ARTICLE |
Correspondence to: A. Nanci, Université de Montréal, Faculty of Dentistry, PO Box 6128, Station Centre-Ville, Montreal, Quebec, Canada H3C 3J7. E-mail: nancia@ere.umontreal.ca
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Summary |
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During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acidSchiff- and periodic acidsilver nitratemethanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation. (J Histochem Cytochem 48:10431057, 2000)
Key Words: hypocalcemia, amelogenesis, maturation stage, rat, immunocytochemistry, amelogenin, ameloblastin, albumin
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Introduction |
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Calcium regulates many cellular activities, such as cell communication, signal transduction, activation of enzymes, and polymerization of cytoskeletal proteins (
Despite the importance of calcium in amelogenesis, there have been relatively few studies on the influence of dietary calcium on the formation of enamel (
The aim of the present investigation was to examine the effect of chronic hypocalcemia on the integrity of the enamel organ and the distribution of enamel proteins during the early maturation stage, using histochemical staining for glycoconjugates, in situ hybridization, and postembedding colloidal gold immunocytochemistry.
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Materials and Methods |
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Animals and Diet
Twenty-six male Wistar rats weighing approximately 45 g (Charles River; St- Constant, QC, Canada) were used for the experiment. They were housed two per cage, maintained on a 12-hr light/12-hr dark cycle, and given free access to food and deionized water. The animals were divided into a treated (n = 18) and a control (n = 8) group. The treated group was fed a calcium-free diet (Altromin DP1031; Rieper, Vandoies, Italy) and the control group a normocalcic chow (Altromin DP1000; Rieper) for 28 days. Both the normal and calcium-free food formulations contained 1000 IU of vitamin D/kg. Each pair of control and treated animals was given equal daily quantities of food and water. Food consumption was measured every 2 days and water every 4 days. The data were averaged and expressed as the weekly food and water consumption per rat. The difference among groups was assessed by the bidirectional t-test adjusted for multiple comparison. One-way analysis of variance (ANOVA) was performed to determine significant differences. The experimental protocol was approved by the Comité de Déontologie de l'Expérimentation sur les Animaux of the Université de Montréal.
Blood Sampling
The rats were anesthetized with chloral hydrate (0.4 mg/g body weight; Sigma Chemical, St Louis, MO) and approximately 3 ml of blood was drawn from the jugular vein. The serum levels of (a) phosphorus, calcium, and alkaline phosphatase were measured as an indicator of calcification, (b) creatinine of renal status, (c) albumin of intestinal protein absorption, and (d) aspartate aminotransferase and alanine transferase liver activity in relation to alkaline phosphatase levels. These parameters were measured using routine hospital biochemical assays [Pavillon St-Luc, Centre Hospitallier de l'Université de Montréal (CHUM), Montreal, QC, Canada].
Morphological Analyses
After blood was drawn, the anesthetized rats were perfused through the left ventricle first with lactated Ringer's solution (Abbott; Montreal, QC, Canada) for approximately 30 sec and then with fixative for 20 min. The fixative solution consisted of either 4% paraformaldehyde + 0.1% glutaraldehyde (PG) in 0.08 M sodium cacodylate buffer, pH 7.3, or 1% glutaraldehyde (G) in the same buffer. The hemimandibles were removed and immersed in the corresponding fixative for 3 hr (PG) or overnight (G) at 4C. The right hemimandibles were left calcified, whereas the left ones were decalcified in 4.13% disodium EDTA, pH 7.2, for 2128 days at 4C (
Immunocytochemistry
The postembedding protein Agold method was used for immunolabeling (reviewed in
Preparation of mRNA Probes
For mRNA probes, an ameloblastin DNA insert (1900 bp) (
In Situ Hybridization
Sections of the entire hemimandible were cut at 510-µm thickness and mounted on aminoalkylsilane-coated glass slides (Sigma). The paraffin was removed with xylene and the sections were rinsed in PBS and treated at 37C with proteinase K (20 µg/ml; Boehringer Mannheim) for 30 min in a buffer consisting of 100 mM Tris-HCl and 50 mM EDTA, pH 8.0. After digestion, they were rinsed in 0.2% glycine, fixed with 4% paraformaldehyde in PBS for 5 min, and immersed for 10 min in 20 mM triethanolamine containing 0.5 ml concentrated acetic anhydride. The slides were then rinsed with PBS and treated with a prehybridization solution consisting of 2 x SSC (300 mM NaCl + 30 mM sodium citrate) containing 50% deionized formamide, for 60 min at 50C. Hybridization was carried out by incubating the slides overnight at 50C in a humidified chamber with 50% formamide, 2 x SSC, 1 x Denhardt's solution, 10% dextran sulfate, 500 µg/ml herring sperm DNA, and 250 µg/ml yeast tRNA containing ~0.5 ng/µl of the antisense or sense probe. After hybridization, they were washed several times with 4 x SSC. Nonhybridized transcripts were digested for 30 min at 37C with 20 µg/ml RNAse A (Boehringer Mannheim) in 500 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Digested sections were washed with decreasing concentrations of SSC (4 x, 2 x, 1 x and 0.1 x), for 30 min each at 4C. The hybridized probe was then detected by incubating with a sheep anti-digoxigenin antibody conjugated to alkaline phosphatase (Boehringer Mannheim) for 2 hr at 4C. Phosphatase activity was revealed with 450 µg/ml nitroblue tetrazolium and 175 µg/ml of 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-HCl, pH 9.5, containing 100 mM NaCl and 50 mM MgCl2.
