D o W e N eed to be C oncerned about D ental C aries in the C oming M illennium ?

Abstract

Dental caries continues to be a pubic health problem despite claims that 50% of schoolchildren are caries-free. There are widespread variations in the prevalence of caries worldwide. Caries lesions are the clinical manifestation of a pathogenic process that may have been occurring on the tooth surface for months or years. Acid production by bacteria embedded in a biofilm termed "dental plaque" is a key aspect of the pathogenesis of dental caries; nevertheless, the ability of micro-organisms to survive in a hostile acid milieu and the influence of fluoride and additional agents on this acid tolerance receive scant attention. Study of cariogenic micro-organisms largely has been limited to observations made on them in the planktonic state; clearly dental caries is essentially a surface phenomenon, and micro-organisms behave distinctively when grown on a surface. Although significant progress has been made in our understanding of the etiology, pathogenesis, and prevention of dental caries, it still remains a scientific and clinical enigma worthy of the attention of the best scientists.

Controversy

Caries is an unique dieto-bacterial disease and not simply an infection. The disease occurs on essentially an inanimate surface, and therefore the traditional methods of studying diseases may not apply. The following article brings into focus that, for decades, investigation of the pathogenesis of dental caries has been largely restricted to acid production. Now there is clear evidence that glucan production by bacteria from dietary substrate plays a critical role, and growing evidence that lack of alkali production by dental plaque is critical in the pathogenesis of this still-ubiquitous disease.

–Olav Alvares, Editor

Introduction

Dental caries continues to plague most of the world's populations despite overly optimistic claims of success in the elimination of this disease (Mandel, 1993; Stephen, 1993). It is indeed true that the prevalence of dental caries has declined in some segments of the population in developed countries (Glass, 1982), but the level of decline has been exaggerated (NIH, 1987, 1989). Claims have been made that 50% of the 12-year-old children in the United States are free of tooth decay. This statement has been repeated so often, although not based on fact, that it has assumed a mantle of truth. The national survey (NIH, 1989) upon which the statement is allegedly based shows clearly that deciduous teeth were not examined for caries, and furthermore that 85% of the 17-year-old children who were examined had one or more carious teeth. A mean was used to calculate the number of children who were caries-free (Edelstein and Douglass, 1995), which is not an acceptable approach, since the data were not normally distributed. Furthermore, even if 50% of children are caries-free, this, of course, means that 50% have caries—hardly grounds for euphoria. There are certainly no reasons for believing that those who have been determined to have no caries lesions at a particular age will maintain that status forever (DePaola, 1993).

National surveys over the years have revealed wide disparities in caries experience from one region of the United States to another. For example, when regional caries prevalence was first recorded, the highest prevalence was observed in the Northeastern part of the United States and the lowest in the Southwestern region. The differences observed in prevalence between the Northeastern United States and the Southwest region, if reported in a clinical trial, would be reason for excitement. Nevertheless, reasons for this disparity have received scant attention. Even within states, pockets of unusually low levels (Bowen, 1991) or high levels of caries have been reported—observations which have rarely been pursued (Curzon, 1983).

Nature of the Disease, Dental Caries

A caries lesion is the clinical manifestation of a pathological process that may have been occurring on the tooth surface for months or years. Caries lesions are often confused for the disease process. Reducing the prevalence of caries lesions through, for example, topical application of fluoride may have little or no effect on the disease process per se. Caries lesions result from the interaction of the bacteria which colonize or infect the tooth surface with constituents of the diet, usually sucrose. The appearance of dental plaque is the first overt clinical evidence of this interaction. Nevertheless, the processes involved in the initiation and formation of plaque are complex and diverse. Plaque is initiated by the formation of a salivary pellicle on the tooth surface. Although most attention has been focused on the mammalian constituents of pellicle, there is unequivocal evidence that bacterial constituents are present from the earliest stages of formation (Rølla et al., 1983a,b; Vacca Smith et al., 1996b). Numerous studies have been carried out on the biochemical composition of pellicle, and many of the constituents have been identified; nevertheless, the sequence in which the constituents are adsorbed has received scant attention (Hay and Moreno, 1993). The orientation of the components one to another, the conformational changes they undergo when adsorbed, and the modifications that pellicle undergoes with time have been investigated sparsely, even though each one could influence the subsequent attachment of bacteria. Perhaps even more surprising is the apparent lack of interest on the influence of dietary constituents, directly or indirectly, on the formation of pellicle (Vacca Smith et al., 1996a).

