Archives of Oral Biology (2006) 51, 252—261 www.intl.elsevierhealth.com/journals/arob SHORT COMMUNICATION The dentin sialoprotein (DSP) expression in rat tooth germs following fluoride treatment: An immunohistochemical study Izabela Maciejewska a,*, Jan Henryk Spodnik b, Sławomir Wójcik b, Beata Domaradzka-Pytel b, Zdzisław Bereznowski a a Medical University of Gdańsk, Department of Oral Implantology, 18 Orzeszkowa Str., 80-208 Gdansk, Poland b Medical University of Gdańsk, Department of Anatomy & Neurobiology, 18 Orzeszkowa Str., 80-208 Gdansk, Poland Accepted 12 July 2005 KEYWORDS Dentin sialoprotein; Immunohistochemistry; Fluorides; Tooth development Summary Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications. Objective: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride. Design and methods: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry. Results: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (FL) but not in the group supplemented with the high fluoride concentration (FH). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the * Corresponding author. Tel.: +48 58 349 16 20; fax: +48 58 349 16 20. E-mail address: Izabelam@amg.gda.pl (I. Maciejewska). 0003–9969/$ — see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2005.07.003 DSP expression in rat tooth germs after fluoride treatment 253 positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong. Conclusions: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs. # 2005 Elsevier Ltd. All rights reserved. Introduction Since dental fluorosis has been recognized as a side effect of fluoride caries prevention, research efforts have been focused on identifying fluoride-susceptible points of odontogenesis.1—4 Fluoride delays proteolytic degradation and removal of amelogenin, an enamel non-collagenous protein, during the maturation stage of amelogenesis.5 Amelogenin removal is a prerequisite for proper enamel mineralization; however, the manner in which fluoride influences the metabolism of amelogenin remains unclear. It is postulated that fluoride may change the molecular conformation of the protein and subsequently enhance its binding to fluoridated enamel crystals.6,7 It is also suggested that fluoride diminishes the free Ca2+ concentration in the vicinity of the crystals, thus decreasing Ca2+-sensitive protease activity.8 The impact of fluoride on ameloblasts is also considerable. It has been shown that even low concentrations of fluoride slow down ameloblast proliferation9 as well as the transformation of smooth-ended into ruffle-ended ameloblasts,10 which alters their functional efficiency. Fluoride adversely influences dentin formation as well. Although dentin, unlike enamel, is derived from mesenchyme, dentin fluorosis may be caused by local disturbances that take place in the dentin extracellular matrix. Dentinogenesis involves secretion of mainly type I collagen and several noncollagenous proteins to form a collagenous scaffold. Next, carbonate apatite crystals are deposited on the existing scaffold. Dentin phosphoprotein (DPP) and dentin sialoprotein (DSP) are two types of dentin non-collagenous matrix proteins. These proteins were originally thought to be dentin-specific until Qin et al.11 described their presence in bone tissue. The bone concentration of DSP was found to be four hundred fold lower than that in dentin.11 Nucleotide sequences of DPP and DSP are localized next to each other and encode the single DSPP transcript. The DSPP gene was found on chromosome 5 in humans12 and chromosome 4 in rats.13 The secreted parent DSPP protein is then proteolitically cleaved to yield DPP and DSP. DPP has been confirmed as the main non-collagenous dentin specific matrix protein, responsible for the nucleation and mineralization of dentin.14—17 Recent studies indicate that fluoride drastically decreases the phosphorylation of DPP.18,19 Most likely, fluoride does not affect DPP directly. Available data indicate that fluoride impairs action of casein kinase II and alkaline phosphatase, two enzymes which are involved in DPP phosphorylation.19 The decrease in DDP phosphorylation leads to the reduction of Ca2+-binding abilities and ultimately to hypomineralization of dentin. DSP is the second most abundant non-collagenous dentin protein.20,21 Its function in odontogenesis is still obscure. However, DSP’s unique expression in odontoblasts and predentin just before the onset of mineralization suggests that it may participate in dentin formation.22,23 DSP is also thought to be a factor in the conversion of unmineralized dentin matrix into mineralized tissue.24 The fact that DSP is coded together with DPP as one transcript known as DSPP25 could be regarded as an argument in favor of these presumptions. However, there is evidence that ‘‘all of the phosphorylation sites were located at the parts of transcripts containing the DPP coding information.’’12 Recent data demonstrate that DSP has a limited effect on the formation of hydroxyapatite,26 suggesting that DSP can play a regulatory role in dentinogenesis. Considering DSP’s role in dentin formation and assuming that fluoride given during pregnancy is freely transferred