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Parotid Gland Function and Dentin Apposition in Rat Molars

¹ Department of Physiology/Pharmacology, Risley Hall, and
² Department of Internal Medicine, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA; and
³ Institute of Dentistry, University of Oulu, Oulu, Finland, and Department of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada;

*corresponding author, Jleonora{at}som.llu.edu

	ABSTRACT

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Previous studies have clearly established the down-regulating effect of a sucrose-rich diet on primary dentinogenesis in rat molars. Earlier observation of the negative effect of a high-sucrose diet on a parotid function involved in the control of intradentinal solute movement led us to hypothesize that parotid gland function(s) may have a role in regulating dentinogenesis. Dentin apposition in 1st and 2nd molars of young rats was measured by planimetry in sagittal sections. The following experimental variables were tested: standard and high-sucrose diets, removal of the parotid or the submandibular/sublingual glands, and diets in powder or pellet form. Removal of the submandibular/sublingual glands and changes in diet consistency did not significantly affect dentin apposition. Dentin apposition was significantly depressed by the high-sucrose diet or following parotidectomy. A further decrease followed the combination of the two treatments. Parotid glands appeared to exert a positive effect on dentin apposition in rat molars.

KEY WORDS: dentinogenesis • parotid hormone • salivary function • odontoblasts

	INTRODUCTION

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Ingestion of a sucrose-rich diet is associated with several deleterious effects, the best-documented of which is an increase in dental decay (Moynihan, 1998). Other debilitating metabolic effects have been observed, such as increased insulin resistance (Kim et al., 1999), hyperinsulinemia and decreased glucose tolerance (Thresher et al., 2000), and elevation of coronary artery risk linked to a decrease in high-density lipoproteins (Archer et al., 1998). A high-sucrose intake has also been linked to alterations in bone composition, mechanical strength, and metabolism (Tjäderhane and Larmas, 1998). Several studies in rats have confirmed the deleterious effect of a sucrose-rich diet on dentin apposition during primary dentinogenesis (Tjäderhane et al., 1994; Hietala and Larmas, 1995; Pekkala et al., 1998, 2000a), and this effect appears to be concentration-dependent (Huumonen et al., 1997). Recent findings suggest that the effect of sucrose in reducing dentin apposition is, at least in part, mediated through a systemic mechanism (Pekkala et al., 2000b). This mechanism, however, has not yet been identified and deserves attention.

In our laboratory, the ingestion of a high-sucrose diet has been linked to a decrease in parotid gland function. The basal plasma titer of immunoreactive parotid hormone, a factor isolated from porcine parotid gland that stimulates intradentinal dye penetration in rat molar teeth (Tieche et al., 1980), is significantly reduced in pigs fed a high-sucrose diet (Tieche et al., 1994; Tieche and Leonora, 1995). Similarly, under a high-sucrose regimen, intradentinal dye penetration in rats becomes depressed (Steinman and Leonora, 1971; Tieche et al., 1994). The observations of decreased dentinogenic function and of decreased parotid function that resulted from ingesting a high-sucrose diet raised the possibility that the parotid glands may have a role in regulating dentinogenesis. The latter function may be down-regulated in response to ingestion of a high-sucrose diet.

Dentinogenesis consists of highly controlled extracellular events orchestrated by a single layer of mature odontoblasts that secrete the fibrillar protein matrix of predentin and subsequently initiate its mineralization (Butler, 1998). The physiological control mechanisms regulating primary dentin deposition and mineralization are, however, still poorly understood. Thus, on the basis of the above observations, we hypothesized that normal dentinogenesis is, in part, under the control of a parotid gland function. The objective of this study was to provide evidence for the existence of a regulatory link between parotid gland function and dentin formation.