The presence in pellicle of glucosyltransferases and fructosyltransferases in an active form, and the presence other unidentified bacterial products, has been known for more than a decade (Rølla et al., 1983a; Schilling and Bowen, 1988). Nevertheless, reports continue to appear referring to the mammalian constituents only. Clearly, an important facet of pellicle formation is being ignored, despite the fact that mutans streptococci and other oral micro-organisms adhere effectively to glucan surfaces formed when pellicle is exposed to sucrose. Furthermore, there appears to be a considerable degree of selectivity in the surfaces to which the various glucosyltransferases bind. For example, glucosyltransferase C binds avidly to saliva-coated hydroxyapatite in vitro (***Venkitaraman et al., 1995). An enzyme with similar properties is found in pellicle formed in vivo, although the exact source of the enzyme remains to be determined. In contrast, glucosyltransferase B binds preferentially to the surfaces of bacteria (Vacca-Smith and Bowen, 1998). The phenomenon of glucosyltransferases binding to oral bacteria was well-described by McCabe and Donkersloot (1977) and Rølla et al. (1983b). They observed that cell-free glucosyltransferase (Gtf) adheres equally as well to bacteria that produce Gtf as to those that do not make the enzyme. Thus, micro-organisms that bind the enzyme become de facto glucan producers. Clearly this phenomenon is highly relevant in the biology of dental plaque. Transmission electron microscopy with appropriate staining clearly shows that micro-organisms are enmeshed in a matrix of polysaccharide (Critchley et al., 1967). Furthermore, chemical analyses of dental plaque from humans and animals reveal that approximately 20% of the dry weight of plaque is composed of carbohydrate (Wood and Critchley, 1966; Hotz et al., 1972; Bowen et al., 1977). There is clear and unequivocal evidence that glucan production is essential for the expression of virulence by mutans streptococci. Deletion, from mutans streptococci, of the genes expressing enzymes responsible for glucan production leads to a dramatic reduction in the ability of the organism to induce dental caries in experimental animals (Yamashita et al., 1993). Despite the proven importance of glucan production in the pathogenesis of dental caries, virtually nothing has been published since 1972 on the formation and structure of glucan in dental plaque (Hotz et al., 1972). The research efforts expended on the formation and structure of glucan in vitro have also been sparse. Furthermore, efforts to inhibit glucosyltransferase as a means to prevent caries have been infrequent, despite the now-obvious importance of glucosyltransferases in the formation of dental plaque and in the pathogenesis of dental caries.

By aiming to prevent glucan production, therapeutic approaches to the prevention of caries would be precise: Unlike broad-spectrum antimicrobials, the oral flora would not necessarily be suppressed. The physical properties of dental plaque would certainly be altered by such an approach.

Dental cavitation results from a series of interactions which occur on a tooth surface. Understandably, most research on the physiology of cariogenic micro-organisms has been carried out with organisms in the planktonic state. However, most micro-organisms behave quite differently when they are attached to a surface. For example, Burne et al. (1997) have showed, using gene fusions to the gene promoter region of the glucosyltransferase B,C genes, that the polysaccharide synthesis pathway of S. mutans is expressed distinctively in biofilms. It was further shown that urease activity in biofilms of S. salivarius was enhanced by as much as 130-fold the level observed in fluid chemostat cultures cultivated under comparable conditions (Li et al., 2000).

Given that dental plaque is a biofilm, it is apparent that the biology of micro-organisms in dental plaque may differ in many ways compared with that observed in the planktonic state.

Acid Production

Since 1940, a considerable amount of caries-related research has centered around the ‘Stephan curve’. There is little doubt that Stephan's observation (1944) at the time contributed considerably to investigators' understanding of the pathogenesis of dental caries. Unfortunately, too many researchers have become enamored of the curve and have not proceeded beyond its acid production phase. It is clear that the resting pH in the low-caries group in Stephan's curve is higher (more alkali) than in the caries-active group; furthermore, it is apparent that the pH of plaque in caries-free subjects returns to neutrality or alkaline pH much more rapidly than that in caries-active subjects (Geddes, 1975). Alkali generation and disposal of acid play an essential role in plaque physiology (Bowden, 1993). Many oral micro-organisms, in contrast to mutans streptococci or lactobacilli, display poor acid tolerance; nevertheless, they are found in relatively large numbers in dental plaque (Marquis et al., 1987). Clearly, these micro-organisms have evolved methods to overcome the inimical influences of acid in their environment (Quivey et al., 2000). Interest in alkali generation, and disposal of acid by plaque micro-organisms, has been sporadic. Capitalizing on the ability of micro-organisms within plaque to dispose of acid as a means for the prevention of caries has received scant attention (Bowden, 1993).

