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Enamel mineral concentration in diabetic rodents.

Abstract

Aim: This was to assess the mineral composition and 3-D structure of enamel and bone in the teeth and skulls of diabetic rodents. Methods: Three-dimensional images of the skull were reconstructed using x-ray microtomography (XMT) computer-generated false colour to highlight different levels of mineralization in bone and enamel. Results: These showed that diabetic rodents exhibited more wear in their teeth. Deformities were observed in the alveolar process of the mandible and maxilla. Regions of extensive hypomineralization were found in the calvarial bone of skulls. The maximum mineral concentrations and the time constants for diabetic rodents were similar to the controls. The diabetic mice appeared to have random regions of hypomineralization and one diabetic rat had areas of hypoplasia in the mandibular incisors. Conclusions: Diabetes may have a profound detrimental influence on the function of ameloblasts in laying down enamel.

Key words: Diabetes mellitus, enamel, bone, dental hard tissues, mineral concentration, x-ray microtomography

Introduction

Diabetes mellitus is an endocrine disease leading to complications in the vasculature of developing tissues [Cooper et al., 2001]. Maternal diabetes mellitus influences the developing teeth of the embryo or premature children, with reduced mineralization of bone found to be more common in the children of diabetic mothers compared with controls [Grahnen and Edlund, 1968; Noren, 1984; Seow and Perham, 1990; Schwartz, 2003; Carnevale et al., 2004]. Macroscopic and microscopic investigations have revealed defects in the primary teeth of both diabetic children and children of diabetic mothers [Adler et al., 1973; Noren et al., 1978; Albrecht et al., 1991).

Qualitative ultrastructural differences of enamel between diabetic and control rodents have been demonstrated, suggesting that diabetes mellitus has a destructive influence on the developing enamel ultrastructure [Atar et al., 2004]. In addition, mineral analysis by energy dispersive x-ray (EDX) showed a decrease in the two mineral compounds of enamel, calcium and phosphorus, altering the calcium to phosphorus ratio. The present study takes this further by studying the quantitative effect of diabetes on the mineral composition and 3-D structure of bone and enamel in rodents using x-ray microtomography (XMT).

XMT allows the non-destructive and quantitative high resolution visualization of internal microstructure, the creation of three-dimensional images and the measurement of the linear attenuation coefficient, thus allowing mineral concentration to be analyzed [Davis and Wong, 1996]. XMT investigations have shown higher mineral concentrations near the incisal tip of the tooth than at the root apex in rodent enamel and in the outer enamel than the enamel nearer the amelo-dentinal junction [Anderson et al., 1996; Davis and Wong, 1996; Elliott et al., 1998; Dowker et al., 1999; Wong et al., 2000]. The aim of this study was to determine the mineral concentration of bone and enamel in the teeth of rodents using XMT.

Materials and Methods

Animal samples. Two rodent strains which suffer from a genetically induced obesity leading to diabetes mellitus and relevant controls were used (Hoffmann-La Roche Ltd., Switzerland). B6.Cg-m +/+ Leprdb mice, homozygous for the diabetes spontaneous mutation [Lepr.sup.db], become obese around 3 to 4 weeks of age with elevations of plasma insulin from 10 to 14 days and blood sugar from 4 to 8 weeks. Three mice (md1-3) were killed at 23 weeks by decapitation, having been diabetic for about 17 weeks. The ZDF/Gmi-fa rat spontaneously develops obesity and diabetes and hyperphagia. Hyperglycemia begins to develop at about 7 weeks and glucose levels typically reach 500 mg% by 10 to 11 weeks of age. These suffer from a non-insulin dependent diabetes mellitus and develop insulin resistance. Three rats (rd1-3) were killed when 19 weeks old, having been diabetic for about 12 weeks. BL/6 mice (mc1-3) and Wistar rats (rc1-3) were used as controls and fed standard diet. No signs of obesity or diabetes mellitus were seen and they were killed when 8 weeks old.