	MATERIALS & METHODS

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Dentin Apposition: Partial Desalivation and Standard Rat Diet
Sprague-Dawley 21-day-old male rats (Harlan, Indianapolis, IN, USA), weighing from 40 to 50 g each, were used in this and subsequent experiments. The rats were weaned on day 20 and delivered to our Animal Care Facility on day 21. They were assigned to four experimental groups: (1) control, intact; (2) control, sham-operated (sham); (3) parotidectomized (Px); and (4) submandibular/sublingual glands removed (Smx/Slx). Bilateral parotidectomy and removal of the submandibular/sublingual glands were done aseptically by blunt dissection while the animals were under general anesthesia, with Ketamine (40 mg/Kg) and Xylazine (10 mg/Kg) combined. Sham-operated animals were similarly treated, except that the glands were left in place. On day one of the experiment, the animals received an intraperitoneal injection of oxytetracycline-HCl (O-5875, by Sigma, St. Louis, MO, USA) in saline at the rate of 30 mg/Kg of body weight. Oxytetracycline served as a time marker for dentin apposition. The animals were fed a powdered standard rat chow for 5 wks (4% mouse-rat diet; Teklad, Indianapolis, IN, USA). This diet, which is manufactured in pellet form, was converted into a powder form by being passed through a food grinder. All rats were housed under normal atmospheric condition, at 21°C, and subjected to a 12-hour light/12-hour dark cycle. They had free access to food and water at all times. Body weight and food consumption were monitored twice a week throughout the experiment. On day 56, the food was removed by 8:00 a.m. Between 9:00 a.m. and 2:00 p.m., the animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (4 mg/100 g body weight), and the salivary glands were dissected out and weighed. The animals were then decapitated, and the mandibular jaws were removed, defleshed, and preserved in absolute ethanol. The jaws were coded, and further processing was done without the technicians' knowledge of treatment. First and second molars from each mandible were sectioned sagittally by the method of Keyes (1958). Dentin formation was measured planimetrically under the main central transverse fissure of each molar according to methodologies previously described in detail (Tjäderhane et al., 1994).

Dentin Apposition: Partial Desalivation and High-sucrose Diet
In a second experiment, the effect of partial desalivation was examined in rats fed a high-sucrose diet. The experimental design included 4 treatment groups. Group 1, serving as an internal control, was sham-operated and fed the standard rat diet. Groups 2 to 4 were maintained on the high-sucrose diet and consisted of groups that were either sham-operated, parotidectomized, or had their submaxillary/sublingual glands removed. The sucrose diet was a powdered diet containing 50% sucrose (# 8056, AIN 76A semi-purified meal, by Purina Test Diet, Richmond, IN, USA). The other experimental conditions were identical to those described above. After 5 wks, dentin formation was measured in mandibular molars by the same procedure.

Dentin Apposition and Diet: Composition vs. Texture
Because diet texture and composition can affect parotid gland function (Johnson et al., 1995), we tested the effects of both on dentin apposition. Using a 2 x 2 factorial experimental design, we fed weanling rats the standard rat diet or the high-sucrose diet in either a solid or powdered form. To create a similar solid consistency between the standard rat diet and the high-sucrose diet, we mixed each powder diet with water, using a diet:water ratio of 1:0.75 and 1:0.08, respectively. Each diet mixture was then extruded through a commercial meat grinder, yielding hard irregular pellets after forced-air-drying. Dentin apposition in mandibular molars was measured after 5 wks of treatment following the same procedure as above.

The experimental protocols for these studies were approved by the Loma Linda University Animal Research Committee, and animal treatments conformed at all times to the NIH guidelines for the care and use of laboratory animals.

Data Analysis
For each animal, the dentin surface areas deposited during the experimental time were averaged for each molar between the right and left mandibles. Mean dentin apposition values and standard error of the mean (mm² ± SEM) were computed for each treatment group. Terminal body weights and salivary gland weights were similarly computed and expressed as g ± SEM, and mg/100 g body weight ± SEM, respectively. Statistical difference between treatment means was assessed by one-way ANOVA. Further comparisons between individual treatment groups were carried out by the multiple-comparison post hoc test of Bonferroni when overall differences were indicated by ANOVA. Values of p < 0.05 were considered significant.

