Dental caries continue to represent one of the most widespred human diseases in the world. Dietary factors, in addition to bacterial inoculation, play a primary role. A large number of experimental and epidemiologic studies have demonstrated direct relationships between the frequency of intake and total consumption of sucrose and between that total consumption and dental caries (Amsbaugh and Bowen 1981, Gustafsson et al. 1954, Soames and Southam 1993). The form of carbohydrate (Shaw 1981) and the frequency of consumption (König et al. 1968, Weiss and Trithart 1960) may be more important than the absolute amount of sucrose consumed in the initiation and progression of caries.

Most of the caries research is directed toward the initial phases of caries in enamel, which is practically inorganic. Underlying dentin is a living tissue that contains an inorganic extracellular component with an organic matrix and connection to living cells, i.e., odontoblasts and pulpal cells. The pulpo-dentinal complex is capable of responding to caries even in its very initial stage (Brännström and Garberoglio 1972, Läikkö and Larmas 1979), and this process includes both the carious destruction and the defensive reactions in dentin and pulp.

In the understanding of the effects of a high sucrose diet on the etiology of dental caries, it is widely accepted that bacteria utilize sucrose for their metabolism. Polysaccharide production by certain Streptococci enables dental plaque formation, and bacterial acids produced by the fermentation of dietary sucrose destruct dentin. As another possible mechanism, we have reported a reduction in dentin formation and thereby a possible reduction of dentinal response in young rat molars as a result of a high percentage (43%) of sucrose in the diet (Kortelainen and Larmas 1990, Larmas and Tjäderhane 1992). This effect is independent of other dietary constituents as well as the severity of the caries (Tjäderhane et al. 1994). Concomitant reduction in dentin mineralization is also observed (Tjäderhane 1996). We have also reported a negative correlation between caries progression and primary dentinogenesis (normal dentin formation) with a rapid rate of dentin formation (Tjäderhane 1995, Tjäderhane et al. 1994 and 1995a). The mechanisms by which diet alters dentin metabolism are not presently clear. To investigate further the role of dietary sucrose on dentin formation, caries initiation and dentinal caries progression, we conducted an experiment in which we used three different concentrations of sucrose to test whether a dose response existed.

MATERIALS AND METHODS

The rats were randomly divided into four groups of 10. The first group received a 15 g/100 g sucrose diet, the second group a 30 g/100 g sucrose diet, and the third group a 43 g/100 g sucrose diet (Tables 1, 2). The fourth group was fed a commercially available noncariogenic powdered pellet diet, produced especially for growing rats and mice (Brood Stock Feed for Rats and Mice R3, Ewos, Södertälje, Sweden), and served as a reference group. All of the ingredients of the diets were in a fine-powdered form. The sucrose diets were mixed in our laboratory to ensure a uniform constitution of the food. All rats were given free access to diet and tap water. The weight gain was recorded weekly, and food and water consumption were noted at intervals of 2-3 d. Because the main interest was the systemic effects of sucrose on dentin formation instead of caries, no inoculation of cariogenic bacteria was performed. Therefore rats had only their own oral bacterial flora.

Table 1. Composition of experimental diets [View Table]

Table 2. Nutritional values of diets [View Table]

Six weeks after the beginning of the experiment the rats were weighed, anesthetized by a combination of fentanyl-fluanisolone (Hypnorm, Janssen Pharmaceutica, Brussels, Belgium) and midazolam (Dormicum, Roche, Basel, Switzerland) and killed by decapitation. The jaws were defleshed and preserved in absolute ethanol.

All experimental procedures were approved by the Experimental Animal Committee of the Medical Faculty, University of Oulu, Oulu, Finland.

The first and second molars in the sections were also stained with Schiff 's reagent, and the number of intact fissures and the fissures with enamel or dentinal caries lesions was counted by a modification of the method of König et al. (1958). The teeth were scored by number and severity of fissure caries, classified as intact fissure, enamel lesion or dentinal lesion. The maximum potential caries scores would be 10 for the number of lesions (all fissures) and 20 for severity (0 for intact, 1 for enamel lesion and 2 for dentinal lesion) (Tjäderhane et al. 1995a). The size of the dentinal caries lesion, seen as a change of fluorescence (Hietala et al. 1993) under the main fissures, was also measured planimetrically as described above.

One-way ANOVA with Tukey's honestly significant difference test (Tukey 1949) was used to identity differences in dentin formation among rats fed the experimental sucrose diets. Pearson correlation coefficients were calculated to analyze the relationship between increasing dietary sucrose content and the progression of dentinal caries (Ascherio et al. 1992). To analyze the differences between the groups in caries progression, number and severity of carious lesions, the nonparametric Kruskal-Wallis ANOVA (Kruskal and Wallis 1952) was used to determine whether an overall difference existed among the sucrose groups because these data did not meet the assumption of homogeneity of variances required for ANOVA. If the difference was detected, Dunn's method (Dunn 1961) was used to determine differences among the groups. The level of statistical significance was set at P < 0.05.

RESULTS

The 43% sucrose diet significantly reduced dentin formation in both the first and second molars compared with the other sucrose diets (P < 0.05). Some reduction was observed also with the other sucrose diets compared with the reference group (Table 3). The differences obtained from the area measurements were supported by measuring the thickness of the dentin formed during the experiment in the direction of the fissure, although differences were not significant (Table 4).