Perhaps one of the more fascinating physiological reactions which occur in dental plaque is the Stickland reaction. This reaction was first described by Stickland (1934) and was shown to occur in clostridia. A similar reaction was described by Curtis and Eastoe (1978) to occur in dental plaque. The reaction involves opening the proline ring and the acceptance of two protons from lactic acid, giving rise to delta amino valeric acid (Curtis and Kemp, 1983). Delta amino valeric acid has the fourth highest concentration of any amino acid in dental plaque. The peptostreptococci appear to be the major micro-organisms involved in this reaction in dental plaque (Curtis and Kemp, 1978). Clearly, this is a highly effective method of removing protons from the plaque, rendering the plaque less likely to dissolve enamel.

Many micro-organisms which lack acid tolerance mechanisms per se survive in dental plaque through the arginine deiminase system. This pathway has been well-described by Marquis and colleagues (1987) (Curran et al., 1995, 1998) and basically involves the generation of ammonia and carbon dioxide to prevent the pH of the immediate environment from declining to low values. This pathway is inducible at low pH values and is suppressed by the presence of glucose. An attractive approach might be to determine methods to avoid repression and to have the pathway active continuously. There is certainly no scarcity of arginine substrates in saliva (Casiano-Colon and Marquis, 1988).

Generation of alkali through the breakdown of urea has long been recognized as a major source of alkali in the mouth. Small amounts of urea (Kleinberg, 1967) are constantly found in saliva. In addition, many micro-organisms—e.g., S. salivarius and actinomyces—produce urease (Sissons et al., 1988). Results from studies conducted in animals show that there is an inverse relationship between dental plaque ureolytic capacity and cariogenicity (Clancy et al., 2000). Persons who have end-stage renal disease have reduced levels of caries experience, despite consuming elevated levels of sugar in their diet; they also exhibit elevated levels of urea in their saliva. The terminal pH of plaque exposed to sugar is also generally higher, even though the total amount of acid produced may be increased (Meyerowitz, 1993). Again, attempts to exploit this reaction clinically have been sparse and would appear to offer some promise for success.

Acid Tolerance

Acid tolerance by micro-organisms plays a critical role in their expression of virulence and in the pathogenesis of dental caries (Quivey et al., 2000). Following the ingestion of sugars, pH values lower than 4 are frequently recorded. Clearly, micro-organisms are unable to survive in such a milieu unless they have adopted mechanisms to alleviate the inimical influences of acid. Mutans streptococci and lactobacilli, in particular, have developed effective proton-translocating ATPase which enables the micro-organisms to maintain an intercellular pH which is 0.5 to 1 unit higher than the extracellular environment. Furthermore, the optimum pH for ATPase in S. sanguis is 7.5, for S. mutans 6.0, and for L. casei 5.0 (Bender et al., 1986). The amount of ATPase produced by these organisms can be enhanced several-fold if they are cultured at low pH values. Lactobacillus casei harbors 50 times more ATPase protein than does Actinomyces viscosus (Bender and Marquis, 1987). Available evidence clearly shows that disruption of the proton motive force by agents such as gramicidin, benzoate, and ketoprofen results in more alkaline pH values following glycolysis. This most important facet of virulence has not been the focus of therapeutic intervention to prevent dental caries. There are numerous agents readily available which appear to offer some promise as a means to disrupt the tolerance to acid displayed by these organisms. These include, for example, fluoride, alone or in combination with other weak acids, such as those mentioned above (Eisenberg et al., 1980; Belli et al., 1995). Controlling caries by this approach would not disrupt the oral microbiota but would simply result in a less-acid-tolerant microbiota with reduced virulence. All of these agents affect the membrane of bacterial cells, and evidence suggests that the production of glucosyltransferase may also be reduced significantly.

Enhancing the Effects of Fluoride

Fluoride toothpastes were introduced in the 1960s, and their widespread use is generally credited with the decline in the prevalence of caries in some segments of the population (Fischman, 1992). It is important to realize, however, that fluoride is not completely effective, and that, in large measure, it controls caries but does not prevent the disease. Despite its proven clinical effectiveness spreading over 3 decades, it remains virtually alone among cariostatic agents, and its effectiveness has for the most part remained relatively unenhanced. Several avenues of research suggest strongly that the clinical effectiveness of fluoride could be enhanced through improved delivery or use in combination with additional agents.

Available evidence shows clearly that the cariostatic effect of fluoride is heavily dependent on its ambient levels in the mouth (Larson and Mellberg, 1977; Mirth et al., 1982, 1983; Corpron et al., 1986). These observations suggest that low levels of fluoride constantly present in the mouth are critical for the expression of its maximum effect (Duckworth et al., 1987; Meyerowitz and Watson, 1998). Results from several clinical studies support this concept (Duckworth and Morgan, 1991); nevertheless, mechanisms of delivery of fluoride to the mouth have remained relatively unchanged for decades.