X-ray microtomography. A 4th generation XMT scanner was used. In order to eliminate ring artifacts, a time delay integration CCD readout method was employed giving a maximum resolution of 8 [micro]m in the final reconstruction [Davis and Elliott, 1997]. The temperature inside the x-ray was controlled to within 0.1[degrees]C to ensure stability of the system during data acquisition. In order to compensate for the dishing artifacts due to polychromatic radiation a symmetrical seven step wedge made of 99.98% aluminium was used for calibration.

Specimen preparation. Mouse and rat heads were obtained following decapitation and stored in 70 vol% alcohol at 4[degrees]C. A length (approximately 20 mm) of 2 mm diameter 99.98% aluminium wire was inserted into the oesophagus of each head to be scanned for internal calibration. Each head, in its complete form with fur and muscle tissue, was fixed using 70 vol% alcohol before placing them inside the scanning microtomography device. Scanning was performed at 30 [micro]m single steps, taking approximately 47 hours for each head.

Visualization and analysis. Reconstructed images of the rodent skulls were viewed and manipulated using a three-dimensional visualization software package (Volume Graphics, Germany). The mean linear attenuation coefficients in the mid labial region in 1 mm steps from incisor tip to the root apex were measured and calibrated against the aluminium wire standard with a linear correlation coefficient of 1.53 cm-1 at 45 KeV. Mean mineral concentrations were calculated from the calibrated attenuation coefficients by assuming the mineral phase of enamel being hydroxyapatite with a density of 3.15 g cm-3. Mineral concentration at 1 mm intervals from the root apex to the incisor tip were calculated and plotted against the length of the incisors. For each maxillary and mandibular pair of incisors of each rodent, a saturation potential curve was fitted to the data using the formula: Cm = a - b (1 - e-cL), where Cm = mineral concentration, L = length from apex, and a, b, e = constants. The 3-D reconstructed images were presented with computer generated false colour to highlight specific hard tissue morphology areas or segmented in order to look specifically at dental hard tissues, such as enamel for comparison.

Results

Comparison of B6.Cg-m +/+ L[epr.sup.db] mice and ZDF/Gmi-fa rats with relevant control rodents. Using the XMT results, 3-D images of the whole skull were reconstructed for all animals. Pseudo-colour scales representing the level of mineral concentration were used, where the bone was shown in orange (1.35 g cm-3) and red (0.8-1.34 g cm-3), the enamel in green (1.35-2.24 g cm-3), and the dentine in orange underneath (2.25 g cm-3).

Bone. The smooth and uniform colour of the skull of a BL6 control mouse (mc2) indicated that the mineral concentration of the bone was about 1.35 g [cm.sup.3] (Figure 1a). The calvarial bone was predominantly orange in colour and the enamel, shown in green, had a smooth outline. In comparison, in a diabetic mouse skull, the calvaria were covered with a larger area of red colour (0.8 - 1.34 g [cm.sup.3]) indicating that the bone had a lower mineral concentration (Fig 1c). There were clear impairments of the calvarial bone with marked pitting and hypomineralization (red) and extensive thinning of bone structure, particularly around the roots of the teeth. Holes can be seen within the bone surrounding the teeth with destruction of the bone edge (Fig 1d), compared with the smooth and contiguous outline in the normal control animals (Fig 1b). The bone mineralization in the Wistar control rats was shown to be similar to that of control mice with no difference in mineral concentration between diabetic and control rats.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Dental tissues. The curvature of the maxillary incisors was more marked than the mandibular incisors of B6.Cg-m +/+ [Lep.sup.rdb] mice, with the developing apices of the maxillary incisors anterior to the maxillary molars. The mandibular incisors pass underneath the mandibular molars with their apices distal to the molars (Fig 1b). The apices had jagged appearances. The enamel in diabetic animals had a rougher surface which appeared to extend from the apex to the incisal end, compared to controls. All of the diabetic animals showed a blunting of the normally sharp and smooth surface at the incisal end, and, in some cases, complete loss of the incisal edge of the tooth (Fig 1d). When only the enamel of the mice was rendered visible, the incisors of the diabetic mice were shown to have random regions of hypomineralization (Fig 2b) with more loss of enamel from the molars, as compared to control mice (Fig 2a).

