIRED ENAMEL... DENTAL BIOFILM PRESERVES ENAMEL... DYNAMIC PROCESS OF CARIES... MODULATION OF THE DENTAL... CONCLUSIONS REFERENCES

 
Dental caries is an infectious disease that affects most of the population. This multifactorial and complex disease process occurs along the interface between the dental biofilm and the enamel surface.

Many components in saliva are taken up preferentially by dental biofilm and protect the enamel surface. Paradoxically, newly erupted teeth depend on the enamel pellicle for posteruption maturation of acid-susceptible substituted HAP.

S. mutans colonization of the dental biofilm depends on vertical transmission, horizontal transmission or both. S. mutans is acidogenic and aciduric and is considered to be the primary organism that is responsible for enamel caries. The ability of the biofilm to sequester calcium, phosphate and fluoride from the saliva and exogenous sources allows enamel to undergo remineralization after periods of demineralization.

Optimal remineralization depends on prolonged exposure of the enamel surface to low concentrations of calcium, phosphate and fluoride. Exogenous bioavailable calcium, phosphate and fluoride can alter the cariogenicity of dental biofilm. Fluoride plays a vital role in primary and secondary caries prevention and remineralization of enamel lesions.

The caries process may be modulated by using sugar alcohols (that is, xylitol), povidone-iodine, delmopinol, triclosan and chlorhexidine. Finally, the use of probiotics and molecular genetics to replace and displace cariogenic bacteria with noncariogenic bacteria has shown encouraging results, while maintaining normal oral homeostasis.