through the placenta to the offspring,27,28 the aim of our study was to investigate the potential changes in DSP developmental expression and its localization pattern in tooth germs of rats supplemented with different doses of sodium fluoride in drinking water during prenatal life and post delivery. We focused our study on the expression of DSP in newly differentiated cells, e.g., preodontoblasts, young odontoblasts, and preameloblasts. In addition, we investigated the formation of dentin matrix in both early and late bell stages of the lower first molar tooth germs in 1- and 4-dayold animals, respectively. As with other investigators, we defined young odontoblasts as polarized cells associated with early predentin formation. Mature odontoblasts were defined as those whose Tomes’ processes are embedded in mineralized dentin.29,30 The 4-day-old groups allowed us to follow changes in the DSP expression pattern under the same experimental conditions. We decided to investigate the lower first molar tooth germ assuming that it is better model for potential comparison 254 I. Maciejewska et al. to other species than the continuously growing rat incisor. Material and methods Animals Animal care and treatment guidelines outlined by the European Community Council (1986) and protocols approved by the local ethical committee were followed. Nine pregnant albino Wistar rats were randomly divided into three equal groups: control (C), supplemented with a low concentration of sodium fluoride in drinking water (FL), and supplemented with a high concentration of sodium fluoride in drinking water (FH). Dams were fed a standard diet containing a trace amount of fluoride. From the fifth day of pregnancy until the end of the experiment, the three groups drank water that contained: control group–—0.16 F mg/l (tap water), low-concentration sodium fluoride group–—tap water with 11 mg/l of NaF (fluoride content 5.14 mg/l), and high-concentration of sodium fluoride group–—tap water with 110 mg/l of NaF (fluoride content 49.96 mg/l). Sodium fluoride concentrations followed the protocol used in our previous investigations32,33 and others.34 The high sodium fluoride concentration was calculated according to the mean body weight of dams (22.6 mg NaF/kg b.w.) and was the maximal dose at which the risk of spontaneous abortion or preterm delivery was smallest.35 Water consumption was monitored daily. After delivery mothers and their offspring underwent the same water regime. The experiment was performed three times. The mean weight of pups in each group is shown in Table 1. Tissues On days 1 and 4, animals were deeply anaesthetized with Nembutal (80 mg/kg i.p. body weight) and perfused through the ascending aorta with physio- Table 1 Comparison of the body weight of 1- and 4day-old pups in particular groups: C–—control, FL–—the group supplemented with the low fluoride concentration, and FH–—the group supplemented with the high fluoride concentration. P1 P4 C FL FH 7.30  0.93 12.08  1.30 7.96  0.53 10.61  2.90 7.63  0.87 11.57  1.51 Data in grams, mean value  S.D. logical saline (pH 7.4) with heparin, followed by perfusion of 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4; 4 8C). After decapitation, heads were postfixed for 1 h at 4 8C and dehydrated in 15% sucrose followed by 30% sucrose (both in 0.1 M phosphate buffer at pH 7.4 and 4 8C) until sunk. It was not necessary to demineralize the tissue, which is known to abolish immunostaining for DSP.31 The tissue was then frozen and sectioned sagittally into 16-mm-thick serial sections using a cryostat (Young 1800; Leica, Germany). Every sixth section was routinely stained with hematoxylin eosin (H&E), air-dried, and cover slipped with DPX mounting medium (Fluka, Germany). Immunohistochemistry Sections were preincubated in 10% normal goat serum and 0.1% solution of Triton X-100. They were then incubated for 1.5 h at room temperature with rabbit polyclonal antibodies against dentin sialoprotein DSP diluted 1:100.36 Subsequently, they were incubated in secondary antibodies: goat anti-rabbit conjugated with Cy-3 (Jackson, USA) at 1:600 for 2 h at room temperature. Sections were rinsed in PBS, mounted on slides, dried, and coverslipped with Kaiser gelatin (Merck, USA). In the control sections, the primary anti-DSP antibodies were omitted and replaced by buffer. In all the investigated groups either 1 or 4 days of age, the controlled sections stained without anti-DSP antibodies showed none DSP positive immunoreactivity in all the investigated structures (not shown). Data analysis Immunohistochemically stained sections were examined with confocal laser microscopy (Radiance 2100; Bio-Rad, UK). The system was equipped with krypton/argon lasers and mounted on an Eclipse 600 (Nikon, Japan) microscope, using the software LaserSharp 2000 v. 4.0 (Bio-Rad). The confocal laser scanning microscopy (CLSM) images were obtained using 20 and 60 oil immersion objective lenses of NA = 1.3 and 1.4, respectively. Statistical analysis was performed by means of the computer program Statistica v. 5.0 (Statsoft, USA) and Excel 2000 (Microsoft, USA) in the following manner. All calculations were performed