There appeared to be a loss of enamel in the molars of the ZDF/Gmi-fa diabetic rats compared with control animals (Fig 3a,b). One diabetic rat (rd3) showed a large hole in the enamel of the incisors, which was not seen in the other diabetic rats (Fig 3b).

Mineral concentration analysis. There was a steady increase in mineral concentration with increasing distance from the root in all the incisors from the control and diabetic rodents with a plateau reached towards the incisal tip of the teeth (Fig 4 a-d). Overall, the mineral concentration in the mice was higher than in the rat teeth. For each maxillary and mandibular pair of incisors of each rodent, a saturation potential curve was fitted to the data (Table 1). In general, most teeth followed the normal saturation potential pattern as shown by the high correlation coefficient values (around 0.9), except for diabetic rat (rd3) which appeared to follow a sinusoidal pattern. The maximum mineral concentration ranged from 2.15 to 2.71 g cm3 with the maximum mineral concentration for mandibular incisors being slightly higher than that of maxillary incisors, and that of mice higher than rat.

Another measurable parameter is the time constant which can be defined as the time taken for the mineral concentration to change from zero to (1-e-1) of its maximum concentration (Table 1). The time constant in distance ranged from 0.61 to 4.83 g cm3/mm excluding diabetic rat (rd3). These can be converted to a time constant in growth days of 1 to 8.05 g cm3/day assuming the growth of the incisors were 0.6mm/day (Robinson et al., 1974; Smith and Warshawsky, 1975). The time constants for mice were less than that seen in rats, with maxillary teeth being less than mandibular teeth, and control rodents less than diabetic rodents. The trend did not show differences in mineral concentration between normal and diabetic rodents.

[FIGURE 3 OMITTED]

Discussion

In the present study, hard tissues of rodent skull were rendered visible in three-dimensional reconstructions. Visualization of bone tissue showed that the alveolar processes appeared to be thin, discontinuous, and receded, and the calvaria had large areas of red colour in diabetic rodents indicating a lower mineral concentration than the control animals. Similar studies in diabetic rats have shown significantly reduced growth in most mandibles compared to controls, with delays in bone maturation also shown in the foetuses of diabetic rats [Verhaeghe et al., 1999; Giglio and Lama, 2001]. In addition, clinical studies have shown reduced bone mineralization in the children of diabetic mothers, which treatment could partially reverse, with decreased skeletal maturation and cephalometric measurements in patients with juvenile diabetes [Grahnen et al., 1968; Noren et al., 1978; El-Bialy et al. 2000; Gunczler et al., 2001]. It has been suggested that deficient calcium incorporation into bone or enamel may be responsible for alterations in the mineralization patterns of enamel [Sato et al., 1996]. This study has demonstrated microscopic differences in the molar crowns of diabetic rodents which were worn more severely than the controls. Although the diabetic rodents are older than the controls, the attrition was not as noticeable in the incisors, with the controls tending to have sharper incisal edges than the diabetic rodents. The diabetic mice and diabetic rat (rd3) showed distinct areas of hypoplasia/ hypomineralization suggesting that diabetes may have a direct effect on enamel formation. This is supported by a study investigating the ultrastructure of enamel in diabetic rodent incisors by SEM and energy dispersive x-ray (EDX), which found that the enamel prism arrangement and the crystallites were altered in diabetic rodents [Atar et al., 2004].

One of the advantages of the present study is the use of XMT technology, which is capable of scanning specimens with no manipulation or preparation of the samples prior to analysis. Previous studies using 1st generation XMT [Wong et al., 2000] were able to measure specimens that had a diameter less than 5 mm. However the rat incisors had to be extracted from the head prior to analysis and the organic protein-rich developing enamel at the apical end was subjected to drying artifact which was detected in their reconstructions. In the present study, the shrivelling artifact was eliminated because the whole head of each rodent was scanned with the enamel mineral concentration gradient consistently decreasing towards the apical end.