Table 3. The area of dentin formed in first and second mandibular molars of rats fed diets differing in sucrose concentration for 6 wk1,2 [View Table]

Table 4. Thickness of dentin formed in first and second mandibular molars of rats fed diets differing in sucrose concentration for 6 wk1 [View Table]

When only intact fissures were chosen for comparisons among the groups, increasing dietary sucrose concentration resulted in similar reductions in dentin formation (Fig. 2). When only fissures with large caries lesions (10,000 µm² or more) were chosen for the comparisons, the outcome was again the same (Fig. 2). In these cases of selected fissures, statistical analysis could not be performed because selection of fissures with specific outcome would bias the results of the analysis.

Rats fed the 43% sucrose diet had significantly larger dentinal caries lesions than those fed the 15 or 30% sucrose diet in the first molar (P < 0.05)(Table 5). In the reference diet group, all of the fissures, except one, were intact. In the first molar, the area of dentinal caries lesions increased with the increasing concentration of sucrose in the diet (r = 0.3, P < 0.05). In the first molar, the area of caries lesions was ~twofold in the 43% sucrose group compared with the 15 and 30% sucrose groups.

Table 5. The area of dentinal caries lesions in first and second mandibular molars of rats fed diets differing in sucrose concentration for 6 wk1,2 [View Table]

The number (r = 0.5, P < 0.05) and severity (r = 0.6, P < 0.05) of caries lesions increased as the sucrose concentration of the diet increased as visualized by Schiff 's staining. The severity of caries lesions was greater in rats fed 43% sucrose than in those fed 15% sucrose (P < 0.05)(Table 6).

Table 6. Caries scores per rat fed diets differing in sucrose concentration for 6 wk1,2 [View Table]

DISCUSSION

Our overall hypothesis is that after the initiation of caries, the progression of the process is dependent not only on an external bacterial-induced attack via sucrose fermentation, but also on the intrinsic modulation or response by odontoblastic activity (Larmas et al. 1992). This response may be changed by sucrose during the cariogenic challenge. The dentin formation during the experimental period was mainly primary dentin (i.e., normal dentin formation). Therefore, modulation of the dentinogenesis by the test diets seems to be the outcome.

Several previous studies have shown sucrose-induced reduction in dentin formation either with high incidence of caries (Hietala and Larmas 1995, Kortelainen and Larmas 1990, Larmas and Tjäderhane 1992, Tjäderhane et al. 1995a) or with minimal caries development (Tjäderhane et al. 1994). The primary dentin formation may be slowed down by the toxic effects of bacterial metabolites during the progression of dentinal caries lesion, traumatizing or inhibiting the normal function of the odontoblasts. However, this is not the reason in this case, because when the dentin formation was measured in intact fissures, it was clearly reduced in the 43% sucrose group compared with other diet groups. Because the teeth were intact, the bacterial invasion could not disturb the dentin formation. Also, intact and carious (lesions < 10,000 µm²) fissures did not exhibit any differences in the amount of dentin formed during the experiment within the groups, further emphasizing the importance of systemic effects. Therefore it is not caries but the systemic effects of a caries-inducing diet that reduces dentin formation.

This experiment allows us to draw some conclusions about the concentration-dependent nature of the sucrose effect. In this experiment, the critical "threshold" point for the sucrose concentration was between 30 and 43% as can be observed by the following three variables: the significant reduction of dentin formation under the carious lesions; the increase in the number of carious lesions; and the increase in the severity of the carious lesions in the 43% sucrose group. This is supported by the finding that, in the 15 and 30% sucrose groups, there were no significant differences in dentin formation in spite of markedly greater caries scores and area of dentinal caries. This is particularly important because the traditional view holds that the increase in caries progression should increase the dentin formation to prevent pulpal exposure. On the other hand, the amount of sugar is not believed to be as critical to dental caries as is the frequency of intake of sugar (König et al. 1968), which was not the case in this experiment.

The possible mechanisms by which sucrose may reduce dentin formation include modulation of the calcium metabolism. Dietary calcium deprivation reduces dentin formation in rat incisors (Lozupone and Favia 1989, Messer and Guo 1979), in rat molars (Tjäderhane et al 1995b) and in rat molars after the use of calcium antagonists (Larmas and Tjäderhane 1992). Sucrose has been shown to alter bone composition in rats and hamsters (Li et al. 1990, Saffar et al. 1981, Salem et al. 1992, de Tessier and Saffar 1992). It is therefore possible that sucrose-induced alterations in calcium metabolism might also influence the formation of another mineralized tissue, dentin, because bone and dentin are very similar in composition and mode of formation (Linde 1989). This is further supported by the reduction in several mineral elements as well as the total content of minerals in dentin as a result of the high sucrose diet in the molars of growing rats (Tjäderhane 1996).

Diets rich in sucrose have been shown to induce hyperinsulinemia, insulin resistance and an increase in plasma glucose in rats (Grimditch et al. 1987 and 1988, Hara et al. 1992, Lockwood 1992). Therefore the effects on dentin may also be mediated via alterations in insulin metabolism.

Because we wanted to study primarily the effect of the diet on dentin formation and secondarily dentinal caries progression, we did not inoculate bacteria to induce rapid caries formation. The caries-inducing effect of the high sucrose diet in the absence of Streptococcus sobrinus demonstrates the ability of the normal oral flora of rats to produce cariogenic agents such as acids, as is clearly seen in the number of lesions (Table 6). The dependence of the caries progression on the amount of dietary sucrose is also confirmed because no caries-inducing bacterial inoculation was done in this experiment, as is the case in many previous studies in rats. Germ-free animals would be needed to exclude caries totally. However, in this experiment, without sucrose and S. sobrinus, practically no caries developed.

The increase of sucrose in the diet reduced the dentin formation and at the same time increased the number, severity and progression of caries; the critical amount of sucrose seemed to be between 30 and 43 g/100 g.