Cariology Module 2

MODULE 2

Dental caries is a chronic endogenous infection caused by the normal commensal flora. It can also be defined as a localized and chemical loss of the tooth structure caused by the metabolic activity of the biofilm which consists of several microorganisms covering the tooth surface. (Selwitz, 2007)

The carious lesion is the result of demineralization of enamel – and later of dentin – by acids produce by plaque microorganisms as they metabolize dietary carbohyrates. The loss of tooth structure may be reversible or irreversible in nature, depending on the stages of the lesion. It can also develop in any part of the tooth surfaces, but it is more commonly seen in site where the biofilm is more protected (i.e. pits and fissure) allowing them to mature and grow, producing more microorganisms. (Fejerskov, Kidd. 2008)


THE RESIDENT MICROFLORA

It has been estimated that the human body is composed of approximately 1014 cells, of which 105 are mammalian. The majority are the organisms that comprise the resident micrflora of the host. Acquisition of this resident microflora occurs from birth and is a natural process, during which all environmentally exposed surfaces of the body become colonized.

The organisms that establish and predominate on particular surfaces vary, however, depending on the biological and physical properties of each site.  The mouth is no exception during to this process, and distinct species of bacteria can be recovered from the mouth of infants only a few hours old. Once established, the resident microflora gas a diverse composition, consisting a wide range of Gram + and Gram - bacterial species, as well as yeast and other types of microorganism. In addition, the composition of the oral microflora will change as the biology of the mouth alters over time.


SITE AND DISTRIBUTION OF ORAL BACTERIA

Although the mouth is highly selective for the microorganisms that are able to colonize and become established, more than 700 different types have been detected in the mouth. The mouth is not a homogeneous environment for microbial colonization. Distinct micro-habitats (niches) exists and these can serve as reservoir for many species commonly found in dental plaque.



According to a study conducted by Xiaojing et al. in 2000, the anatomic closeness of this microflora to the bloodstream can facilitate the spread of bacterial products, components, and immunocomplexes to the other parts of the body that may be associated with different systemic diseases.


DENTAL BIOFILM: DEVELOPMENT, STRUCTURE, COMPOSITION AND PROPERTIES

In order for the oral microorganisms to persist, they have to attach to a surface and grow: otherwise they will be lost from the habitat. An important relatively recent discovery is that the properties of microbial cells forming a biofilm are distinct from those expressed when microorganisms are growing as individual cells in a liquid culture. This has led to a novel interest in trying to understand the dental biofilm mode of growth.

The development of dental biofilms can be divided into several arbitrary stages, as revealed by experimental studies in situ (Nyvad, 1993)



I. Pellicle Formation

Microorganisms do not colonize directly on the mineralized surface. The teeth are always covered by an acellular proteinaceous film (Pellicle), which forms on the "naked" tooth surface within minutes to hours. In uncolonized areas, the pellicle reaches a thickness of 0.01-1 microns within 24 hours.  The major constituents of pellicle are salivary glycoproteins, phosphoproteins, lipids, and, to a lesser extent, components from the Gingival Crevicular Fluid.

The composition of the pellicle has received considerable interest because of its potential role in determining the composition of the initial microflora. It has been speculated that the surface characteristics of different hard tissues and dental materials may influence the profile of amino acids in the pellicle and thereby modify the number of potential adsorption sites for different bacterial species.


II. Attachment of Single Bacterial Cells (0-24h)

Microbial colonization of teeth requires that bacteria adhere to the surface. The mechanisms involved in adherence are complex and are still being investigated. As the microbial cell approaches the pellicle-coated surface, long-range but relatively weak physicochemical forces between the two surfaces are generated. Irrespective of the type of tooth surface (enamel or root) the initial colonizers constitute a highly selected part of the oral microflora, mainly S. sanguinis, S. oralis and S. mitis.


Coccal bacteria attach to the enamel pellicle as pioneer species (A) and multiply to form microcolonies (B), eventually resulting in confluent growth (biofilm formation) embedded in a matrix of extracellular polymers of bacterial and salivary origin (C). With time, the diversity of the microflora increases, and rod and filament-shaped bacteria colonize (D and E)

The selective manner by which bacteria attach to the tooth surface supports the fact that bacteria contain a recognition system on their surfaces that enables bacterial surface adhesins to bind to complementary molecules (receptors) in the pellicle. Some receptors have been identified as oligosaccharides on the protein backbone of the pellicle glycoproteins.