The quantification and pattern of mineralization using XMT was consistent with previous studies [Wong et al., 2000] with rising mineral concentration towards a maximum from the apex to the incisal tip. No difference was detected in mineral concentration between the mandibular and maxillary incisors of diabetic rodents and controls. However, previous studies using EDX revealed a reduction in the amount of calcium and phosphorus in all teeth in diabetic mice and rats, with phosphorus being more decreased than calcium [Atar et al., 2004]. This may be due to EDX being relatively qualitative and crude, analyzing large areas in contrast to the resolution using XMT. Although the mineral concentration data do not show significant differences between diabetic and control mice, the 3-D reconstruction of the incisors show marked enamel hypomineralization in the diabetic mice. These hypomineralization regions appeared to be random with no specific patterns which measurements taken along the mid regions of the incisors may have missed. Interestingly, these hypomineralized defects were observed in the diabetic mice and not the diabetic rats, the reason for which is unclear.

[FIGURE 4 OMITTED]

A recent study utilizing an in vitro bone nodule formation assay, demonstrated that elevated glucose concentration inhibited osteoblastic calcium deposition and bone maturation [Balint et al., 2001]. Since there is a reciprocal correlation between osteoblast proliferation and maturation, in the presence of elevated glucose concentration, osteoblast proliferation is enhanced while bone formation is inhibited. However, experimentally induced diabetes has been shown to inhibit enamel protein secretion by secretory ameloblasts [Karim, 1983]. This current study would appear to suggest that enamel malformation in the diabetic animals may be due to decreased secretional and maturational function of the ameloblasts, rather than through a deficiency of calcium and phosphorus.

The increase in mineral concentration followed a saturation potential pattern in the rodents except diabetic rat (rd3), which appeared to follow a sinusoidal pattern and with the region of denuded enamel suggested that this rat may have experienced alternating episodes of disease and recovery preventing enamel formation. With the advent of XMT, precise, high resolution and non-destructive measurements can now be made. The results of this study strongly suggest that diabetes mellitus may cause profound and destructive influences on developing dental enamel and bone hard tissue with the effects on the calvarial bones of the skull particularly striking.

Acknowledgements

This study was awarded the EAPD prize in research at the EAPD Congress in Amsterdam, 2006. It has been supported by the Margarete and Walter Lichtenstein Foundation, Swiss National Foundation SNF, Novartis Foundation, Independent Academic Society FAG, Switzerland, in collaboration with Hoffmann-La Roche Ltd., Switzerland.

References

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Anderson P, Elliott JC, Bose U, Jones SJ. A comparison of the mineral content of enamel and dentine in human premolars and enamel pearls measured by x-ray microtomography. Arch Oral Biol 1996;41:281-290.

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Balint E, Szabo P, Marshall CF, Sprague SM. Glucose-induced inhibition of in vitro bone mineralization. Bone 2001;28:21-28.

Carnevale V, Romagnoli E, D'Erasmo E. Skeletal involvement in patients with diabetes mellitus. Diabetes Metab Rev 2004;20:196-204.

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Grahnen H, Moller EB, Bergstrom AL. Maternal diabetes and changes in the hard tissues of primary teeth. II. A further clinical study. Caries Res 1968;2:333-337.

Gunczler P, Lanes R, Paoli M, et al. Decreased bone mineral density and bone formation markers shortly after diagnosis of clinical type 1 diabetes mellitus. J Pediatr Endocrinol Metab 2001;14:525-528.

Karim AC. An ultrastructural study of the effect of streptozotocin on the secretory ameloblasts of the rat incisor. Anat Anz 1983;153:119-136.

Noren J, Grahnen H, Magnusson BO. Maternal diabetes and changes in the hard tissues of primary teeth. III. A histologic and microradiographic study. Acta Odontol Scand 1978;36:127-135.

