Eeva-Liisa Hietala

Dentin Caries Recording with Schiff's Reagent, Fluorescence, and Back-scattered Electron Image

After being weaned, the Wistar rats (12) were fed on a sucrose diet for five weeks to induce dental caries. Tetracycline was injected intraperitoneally into 7 of them to label the mineralizing dentin front. Five rats without tetracycline injection were used to verify spontaneous fluorescence. The evidently carious area under one prominent fissure from each mandibular molar hemisected sagittally in the midline was photographed under ultraviolet light so that autofluorescence would be revealed. The jaws were then stained with Schiffs reagent and photographed under normal light and again under ultraviolet light. The areas of all the lesions were quantified planimetrically as they appeared on the photographs. The mineral contents of the areas were verified with the back-scattered electron images. The caries lesions revealed with Schiffs reagent also exhibited a change in the color of the dentin fluorescence regardless of the tetracycline labeling. The areas of these lesions followed the shapes of the lesions stained by Schiffs reagent but they were greater. Staining with Schiffs reagent was repeatable after the specimen was washed with ethanol for a few weeks. The loss of minerals was seen in the areas stained with Schiffs reaction but was also related to the change in dentin fluorescence, which seems to be a more sensitive indicator of the caries progression than Schiffs reagent, especially in the early phase of the carious process.

Reduction in dentine apposition in rat molars by a high-sucrose diet

The effects of high-sucrose and high-starch diets on dentine apposition were examined. One group of young Wistar rats (20 rats) received a high-sucrose (43%) diet; for the high-starch group (16 rats), sucrose was replaced with potato flour. The control group (18 rats) received standard laboratory rat food. The onset of dentine formation was marked with tetracycline. After 5 weeks lower molars were sectioned sagittally, and the areas of the dentine apposition and those of the dentinal caries were quantified. Dentine apposition was independent of sex. The high-sucrose diet reduced dentine apposition and induced caries progression. A negative correlation between dentine apposition and caries progression in the molars was found. This study suggests that a high-sucrose diet itself reduces primary dentine apposition.

Mineral Element Analysis of Carious and Sound Rat Dentin by Electron Probe Microanalyzer Combined with Back-scattered Electron Image

We recently demonstrated the advantages of back-scattered electron images (COMPO) in the visualization of dentinal caries, and the relationship of the change in the dentin fluorescence pattern in caries lesions. However, the exact nature of these changes is not known. In this paper, the nature of the changes in the areas with reduced mineral content in COMPO images was investigated. We examined the relation of changes in mineral elements and the appearance of soft carious and sound dentin in COMPO images using a scanning electron microscope (SEM) equipped with an electron probe microanalyzer (EPMA). Rat molars with small dentinal caries lesions just under the DEJ were chosen for the study. The Ca, P, Na, Mg, Zn, F, and total contents were determined by EPMA from five different dentin sites, and the Ca/P and Mg/Ca ratios were calculated. Generally, the lowest contents were found in caries lesions and highest in mantle dentin, with the exceptions of Mg and Zn. The Ca/P ratio was lowest in mantle dentin and highest in carious dentin. The results confirm that the change in fluorescence in the dentinal caries lesion is correlated with the very initial changes in mineral content, and that EPMA used in combination with COMPO images is a useful tool for determining small changes in mineral elements in the carious and adjacent areas of dentin.

The effect of ovariectomy on dentin formation and caries in adult rats

Since acute osteoporosis is known to enhance bone remodeling and osteoid formation, it may also affect dentin apposition. We induced osteoporosis in 15-week-old Wistar rats by ovariectomy and dental caries by Streptococcus sobrinus infection in the presence of either a high sucrose (cariogenic) diet or a non-cariogenic diet. Intact animals with the same diets served as controls. After 99 days of cariogenic challenge the rats were killed, and the success of ovariectomy was confirmed by failure to detect ovarian tissue and observation of marked atrophy of the uterine horns. Areas of dentinal apposition during the experiment and carious lesions were quantified with a tetracycline method. Ovariectomy activated dentin formation significantly in rats fed either a high-sucrose or a non-cariogenic diet, indicating enhanced odontoblast function. The rate of dentinal apposition in adult rats was smaller than reported in young animals. The effect of ovariectomy on caries remained negligible.

Localization of Estrogen-receptor-related Antigen in Human Odontoblasts

Estrogen receptors have been demonstrated in many osteogenic cell lines. Recently, we showed that estrogen deficiency induced by ovariectomy caused enhanced dentin formation in adult rats, suggesting that estrogen receptors may be present in dental tissues. Nothing is known about estrogen receptors in human teeth. We used immunohistochemical staining and immuno-blotting to demonstrate the presence of estrogen receptors in human pulp and/or the pulpo-dentinal border. Unerupted human wisdom teeth were surgically removed, frozen in liquid nitrogen, and prepared for immunological studies. Western blot analysis with monoclonal antibodies specific for human estrogen-receptor-related antigens demonstrated an approximately 29-kDa clear double band in the material scraped from the predentin-odontoblast border and in the fluid that emerged into the pulpal chamber, evidently from the odontoblasts. A weaker double band was also present in pulpal tissue samples. By immunohistochemical staining, estrogen-receptor-related antigens were visualized in the predentinal-odontoblast region and in the pulpal blood vessels. Our results suggest the presence of estrogen receptors in human teeth, and thus the previously reported enhancement of the dentin formation in rats after ovariectomy may be mediated via these receptors.

Evidence that high-sucrose diet reduces dentin formation and disturbs mineralization in rat molars.