The principle of microbial succession is, briefly, that pioneer bacteria create an environment that is either more attractive to secondary invaders or increasingly unfavorable to themselves because of lack of nutrients, accumulation of inhibitory metabolic products, and/or increase in anaerobiosis, etc. In this way, the resident microbial community is gradually replaced by other species more suited to the modified habitat. The secondary colonizers also attach to established pioneer species via adhesin-receptor interactions (termed co-aggregation or co-adhesion)


IV. Microbial Succession (and Co-Adhesion) Leading to Increased Species Diversity with Continued Growth of Microcolonies (1-7 days)

As the dental biofilms develop, some of the bacteria produce polysaccharides, especially from the metabolism of sucrose and these contribute to the biofilm matrix. The biofilm matrix is not just a physical scaffold that helps to support the structure of the biofilm: the matrix is also biologically active and is involved in retaining nutrients, water, and key enzymes within the biofilm. As the composition of the developing biofilm becomes more diverse, the bacteria can interact both in a conventional biochemical manner and via specific signaling molecules. As the bacterial deposits become thicker, a lowering of the oxygen concentration is one of the factors that help to drive microbial succession. Thus, in developing coronal plaque, a progressive shift is observed from mainly anaerobic and facultative anaerobic species in the early stages to a situation in which facultatively and obligately anaerobic organisms predominate after 9 days.


V. Climax Community/Mature Biofilm (1 week or older)

The composition of dental plaque is diverse and includes a range of Gram + and Gram - bacteria, most of which are facultatively or obligately anaerobic. Dental caries has a high number of acid-producing microorganisms such as the Gram + S. mutans and S. sanguinis and Gram - Lactobacillus species. However, the acidogenic properties of these bacteria can be reduced by other organisms in plaque.

Novel imaging and molecular techniques have confirmed that dental biofilms display properties that are similar consistent with those biofilms present in other natural habitats. Dental plaque is an example of a biofilm which functions as a microbial community. Biofilms protect bacteria living within their structures making them difficult to eliminate. Bacteria in a biofilm also communicate with each other enabling them to monitor each other's presence and modulates their gene expression response.


Dental plaque is an example of a biofilm, a film of microorganisms adhering to a solid surface. When the plaque is formed in areas of the teeth not exposed to the cleansing action of the tongue, cheeks, and foodstuff, known as stagnation areas, a unique microenvironment immediately adjacent to the tooth surface is created undisturbed because it is partly isolated from saliva.

As mentioned in the previous lesson, the interaction of teeth, bacteria, and diet is the basic factor in caries etiology. While the combination of two factors will produce a contribution (e.g. bacteria + tooth= plaque; bacteria + diet = acid), the interaction of all three is required for caries initiation.