on spreadsheets. Data of the test fields from each case were collected and the mean value calculated. These averaged values were used as raw data for statistical analysis. The oneway analysis of variance followed by post hoc honest significant difference (HSD) test was applied to estimate the differences between results for various age groups, as this test is robust against deviations DSP expression in rat tooth germs after fluoride treatment 255 from the assumed population normality and homogeneity of variances. Because the aim of the study was to analyze the changes of immunoreactivity with increasing age, the differences among only consecutive pairs of means were taken into account (although this multiple comparison test calculates the significance level for all possible comparisons). For each experimental group descriptive statistics were calculated and analyzed as mean values with standard deviation ( S.D.). The alpha-level was set at 0.05 for statistical significance. Results Water consumption Daily water consumption differed in each group. The differences were statistically significant between the control and the group supplemented with low doses of sodium fluoride ( p < 0.05) as well as between the control and the group supplemented with high doses of sodium fluoride ( p < 0.001) (Fig. 1). DSP expression in the early bell stage (1-day-old groups) The first positive signal of DSP expression appeared in the early bell stage of the control group. The signal was detected at the basal pole of preameloblasts from the cervical loop region of the first molar tooth germ (Fig. 2A). The same region stained very weakly in the group supplemented with the low concentration of sodium fluoride (FL) (Fig. 2B) and was almost immunonegative in the group supplemented with the high concentration of sodium fluoride (FH) (Fig. 2C). In the more developmentally Figure 1 Water consumption in C–—control, FL–—group treated with low fluoride concentration, and FH–—group treated with high concentration of fluoride. The significance level according to control group: *p < 0.05 and ** p < 0.001. Figure 2 DSP localization in early bell stage of first molar tooth germ (cervical loop region) from (A) control group, (B) FL group, and (C) FH group. Preameloblasts stain weakly positive in C and FL groups. Preameloblasts from FH group are negative. Abbreviations: ab, alveolar bone; dp, dental pulp; pa, preameloblasts. Scale bar 50 mm. 256 I. Maciejewska et al. Figure 3 DSP expression in 1-day-old first molar tooth germ, the cusp tip region. (A and B) the control group (C): young odontoblasts, mature odontoblasts, dental pulp, and predentin (pd) stain strongly. Mineralizing dentin stains at the moderately (d). Preameloblasts are weakly positive for DSP; (C and D) the group supplemented with the low fluoride concentration (FL): young odontoblasts, odontoblasts, dental pulp, and predentin (pd) stain strongly. Mineralizing dentin (d) stains moderately. Preameloblasts are weakly positive for DSP antibodies. Alveolar bone is negative. (E and F) the group supplemented with the high fluoride concentration (FH): young odontoblasts (yo), dental pulp, and predentin DSP expression in rat tooth germs after fluoride treatment advanced parts of the first molar tooth germ (the cusp tip), young polarized odontoblasts, adjacent to predentin, showed a strong DSP immunopositive reaction similar in all water groups (Fig. 3). This staining could not be detected in the rest of the examined groups. Mature odontoblasts immunostained for DSP in all investigated groups; however, in the FL and FH groups, the staining was more intense. In the control group, preameloblasts associated with early predentin but not dentin were weakly immunopositive for DSP (Fig. 3A and B), while in both fluoride-treated groups (FL and FH), ameloblasts lining dentin were weakly DSP positive as well (Fig. 3C—F). In the FH group, DSP staining of ameloblasts was much more intense (Fig. 3E and F) than those in other groups. The strong positive staining for DSP was also detected in the dental pulp cells in all experimental groups; however, some cells stained more intensely than others. Predentin in all groups showed very intense staining, but the staining decreased in dentin as mineralization progressed. The greater homogeneity in the predentin immunostaining was observed in the control group (Fig. 3A and B) and decreased in the FL (Fig. 3C and D) and FH (Fig. 3E and F) groups, respectively. The widest layer of predentin and dentin was observed in the FH group (Fig. 3E and F), but the structure of both tissues, especially predentin, showed the presence of some specific ‘‘empty spaces.’’ DSP expression in the late bell stage (4-day-old groups) All examined tooth germs at the late bell stages showed changes in the intensity of staining. The positive reaction for DSP antibodies at the cervical loop region of the first molar tooth germs became more visible in all groups (Fig. 4A—C). In the cervical loop region, the positive signal for DSP also appeared in the FH group. In the control group, odontoblasts stained weaker (Fig. 4D) than those from the fluoride-treated groups of the same age and younger controls. The immunopositive reaction for DSP in predentin was still very intense but weaker than in both FL (Fig. 4E) and FH (Fig. 4F) groups. Dentin from the control group stained strongly at the mineralization front; the intensity of staining decreased proportionally as mineralization progressed (Fig. 4D). In the fluoride-treated groups, (Fig. 4E and F) the intensely stained layer 257 of dentin was visibly wider. In all investigated groups dental pulp showed diminished immunoreactivity for DSP in comparison with the 1-day-old groups. However, the least reduction in immunoreactivity was observed in the FH group (Fig. 4F). In all groups, either enamel or ameloblasts were almost completely immunonegative. The only weakly positive reaction for DSP in ameloblasts could be found in the FH group (Fig. 4F). Discussion The DSP expression pattern in tooth germs of rats treated with different concentrations of sodium fluoride during prenatal and early postnatal life differs between early and late bell stages of tooth development. These differences were also observed in the control group and groups supplemented with sodium fluoride in drinking water. In the early bell stage (1-day-old animals), the DSP-positive reaction was detected in young, mature odontoblasts and preameloblasts in all groups. This finding was previously reported,29,31 although the reaction in odontoblasts from the FH group was weaker than that of the C and FL groups. Conversely, at the late bell stage (4-day-old animals) the reaction in odontoblasts from the control group was weaker than that of both fluoride-supplemented groups. Surprisingly, in the 1-day-old FH group, preodontoblasts lining the basement membrane were positive for DSP antibodies. This observation contradicts the classic DSP expression pattern established in previous investigations29,31 as well as our own results obtained from the control group. Additionally, there was no DSP immunoreactivity in preodontoblasts from any of the investigated 4-day-old groups with late bell stages of tooth development. The positive DSP expression in 1-day-old preodontoblasts from the FH group suggests the existence of disturbances in their development. Some recent studies have demonstrated that fluoride has an adverse effect on the morphology, proliferation, and function of ameloblasts.37,38,8—10 In our study, the first positive reaction for DSP was detected in the ameloblastic cell lineage. In the early bell stage, the preameloblasts were found to be weakly stained at the cervical loop region in the control and the FL group. However, DSP expression was not found in the FH group, which is a novel stain strongly. Predentin shows specific ‘‘empty spaces’’ (arrowheads). Mineralizing dentin stains moderately (d). Odontoblasts stain very intensely. Preameloblasts and tall ameloblasts are weakly positive for DSP antibodies. Young odontoblasts stain moderately (yo), preodontoblasts are weakly positive (po). Alveolar bone (ab) is negative. Scale bar 50 mm. 258 I. Maciejewska et al. Figure 4 DSP localization in 4-day-old first molar tooth germ (apical region) from (A) the control group (C): odontoblasts and dentin show moderate staining. Predentin is strongly immunopositive for DSP (pd). Alveolar bone, ameloblasts (a), enamel (e) and dental pulp are negative and (B) the group supplemented with the low sodium fluoride concentration: odontoblasts and predentin (arrow) are strongly immunopositive. Mineralizing dentin (d) stains moderately but more intense than those in the control group. Preameloblast (arrow), ameloblasts, and dental pulp are faintly positive for DSP antibodies. Alveolar bone is negative, (C) the group supplemented with the DSP expression in rat tooth germs after fluoride treatment finding not yet reported. The lack of reactivity to DSP in the early bell stages was probably caused by delay in the embryonic development observed in groups of animals treated with the high sodium fluoride concentration. In the FH group, the positive staining for DSP in preameloblasts from the apical area comparable to other groups (C and FL) became visible at the late bell stage (4-day-old animals). In our study, like others,29,30 preameloblasts at the cusp tip region in all 4-day-old groups were also immunopositive for DSP. It has been previously noted that positive immunostaining for DSP in the ameloblastic cell lineage is transient and rapidly diminishes immediately after the onset of dentin mineralization.31 In our study, the prolonged positive reaction for DSP was observed even after the onset of dentin mineralization in both fluoride-supplemented 4-day-old groups but not in controls. The positive DSP reaction was especially prolonged in the FH group. The prolonged expression of DSP in ameloblasts suggests that fluoride somehow modifies the DSP expression in ameloblastic lineage cells. These changes may reflect a direct effect of fluoride on ameloblasts or an indirect effect of fluoride-dependent changes in DSP posttranslational modifications. Another conspicuous feature observed in our investigation was the height of ameloblasts from the FH group. Ameloblasts were much taller than those