In addition to its caries-promoting effect, a high-sucrose diet reduces the apposition of mineralized dentin in young rats. This study was undertaken to test whether it has a similar effect on the width of the as-yet-uncalcified matrix, predentin. Female Wistar rats were weaned at the age of 3 weeks and fed for 7 weeks with either a high-sucrose diet, a non-cariogenic raw potato starch diet, or a non-cariogenic commercial powdered rat food (for reference). The sucrose diet induced the greatest number of caries lesions. Dentin formation was smaller and the predentin zone wider in rats fed a sucrose diet when compared with rats fed the reference diet. In rats fed a starch diet, dentin formation was smaller than in rats fed a reference diet, but no alterations in the areas of caries lesions or predentin width were observed. Widening of the predentin zone in rats fed a high-sucrose diet may reflect changes in odontoblast function, such as reduced matrix synthesis and possibly disturbed mineralization. Dentinal caries progression may thus be modulated by odontoblast function, not as much by matrix formation but rather by mineralization.

The effect of ovariectomy and high-sucrose diet on dentine formation and caries in growing rats

Ovariectomized (Ovx) or sham-operated (Cnt) and tetracycline-labelled growing rats were fed either a commercial powdered standard rat diet (Ovx and Cnt) or a high-sucrose diet (Ovxsuc and Cntsuc). All animals were inoculated in the mouth with Streptococcus sobrinus. At 11 weeks of age the areas of caries lesions and dentinal apposition of the first and second molars and the areas of the pulpal cross-section of the incisors were determined. The area of dentinal apposition was largest in Cnt animals, the difference being statistically significant only in comparison with the Cntsuc animals. No such difference between the diets was found among the Ovx animals. The cross-section of the pulpal chamber was smallest in the Ovxsuc rats and largest in the Cnt animals, but no statistically significant differences were found between the groups. The caries lesions were significantly larger in Ovx, Ovxsuc and Cntsuc animals than in Cnt ones. Ovariectomy thus increased caries progression in growing rats, but the effect on dentine formation remained negligible. In this respect there is a difference between primary and secondary dentinogenesis.

The effect of two sucrose diets on formation of dentin and predentin in growing rats

The effect of two high-sucrose diets on dentinal caries, dentin formation, and the predentin width was studied in Sprague-Dawley rats. Rats were weaned at the age of 3 weeks and for 4 weeks fed a non-cariogenic commercial rat food (R36) for control, a high-sucrose Stephan-Harris (S-H) diet, or a new high-sucrose (sR36) diet in which most of the barley and wheat flour of the control R36 diet were replaced by sucrose. The areas of dentinal caries, the areas of dentin formation, and the width of predentin and dentin were quantified. Both high-sucrose diets induced dentinal caries, and both reduced dentin formation and increased the width of predentin compared with the control diet. Moreover, rats fed the S-H high-sucrose diet showed significantly greater progression of caries and reduction of dentin formation relative to rats fed the new high-sucrose diet, sR36. The high-sucrose diet thus was a substrate for caries-inducing microbes and a significant, but possibly not the exclusive, substrate for host modulation of odontoblast function.

Morphological analysis of dentine formation in young rat molars during the recovery phase with calcium alone or combined with xylitol following a low-calcium dietary regimen

The effects of dietary calcium deficiency and subsequent replenishment of the diet with calcium alone or with xylitol were studied. Thirty 3-week-old Wistar rats were labelled with an i.p. tetracycline injection. Twenty rats were fed a diet with 0.026% calcium (Ca-deficient); 10 received a 0.5% Ca diet (controls). After 3 weeks the tetracycline labelling was repeated. Replenishment of the diet was introduced for Ca-deficient rats, and 10 received additional 5% xylitol in the diet. After 4 weeks the labelling was repeated and the animals were decapitated. Dentine formation was measured by the tetracycline stripes in the lower first and second molars. Calcium deficiency during the first 3 weeks reduced dentine formation. In the control and xylitol groups, a much smaller amount of dentine was formed during the recovery period. With Ca alone, dentine formation was faster than in the controls or Ca-xylitol group and did not differ from the Ca-deficient period. These results indicate that in rat molars the odontoblasts can accelerate the rate of dentine formation when calcium is restored to the diet, at least during primary dentinogenesis. This effect was not seen when 5% xylitol was added to the replenishment diet.

Effects of a high sucrose diet and intragastric sucrose feeding on the dentinogenesis, dental caries, and mineral excretion of the young rat

Previous studies show that a high sucrose diet reduces the rate of primary dentinogenesis and increases dental caries, although their cause-effect relationship is still obscure. The purpose of this study was to explore whether the effect of sucrose load on the dentinogenesis and dental caries of young rat molars is mediated by systemic (intragastric) or by systemic and local (dietary) factors. At weaning (19 days), animals were randomized into the control, intragastric sucrose, and dietary sucrose groups for 4 weeks. The areas of dentin appositions and dentinal caries lesions were measured planimetrically. Caries was also determined with Shiffs staining and the width of predentin by histology. Urinary Ca, K, and Na levels were measured by flame photometry, urinary P levels using an UV method, and serum insulin levels using radioimmunoassay. Systemic and local sucrose load reduced dentin appositions and intragastric sucrose increased urinary Ca excretion. No differences in the width of predentin were noticed. Only dietary sucrose enhanced the occurrence and progression of caries. The present findings show that sucrose load reduces dentinogenesis by impairing the synthesis of dentin matrix, but also point out the crucial importance of the local sucrose challenge in the initiation of dental caries.