	RESULTS

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Parotidectomy was associated with a significant reduction in dentin apposition (Fig. 1). In rats fed the standard rat diet, 21% and 16% reductions, respectively, were observed in 1st and 2nd molars following parotidectomy (Fig. 2A). In rats fed the high-sucrose diet, parotidectomy was accompanied by a significant reduction (41%) in 1st molar dentin apposition, but the reduction in 2nd molars was not statistically significant (Fig. 2B). In contrast, removal of the submandibular/sublingual glands had no significant effect on dentin apposition in 1st or 2nd molars of animals fed either diet (Figs. 2A and 2B, respectively). Dentin apposition in the 1st and 2nd molars of animals who were sucrose-fed and sham-operated was significantly lower than in their counterparts fed the standard diet (Fig. 2B).

View larger version (168K): [in this window] [in a new window]   Figure 1. Representative photomicrographs of sagittal sections of 1st (A,B) and 2nd (C,D) molars of rats that were either sham-operated (A,C) or parotidectomized (B,D) at 22 days of age, and then maintained on a standard rat diet for 5 wks. The dentin formation was measured between the tetracycline label, marking the onset of the experiment (arrows), and the dentin-pulp interface. The dentinal area formed during the experimental period was notably smaller in the parotidectomized animals, especially in the 1st molars.

View larger version (56K): [in this window] [in a new window]   Figure 2. Effects of selective desalivation on dentin apposition in 1st and 2nd molars of young rats (A,B) and their terminal salivary gland and body weights (C,D). Twenty-one-day-old rats were surgically treated: sham-operated (sham), parotidectomized (Px), or submandibular/sublingual glands removed (Smx/Slx). They were maintained for 5 wks either on a standard rat diet (Std diet) (A,C) or on a high-sucrose diet (HSD) (B,D). The results for each treatment group are expressed as the mean ± SEM. Statistical difference among treatment groups was assessed by ANOVA, followed by the Bonferroni post hoc test, and group pairs found significantly different are indicated by double-headed arrows.

Dentin apposition in animals fed the powdered diet was slightly but not significantly lower than in animals fed the same pellet diet. This pattern was similar for both diets (Fig. 3A). Similarly, within each diet, texture did not affect parotid gland weight (Fig. 3B). The critical factor proved to be diet composition. Animals maintained on the high-sucrose diet, regardless of diet texture, experienced not only a decrease in dentin apposition but also a significant decrease in parotid gland weight (Figs. 3A, 3B). In comparison, the submandibular/sublingual glands were affected by diet texture. Without affecting dentin apposition, the glands from animals fed the powder form of either diet were significantly larger than those fed the pellet form of their respective diet (Figs. 3A, 3B). Finally, neither texture nor composition of the diets had any significant effect on body weight (Fig. 3B).

View larger version (42K): [in this window] [in a new window]   Figure 3. Effects of diet texture and composition on dentin apposition in molars of growing rats (A) and their terminal salivary gland and body weight (B). Twenty-one-day-old rats were maintained for 5 wks on standard rat diet (Std diet) or on a high-sucrose diet (HSD), and each diet was presented in the form of either pellet or powder. The results for each treatment group are expressed as the mean ± SEM. Statistical difference between treatment means was assessed by ANOVA, followed by the Bonferroni post hoc test. Significant differences resulting from either the effect of diet texture (pellet vs. powder) or diet composition are indicated by double-headed arrows.

	DISCUSSION

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Presently, our data do not permit definitive conclusions to be drawn regarding the mechanisms involved in the parotid control of dentinogenesis. However, the removal of the submandibular/sublingual glands that did not significantly affect dentin apposition emphasizes the specificity of parotid involvement (Fig. 2B). Hence, it appears that removal of parotid function(s) resulted in down-regulation of dentin apposition. In biological systems, control mechanisms imply some type of signaling process(es) between the moderator and the target tissues—specifically, here, the parotid gland and the pulp/dentin tissues. With the known functions of the parotid salivary glands being primarily exocrine, it is difficult to conceive, rationally, a route through which the parotid salivary components could reach and affect odontoblast cell functions. We propose that the parotid effect on dentin apposition could be the result of a parotid factor having a dentinogenic stimulatory effect on odontoblasts through an endocrine pathway. The parotid effect could be either direct or it could involve an undefined intermediate factor. Such a putative parotid factor could be as yet unidentified, or possibly it could be the parotid hormone that has been implicated in the control of intradentinal dye penetration (Leonora et al., 1993). If the latter is true, we may envision the control of dentinogenesis by parotid hormone as either a direct systemic effect upon the dentinogenic functions of odontoblasts, or as an activation of dentinal fluid movement (as measured by intradentinal dye penetration), which would facilitate the extracellular transport of dentinal matrix proteins. The result would be the enhancement of dentin apposition.