The antibacterial effects of fluoride have been largely ignored, despite clear evidence that fluoride, even in extremely low concentrations, can affect bacterial metabolism (Marquis, 1990). For example, fluoride in combination with aluminum is a potent inhibitor of ATPase, which plays a critical role in maintaining intracellular pH (Sutton et al., 1987; Sturr and Marquis, 1990). Fluoride also inhibits enolase at low pH values and, in addition, inhibits the uptake of sugars. Finally, it has been shown that fluoride affects the production of glucosyltransferase, which plays an essential role in the etiology and pathogenesis of dental caries (Bowen and Hewitt, 1974; Marquis, 1995). Fluoride behaves as a weak acid at low pH values: That is, in the protonated form, it can diffuse into cells as HF, where it can dissociate and affect the delta pH (Belli et al., 1995).

Recently, evidence has been gathered which shows that additional weak acids such as ketoprofen, benzoate, salicylate, and sorbate all enhance the weak acid effect of fluoride on bacterial metabolism (Ma et al., 1999; Bowen et al., 2000). Most of these agents are widely used and are regarded as safe. This area of research appears to offer exceptional promise as a means to enhance the clinical effect of fluoride.

Vaccines

Following the identification of specific micro-organisms associated with the etiology of dental caries, the possibility of the development of an effective vaccine became particularly attractive (Bowen, 1976; Russell and Colman, 1981; Taubman and Smith, 1993). However, there is no evidence that natural resistance to dental caries (a very rare phenomenon) is associated with elevated levels of antibodies to, or reactive with, specific oral micro-organisms. It is true that some modest success has been reported from studies conducted in animals. How this level of protection compares with the effects experienced with the use, for example, of fluoride remains a matter for speculation. Many animal-based studies purporting to show a protective effect from vaccination have used animals of inappropriate age, have not used double-blind techniques, have not used correct statistical methodology, and have not monitored eating patterns of the animals, thereby rendering interpretation of the data very difficult. Furthermore, even in those reporting success, the level of protection is unimpressive.

Significant difference of opinions prevails over whether antibody for protection against caries should reside in the IgG or the IgA class of antibody studies (Bowen, 1996). It is argued that sIgA is the dominant immunoglobulin in the mouth, is resistant to proteases, and is the ‘mucosal’ immunoglobulin. IgG, in contrast, is found in relatively low concentrations in saliva (but in high concentrations in the gingival fluid) and is susceptible to proteases (Brandtzaeg, 1983).

Universal agreement does not exist on the most appropriate immunogen to use as a vaccine. SpaA, antigen I/II (and additional names for the same immunogen), has been promoted over the years as the optimum antigen, based on the observation that the protein is involved in the adherence of mutans streptococci to saliva-coated hydroxyapatite. However, it has been shown that mutans streptococci which lack this protein through genetic manipulation are equally as cariogenic as the parent strain in rats (Yamashita et al., 1993). This observation certainly reduces the likelihood that this could serve as a protective immunogen.

Attention has also been focused on glucosyltransferase(s) as possible immunogens. Most mutans streptococci produce several different types of Gtf, each apparently with a distinct role in plaque physiology. Although antibody to one enzyme usually cross-reacts with other Gtfs, the level of inhibition may vary considerably.

Although the concept of vaccination against caries is attractive, several major problems continue to plague the field, including those listed above. Vaccination against caries is based on the idea that the same principles that apply to mucosal immunity are applicable to protection against caries. However, the disease dental caries occurs not on a mucosal surface but on a hard, non-shedding, largely non-reactive, surface: Protective antibody is required to react on a solid surface in a largely hostile environment with large variation in pH values, active proteases, and limited diffusion into and out of plaque. Furthermore, antibodies which react with epitopes on putative protective bacterial proteins in solution may not identify the same epitopes when the proteins adsorb to a surface and undergo conformational changes. Such changes are known to occur, for example, with glucosyltransferases adsorbed to saliva-coated hydroxyapatite (Vacca-Smith et al., 1996b).

It is assumed that even partial inhibition of glucosyltransferases by antibody may be beneficial. However, it is now clear that, in the presence of antibody which partially inhibits or simply reacts with the enzyme, a glucan of novel structure may be formed, thereby providing a distinctive structure to which micro-organisms may bind.