Noren JG. Microscopic study of enamel defects in deciduous teeth of infants of diabetc mothers. Acta Odontol Scand 1984;42:153-156.

Robinson C, Hiller CR, Weatherell JA. Uptake of 32P-labelled phosphate into developing rat incisor enamel. Calcified Tissue Research 1974;15:143152.

Sato K, Hattori M, Aoba T. Disturbed enamel mineralization in a rat incisor model. Adv Dent Res 1996;10:216-224.

Schwartz AV. Diabetes mellitus: Does it affect bone? Calcif Tissue Int 2003;73:515-519.

Seow WK, Perham S. Enamel hypoplasia in prematurely-born children: A scanning electron microscopic study. J Pedod 1990;14:235-239.

Smith CE, Warshawsky H. Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine. Anatomical Record 1975;83:523-562.

Verhaeghe J, van Bree R, van Herck E, et al. Pathogenesis of fetal hypomineralization in diabetic rats: Evidence for delayed bone maturation. Pediatr Res 1999;45:209-217.

Wong FSL, Elliott JC, Davis GR, Anderson P. X-ray microtomographic study of mineral distribution in enamel of mandibular rat incisors. J Anat 2000;196:405-413.

M. Atar *, G. R. Davis **, P. Verry ***, F. S. L. Wong *

* Depts. Oral Growth and Development, Section of Paediatric Dentistry, ** Biophysics in Relation to Dentistry, Barts and The London, Queen Mary University of London, School of Medicine and Dentistry, London, England; *** Hoffmann-La Roche Ltd., Pharmaceuticals Division, Metabolic Preclinical Research, Vascular and Metabolic Diseases, Basel, Switzerland

Postal address: Dr M. Atar, Dept. Oral Growth and Development Section of Paediatric Dentistry, Barts and The London, Queen Mary University of London, School of Medicine and Dentistry, Turner Street, Whitechapel, London E1 2AD, England.

Email: atardrs@yahoo.com
Table 1. Time constant parameters in a study on enamel mineral
concentration in diabetic rodents.

 Parameters

 MICE a b e R2

 mc1-3 maxillary 1.12 1.35 1.43 0.93
 mean

 md1-3 maxillary 1.41 1.06 1.3 0.95
 mean

mc1-3 mandibular 1.09 1.51 1.21 0.97
 mean

md1-3 mandibular 1.37 1.24 0.54 0.94
 mean

 RATS

 rc1-3 maxillary 1.36 0.89 0.67 0.88
 mean

 rd1-3 maxillary 1.8 0.41 0.72 0.36
 mean

rc1-3 mandibular 1.27 1.1 0.42 0.95
 mean

rd1-3 mandibular 1.55 0.66 23.9 0.43
 mean

 rd3 maxillary 1.92 0.23 0.79 0

 rd3 mandibular 1.47 0.59 47.56 0.056

 Time Maximum
 mineral
 Maximum mineral concentration
 concentration (g [cm.sup.3])
 (g [cm.sup.3]) in distance

 MICE a+b 1/c

 mc1-3 maxillary 2.47 0.70
 mean

 md1-3 maxillary 2.48 0.77
 mean

mc1-3 mandibular 2.59 0.82
 mean

md1-3 mandibular 2.62 1.86
 mean

 RATS

 rc1-3 maxillary 2.25 1.49
 mean

 rd1-3 maxillary 2.21 1.40
 mean

rc1-3 mandibular 2.37 2.36
 mean

rd1-3 mandibular 2.21 0.04
 mean

 rd3 maxillary 2.15 1.25

 rd3 mandibular 2.06 0.021

mc = mouse control; md = mouse diabetic; rc = rat control;
rd = rat diabetic.
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Author:Atar, M.; Davis, G.R.; Verry, P.; Wong, F.S.L.
Publication:European Archives of Paediatric Dentistry
Geographic Code:4EUUK
Date:Dec 1, 2007
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