from other groups. We believe that this observation is the result of disturbances in ameloblast development. Unfortunately, we were not able to assess the exact intracellular changes with the confocal microscope. The presumption that fluoride could alter ameloblast development is in agreement with previous findings by Smith et al.,37 who observed that ameloblasts associated with developing fluorotic enamel remain ruffle-ended longer than controls. Similar to Linde,39 we detected a strong immunopositive reaction for DSP in cells of dental pulp for all early bell stages. In our investigation, the staining in dental pulp cells decreased consistently in the late bell stage from all investigated groups, although specific cells stained more intensely than others. Nevertheless, because of the complete lack of differences in the staining intensity in investigated 1- and/or 4-day-old groups, as well as the proved absence of DSP-mRNA transcripts in the dental pulp cells in all developmental stages,30 we believe that some of the proteins present in dental pulp cells may contain epitopes recognizable by polyclonal DSP antibodies that we used in 259 our experiment. In 1-day-old animals from all groups both predentin and dentin showed positive immunostaining to DSP comparable to those observed in the previous study29,30 however, the homogeneity of the tissues differed. In dentin from the FH group of both ages we observed unusual, specific empty gap like structures, which disturbances in tissue formation similar to those observed by Appleton.40 Conversely, in the 4-day-old control group, DSP expression in predentin and dentin was still consistent with its developmental pattern (the positive staining decreased in dentin proportionally to its mineralization progress), while in both groups treated with fluoride the width of strongly positively stained dentin was conspicuously greater. Based on the findings of our study, we conclude that fluoride supplementation during prenatal and early postnatal life can induce the modifications in the DSP developmental expression pattern in rat tooth germs. High-dose fluoride supplementation in the earliest stages of odontogenesis is most likely responsible for the positive immunoreactivity for DSP antibodies in preodontoblasts. Concurrently, it appears that fluoride causes the prolonged expression of DSP in ameloblasts, most likely due to fluoride-dependent disturbances in their development. This may affect reciprocal mesenchymal— epithelial interactions and leads subsequently to altered dentin and/or enamel mineralization pattern. It is still not known whether prolonged DSP expression in ameloblasts is caused by direct, fluoride-dependent cellular impairment or whether fluoride influences DSP posttranslational modifications. The role of DSP in odontogenesis remains unknown. Thus, it is difficult to hypothesize about the direct impact of prolonged DSP expression on dentin formation. However, resent studies indicate a chaperone function of DSP in folding and transporting parental DSPP molecule. DSP also may play a role in downregulating the amount of DPP delivered to the mineralization front.41 This may suggest that the fluoride-dependent prolonged expression of DSP could possibly result in lower concentration of DPP at the mineralization front causing a delay in nucleation of apatite crystals and dentin mineralization. Thus, further investigations should focus on elucidating the role that DSP plays in dentinogenesis and possible fluoride-induced changes in the molecular conformation of DSP. An additional question to be answered is whether fluoride’s effect on DSP expression is dose-dependent. high sodium fluoride concentration. Odontoblasts (o) and predentin (pd) stain strongly. Dentin (d) and dental pulp (dp) stain moderately. Ameloblasts (a) are tall and faintly positive for DSP. Alveolar bone (ab) is negative. Scale bar 50 mm. 260 Acknowledgements The authors wish to thank Dr. W.T. 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Al-Hiyasat AS, Elbetieha AM, Darmani H. Reproductive toxic effects of ingestion of sodium fluoride in female rats. Fluoride 2000;33:79—84. 36. Butler WT, Bhown M, Brunn JC, D’Souza RN, Farach-Carson MC, Happonen RP, et al. Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP). Matrix 1992;12:343—51. DSP expression in rat tooth germs after fluoride treatment 37. Smith CE, Nanci A, DenBesten PK. Effects of chronic fluoride exposure on morphometric parameters defining the stages of amelogenesis and ameloblast modulation in rat incisors. Anat Rec 1993;237:243—58. 38. Monsour PA, Harbrow DJ, Warshawsky H. Effects of acute doses of fluoride on the morphology and the detectable calcium associated with secretory ameloblasts in the rat incisors. J Histochem Cytochem 1989;37:463—71. 261 39. Linde A. The extracellular matrix of the dental pulp and dentin. J Dent Res 1985;64:523—9. 40. Appleton J. Formation and structure of dentin in the rat incisor after chronic exposure to sodium fluoride. Scanning Microsc 1994;8:711—9. 41. Butler WT, Brunn JC, Qin C, McKee MD. Extracellular matrix proteins and dynamics of dentin formation. Connect Tissue Int 2002;43:301—7.