As humans consume a variety of carbohydrates that are metabolized by plaque bacteria this results in the accumulation of organic acid end products and hence causes a temporary reduction in plaque pH. Such an episode can pose a "cariogenic challenge" since, if the plaque pH falls low enough, minerals within the underlying dental hard tissue can dissolve. The progressive loss of minerals through dissolution by plaque acid (demineralization) during repeated cariogenic challenges is the primary process in dental caries. From the Chemico-parasitic theory of Miller evolves newer hypotheses on the etiology of caries development related to the dental biofilm or plaque. These are the Specific Plaque hypothesis, the Non-specific Plaque hypothesis, and the Ecological Plaque hypothesis. Microbiology of Caries In his chemo-parasitic theory (1890), Miller postulated that caries was caused by acids produced in the mouth by bacteria metabolizing dietary carbohydrate in food particles retained between the teeth. Until the 1960s lactobacilli were favored as the likely pathogens because they are highly acidogenic (capable of rapidly converting sugar to acid) and aciduric (capable of withstanding low pH conditions). Then, from a series of classic experiments with rodents, it was concluded that caries was an infectious, transmissible disease and attention shifted to streptococci, especially Streptococcus mutans, first isolated in 1928. Besides being acidogenic and aciduric, S. mutans synthesizes an insoluble, sticky extracellular polysaccharide from sucrose which promotes adhesion of the organism. Since the 1960s, an enormous body of research on the microbiology of caries has accumulated and many observational, longitudinal, and intervention studies have provided strong evidence for an association of S. mutans with caries. Indeed, many workers have concluded that S. mutans is the sole pathogen involved in caries. Usually, this is extended to include other members of the taxonomic group to which S. mutans belongs—the mutans streptococci particularly S. sobrinus, which is also isolated from cariogenic plaque of humans, although less frequently and in smaller numbers than S. mutans. The hypothesis that caries is caused by infection with S.mutans or mutans streptococci, is known as the specific plaque hypothesis. However, S. mutans usually makes up only a very small proportion of the plaque flora, is not always detectable in plaque associated with caries, and can occur in plaque without caries developing. Further, while S. mutans is particularly acidogenic and aciduric, these properties are also exhibited to some extent by a variety of plaque bacteria. These include not only S. sobrinus but several “low pH” members of the Streptococcaceae, such as strains of S. oralis. Other acidogenic/aciduric plaque bacteria include strains of Actinomyces, such as A. israelii and A. gerencseriae, bifidobacteria, and lactobacilli. Recognition of this fact underlies the nonspecific plaque hypothesis, which suggests that acidogenic, acid-tolerant bacteria besides S. mutans contribute to the caries process and, in the absence of S. mutans, could be the sole agents of caries initiation. A third hypothesis, the ecological plaque hypothesis, emphasizes the importance of the oral environment in determining the composition and properties of the plaque microflora. According to this hypothesis in the mouths of persons consuming a low-sugar diet the plaque bacteria would derive their energy predominantly from slow breakdown of complex salivary and dietary molecules, so would experience only small and infrequent drops in pH. An increased frequency of sugar intake disrupts the homoeostasis of such a plaque because it favors growth of acidogenic, aciduric bacteria and hence promotes low-pH conditions. Bacteria which are sensitive to low pH grow less well under these conditions and are selected against. Thus an increased availability of sugar causes an ecological shift in the plaque microflora which establishes caries-conducive conditions. Since bacteria are selected solely on the basis of their ability to produce acid and to withstand low pH, this process is nonspecific and the bacteria which increase in a high-sugar environment can include a range of species, as noted above. However, if S. mutans has colonized the mouth its growth will certainly be favored, especially under conditions of very high sugar intake, which will result in the creation of extremely acidic conditions which give this species a competitive advantage. Such a sugar-rich, low-pH environment would also favor colonization by lactobacilliand by the fungus Candida.The ecological plaque hypothesis is supported by considerable evidence. The microflora of plaque from theapproximal region is complex and dominated by Gram-positive, rod-shaped bacteria (mainly Actinomyces) andstreptococci, of which the most abundant is typically S. sanguinis, with smaller proportions of other bacteria,such as Bacteroides, Neisseria, Veillonella, Fusobacterium, Rothia, and Lactobacillus. However, the compositionof the flora varies considerably between different sites on the tooth surface. An increased sugar intake results ina higher proportion of acidogenic/aciduric bacteria, and increases in S. mutans, Lactobacillus, and others havebeen observed, along with a decrease in the less acid-tolerant S. sanguinis. Plaque from caries-active peoplehas a higher proportion of acidogenic bacteria than that from caries-free people: these changes affect plaque ingeneral, not just on surfaces on which lesions form. Changes in the plaque flora can be difficult to identifybecause of extensive intra- and inter-individual variation, but in caries-active people the proportion of S.sanguinis and of Actinomyces naeslundii typically fall. During the initial stages of caries, the abundance of S.mutans, S. oralis,acidogenic actinomyces, such as A. gerencseriae, and lactobacilli increase. In advanced(cavitated) lesions, there may be moderate increases in S. mutans, but the flora is dominated by lactobacilli,Bifidobacterium, and Prevotella.Proponents of the specific plaque hypothesis recognize the powerful ecological effect of dietary sugar indetermining the composition of plaque microflora, but would argue that only the increases in abundance of S.mutans are etiologically significant. However, while there is little doubt that S. mutans is a major agent of cariesinitiation, it is very likely that other acidogenic/aciduric bacteria play important roles in both initiation andprogression of lesions.

What is saliva?

  • Saliva is composed of more than 99% water and less than 1% solids, mostly electrolytes, and proteins, the latter giving saliva its characteristic viscosity.

  • The term ‘saliva’ refers to the mixed fluid in the mouth in contact with the teeth and oral mucosa, which is often called ‘whole saliva’.

  • Normally, the daily production of whole saliva ranges from 0.5 to 1.0 liters.

  • Ninety percent of whole saliva is produced by three paired major salivary glands, the parotid, submandibular and sublingual glands. Secretions from the many minor salivary glands in the oral mucosa also contribute, although only somewhat less than 10%.

Salivary protective mechanisms that maintain the normal oral flora and tooth surface integrity include:

1. Oral clearance

  • An important function of saliva is to dilute and eliminate substances, this can be fast or slow. This is a physiological process usually referred to as salivary clearance or, more commonly, oral clearance. A slow clearance rate is more harmful to the teeth than a faster clearance rate.

  • The flushing effect of salivary flow is, by itself, adequate to remove virtually all microorganisms not adherent to an oral surface.

  • The flushing is most effective during mastication or oral stimulation, both of which produce large volumes of saliva.

2. Direct antibacterial activity

  • Salivary glands produce an impressive array of antimicrobial products.