The concept that the parotid glands may possess an endocrine function is not new. In addition to our own work (Leonora and Steinman, 1968; Tieche et al., 1980; Leonora et al., 1987; Tieche and Leonora, 1989), Yamamotoet al. (1986a,b) isolated, from bovine and rat parotid glands, a protein that also stimulated intradentinal dye penetration. A significant body of published literature also supports the concept. Ogata (1955) isolated a biologically active protein from bovine parotid glands and called it parotin. Since 1960, more than 130 papers have been published which characterize the parotin molecule both chemically and biologically (MEDLINE search). Some of the reported effects from the administration of parotin to rodents and rabbits include: induction of hypocalcemia and leukocytosis; maintenance of bone, cartilage, and other connective tissues; and enhanced mineralization of incisor dentin (Iwasaki et al., 1984). To our knowledge, no evidence has been published showing that parotin affects dentin apposition.

Parotid gland weight has been linked to their salivary function (Johnson and Sreebny, 1982; Sheetz et al., 1983; Johnson et al., 1995). The possibility that parotid control in dentinogenesis may be related to salivary function was considered by a study of parotid gland weight in relation to treatments and dentin apposition. In our study, a consistent relationship between parotid gland weight and dentinogenic function was not apparent. In animals fed the standard rat diet, removal of the submandibular/sublingual glands resulted in a modest but significant increase in parotid gland weight (Fig. 2C) with no concomitant change in dentin apposition (Fig. 2A). In high-sucrose-fed animals, removal of the same glands was accompanied by a nearly 100% increase in parotid gland weight (Fig. 2D), and again without any change in the rate of dentin apposition (Fig. 2B). We conclude that, most likely, the changes in parotid gland weight reflect adaptive compensatory changes in salivary function in response to the removal of the other salivary glands (Sheetz et al., 1983).

From a different perspective, salivary function in rats can be modified by the texture of the diet: pellet vs. the powdered form (Johnson et al., 1977; Johnson and Sreebny, 1982). Converting a pellet diet into its powdered form caused a significant decrease in parotid gland weight (Johnson et al., 1977). However, the addition of a non-nutritive bulk to a powdered diet caused significant enlargement of the parotid glands (Johnson and Sreebny, 1982). In contrast, the effect of diet texture on the submandibular/sublingual glands was reported to be minimal (Hall and Schneyer, 1964). In view of these previous observations, we examined the possibility of whether the conversion of the pellet form of our diets into the powdered form might possibly have an effect on dentin apposition. We found that the primary determinant affecting dentin apposition was the composition of the diet. Changing the texture from the pellet to the powdered form did not alter the effect obtained with the pellet diet. This was also true for the effect on parotid gland weight (Fig. 3B). In contrast, the same conversion from pellet to powder form caused a significant increase in the weight of the submandibular/sublingual glands (Fig. 3B). Analysis of these data suggests that parotid control of dentinogenesis may work through function(s) other than the secretion of saliva. The discrepancies between our work and that previously reported could be the result of differences in conditions, such as the duration of the experimental period or the use, in earlier work, of diets similar but not identical in composition.

In conclusion, the parotid glands appear to have a positive effect on primary dentin apposition in rat molars. We propose that if a parotid factor exists which is involved in the control of dentin apposition, this factor may not be related to salivary function, but rather, it could be derived from a parotid endocrine function.

	ACKNOWLEDGMENTS

 
The authors thank Mr. Stacy Petersen (Department of Physiology, Loma Linda University) for his excellent assistance with animal works, and Ms. Eeva-Maija Kiljander (Institute of Dentistry, University of Oulu) for her skillful laboratory work. Funding from the School of Medicine, Loma Linda University, supported this work.

Received May 1, 2001; Last revision February 1, 2002; Accepted February 7, 2002

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