There has been an increasing interest in the possible use of topically applied antibodies as a means of controlling dental caries (Ma et al., 1995). This approach certainly has attractions in that immunogens do not have to be administered systemically. Nevertheless, although the approach is technically feasible, it shares many of the same problems mentioned above. In addition, depending on the method of administration, it may suffer from the same problem as any other mouthrinse or topical application, in that it does not remain in the mouth for a sufficient time to exert its therapeutic effect. Perhaps antibody could be used as a “homing agent” to deliver therapeutic substances to specific areas of the mouth.

Early Diagnosis of Dental Caries

The need for early diagnosis of the disease dental caries is indeed pressing. The presence of a white spot in enamel is regarded clinically as an early lesion but, in fact, is a comparatively late stage in the pathogenesis of dental caries (Eggertsson et al., 1999; Lussi et al., 1999). Populations of Streptococcus mutans and lactobacilli alone or in combination in saliva have been used as predictors of lesion development, with mixed results. The addition of measurements of calcium and fluoride improves the reliability (Leverett et al., 1993) significantly. Clearly, the need for numerous laboratory tests to determine susceptibility is a major disincentive to their widespread use.

The development of a simple, reliable method to diagnose caries, including, for example, non-clinical overt loss of tooth structure, could open the way for innovative approaches to the treatment of this ubiquitous disease. Preventive therapy could be initiated before cavitation, thereby reducing the cost of treatment and preserving tooth structure.

The current methods of conducting caries clinical trials are expensive, cumbersome, and time-consuming, and are certainly an impediment to the introduction of new therapeutic agents and the refinement of existing products. Enhanced methods for detecting caries activity, including detection of pre-clinical enamel loss, would certainly accelerate the conduct of trials and would probably give rise to more meaningful clinical results. Trials as currently conducted measure caries lesions which may have been initiated before the study commenced. In general, they do not inform us of the effect, if any, that the agent has on the disease; furthermore, it is widely assumed that if an agent slows the development of lesions for 1-2 years, it will be clinically effective for the subject's life.

Animal-based Research

The contributions of research conducted in animals to the understanding of the etiology, pathogenesis, and prevention of dental caries have been enormous (Tanzer, 1981). Nevertheless, in parallel with the decline in interest in caries research, there has been a reduction in the number of investigators using animals in their research. This reduction has been compounded by a reluctance by many industrial companies to conduct or support animal research because of pressure from a variety of animal rights groups. It appears inconceivable that the research agenda of a society can be dictated by self-righteous pressure groups.

Nevertheless, the need for high-quality animal models to study dental caries is likely to increase. With the development of new and more sophisticated animal models such as transgenics, it will be possible to define, very precisely, the role of various salivary constituents in preventing or enhancing susceptibility to dental caries. Furthermore, it is conceivable that truly caries-resistant enamel and even replacement teeth could be developed.

The need for more conventional animal models will continue, despite efforts to replace them by in situ demineralization and remineralization models. The in situ models may be appropriate for the study or development of agents where the mode of action is similar to that of fluoride. Such “tunnel vision” on the development of new products is not in the best interests of the public at large, and has the potential to stifle the development of new anti-caries agents.

Translational Research

There have been several approaches to the prevention of dental caries that have shown promise but have not been exploited effectively. For example, it is now widely accepted that dental caries is an infectious and transmissible disease, but with major dietary overtones. Nevertheless, for the most part, treatment of the disease is largely limited to surgical removal of the diseased part of the tooth, and scant attention is paid to controlling the disease itself. The primary caregiver (usually the mother) is the major source of infection, and yet steps to prevent transfer of infection from mother to offspring are not part of routine prevention. Dental caries is, for the most part, a family problem and has been recognized as such for over 50 years (Klein, 1946). Unfortunately, recognition has not led to what appears to be logical action.

The effectiveness of the sustained release of fluoride in the mouth has been shown in animals and in humans. Nevertheless, introduction of this procedure as a means to prevent cavitation has been tardy at best (Mirth et al., 1982; Corpron et al., 1986; Meyerowitz and Watson, 1998). This approach to the therapeutic use of fluoride offers numerous advantages in specialized patients compared with conventional approaches to fluoride therapy. For example, reduced amounts of fluoride are needed to achieve a given result, and compliance by the patient is complete.

Conclusion

Exaggerated claims about the reduction in the prevalence of caries have fostered the perception that dental caries is no longer a public health problem. Unfortunately, this misperception has further led to the belief that there are no more interesting research challenges left to attract and excite young investigators. Major efforts are necessary to re-focus attention on this ubiquitous disease, which continues to affect people worldwide, rich and poor alike, but most often those least able to bear the burden. Unfortunately, we very much need to be concerned about dental caries in the coming millennium.

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