  • Lysozyme, lactoperoxidase, lactoferrin, and agglutinins possess antibacterial activity. These salivary components are not part of the immune system but are part of an overall protection scheme for mucous membranes that occur in addition to immunologic control. These protective proteins are present continuously at relatively uniform levels. The normal resident oral flora apparently has developed resistance to most of these antibacterial mechanisms.

3. Buffer Capacity

  • The buffering capacity of the saliva is determined primarily by the concentration of bicarbonate ions. The benefit of buffering is to reduce acid formation.

  • In addition to buffers, saliva contains molecules that contribute to increasing biofilm pH. These include urea and sialin, which is a tetrapeptide that contains lysine and arginine. Hydrolysis of either of these basic compounds results in the production of ammonia, causing the pH to increase.

  • Because saliva is crucial in controlling the oral flora and the mineral content of the teeth, salivary testing should be done on patients with high caries activity.

4. Remineralization

  • Saliva and biofilm fluids are supersaturated with calcium and phosphate ions. Without a means to control the precipitation of these ions, the teeth literally would become encrusted with mineral deposits.

  • Saliva contains statherin, a proline-rich peptide that stabilizes calcium and phosphate ions and prevents the excessive deposition of these ions on teeth.

  • This supersaturated state of saliva provides a constant opportunity for remineralizing enamel and can help protect teeth in times of cariogenic challenges.

Hyposalivation- is a diagnosis made when the unstimulated salivary flow rate is less than 0.1mL/min and/or when the stimulated flow rate is less than 0.7ml/min.

The following conditions can influence the flow rate and lead to hyposalivation:

  • medications - e.g., antidepressants, diuretics, antihistamines, antihypertensives, antiemetics, narcotics

  • radiation

  • autoimmune diseases, AIDS, diabetes mellitus

  • menopause

  • eating disorders

  • salivary gland stones

Xerostomia is the subjective feeling (symptom) of sensation of oral dryness, which often impairs oral function and even the overall quality of life. A salivary flow rate below 0.16mL/min increases the risk of developing caries.

Of the many factors that contribute to the development of dental caries, diet plays an important role. There is today overwhelming evidence that frequent consumption of fermentable carbohydrates is associated with the prevalence of dental caries.

High frequency exposure of fermentable carbohydrates such as sucrose may be the most important factor in producing cariogenic biofilm, and ultimately, caries lesions.

Frequent ingestion of fermentable carbohydrates begins a series of changes in the local tooth environment that promotes the growth of highly acidogenic bacteria and eventually leads to caries. In contrast, when ingestion of fermentable carbohydrates is severely restricted or absent, biofilm growth typically does not lead to caries.

↑ CHO  =   ↑ acidogenic bacteria   =  ✓ caries

↓ CHO  =   bacteria has no food to digest = ↓ acid = ✕ caries

Dietary sucrose plays a leading role in the development of pathogenic biofilms and may be the most important factor in disruption of the normal healthy ecology of dental biofilm communities. Because the eventual metabolic product of cariogenic diet is acid, and the acid leads to development of caries, the exposure to acidity from other sources (e.g., dried fruits, fruit drinks, other acidic food and drinks) also may result in caries.

For detailed information, read Chapter 19 The Role of Dietary Control (from Dental Caries: The Disease and Its Clinical Management 2nd Edition).

STEPHAN CURVE

The Stephan Curve has played a dominant role in caries research over the past several decades. What is so remarkable about the Stephan Curve is the plethora of interactions it illustrates and yet acid production remains the dominant focus.


The curve on a graph, first described by Robert Stephan in 1943, showing the fall in pH below the critical level of pH 5.5, at which demineralization of enamel occurs following the intake of fermentable carbohydrates, acidic liquids, or sugar in the presence of acidogenic bacteria. After consumption, there is an elimination of the acid and a return to normal saliva or plaque pH, at which point repair of any destruction of the enamel structure takes place (remineralization). Repeated intakes of fermentable carbohydrates cause the low pH to be maintained for longer periods, thereby not allowing remineralization to take place. The use of fluoride is a major breakthrough in public health. Controlled addition of fluoride to drinking water supplies in communities where fluoride concentration is below optimal levels to have a cariostatic effect began in the 1940s and since then extensive research has confirmed the successful reduction in dental caries in many countries. Fluoride works to reduce the prevalence and severity of dental caries. Aside from community water supplies, fluoride is also in toothpaste and in products for professional application, including gels, varnishes, and restorative materials. It can also be found even in milk. However, an excess amount of fluoride in the body is manifested in the dentition as fluorosis or mottled enamel, and there is also a controversy regarding fluoride toxicity.