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Results |
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Measurement of Metabolic Parameters
Measurements of the various metabolic parameters analyzed are summarized in Fig 1. During the last 2 weeks of the experiment, consumption of food by animals receiving the calcium-deficient diet declined to almost half that of controls. Body weight in treated animals paralleled their pattern of food intake and, at Day 28, they weighed on the average 49% less than control rats. There was no correlation between food and water intake.
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Absence of calcium in the diet resulted in a significant reduction in serum calcium. Serum phosphorus, creatinine, albumin, and aspartate aminotransferase showed no statistical difference between the two experimental groups. Serum alkaline phosphatase and alanine transferase increased in animals receiving the calcium-deficient diet.
During the third week of the experiment, two treated rats died as a consequence of bone fractures and general debility. The surviving animals showed no distinctive physical features aside from their lesser body weight and some degree of pili erection. Behavior was not systematically assessed. However, rats were more sensitive to general animal room handling. In addition, towards the end of the diet period, the rats in the treated group were generally less active. There were no deaths in the control group.
Light Microscopy
Total absence of calcium in food and water for 28 days produced significant changes in the organization of both the enamel organ and the enamel (Fig 2A2D). Maturing enamel was hypoplasic and its surface irregular compared to that of control rats (compare Fig 2A and Fig 2E). Alterations of enamel were most prominent in the outer portion, where there were accumulations of organic material (Fig 2B2D). Although smooth- and ruffle-ended ameloblasts were distinguishable, the enamel organ appeared disorganized and there were regions showing accumulations of cells (Fig 2A2C). In some regions, ameloblasts were clearly shorter and occasionally appeared cuboidal.
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The enamel organ of hypocalcemic rats frequently showed the presence of cyst-like structures that varied in size and shape. Some of them contained an extracellular matrix that stained uniformly with toluidine blue, methylene blueazure II and PASNM (Fig 2A2C). In others, only the periphery of the matrix reacted to the stains (Fig 2A2C). The central unstained portion was von Kossa-positive (not shown). Masses of matrix with similar texture and staining properties were also found at the enamel surface in areas where the ameloblast layer was invaginated (Fig 2A and Fig 2B).
In control animals, PASNM staining clearly revealed the basal lamina present at the interface between the ameloblasts and enamel (Fig 2E). In hypocalcemic rats, this basal lamina was obscured by accumulations of PASNM-, PAS-, and methylene blueazure II-positive material near the ameloblast surface (Fig 2A2D).
In Situ Hybridization
In hypocalcemic rats, amelogenin and ameloblastin mRNAs were detected in ameloblasts, cells constituting the cyst-like structures, and groups of cells accumulating in some areas of the enamel organ (Fig 3). Papillary layer cells proper showed no signal. In control animals, only ameloblasts expressed amelogenin and ameloblastin signals. Odontoblasts and bone cells in both groups of animals were not reactive. Control incubations with sense probes resulted in no significant staining.
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Electron Microscopy
Except in areas where there were cyst-like structures in the enamel organ, ameloblasts in hypocalcemic rats showed a normal intracellular organization. They contained well-developed protein synthetic organelles, endosomal/lysosomal elements, and occasionally secretory granules (Fig 4). However, the enamel layer was hypoplasic (Fig 5). Rod and inter-rod enamel were generally distinguishable in the deeper portions of the enamel layer but not near the surface (Fig 5 and Fig 6). At this site, crystal packing was disorganized, there were accumulations of organic material among the crystals, and cell processes and remnants of Tome's processes were frequently encountered (Fig 5 and Fig 7). Small vesicular profiles were present between crystals and around ameloblast processes (Fig 8A). The enamel did not always extend up to the ameloblasts; a layer poor in organic matrix or patches of amorphous material were sometimes found at the ameloblast surface (Fig 5, Fig 7D and Fig 9). In areas where there was no basal lamina at the interface between the ameloblasts and the enamel, enamel crystals or tubular crystal "ghosts" (in decalcified specimens; see
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The cyst-like structures in the enamel organ were generally formed by cells with ameloblast characteristics, often showing a ruffled border (Fig 10) and less frequently smooth ones (Fig 11B). Processes extended from the cells into the matrix they englobed; in some cases, a basal lamina was present between the cells and the matrix (Fig 10A and Fig 10C). Tubular "crystal ghosts" were sometimes identifiable in the matrix, whereas in other cases it appeared finely granular or homogeneous (Fig 10 and Fig 11B). In decalcified teeth, plate-like "ghosts" like those seen in decalcified bone (
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None of the above altered cell and matrix characteristics was observed in control animals (data not shown).
Immunolocalization of Ameloblastin, Amelogenin, and Albumin
Early maturation stage ameloblasts from control rats showed labeling for ameloblastin and amelogenin over organelles involved in protein synthesis and secretion, and over endosomal/lysosomal elements. Immunoreactivity for amelogenin was found throughout the enamel layer. There was a concentration of intact ameloblastin near the cell surface and labeling for albumin was weak and mostly restricted to the superficial portion of the enamel layer. These immunolabeling results in the control group are similar to what has been extensively documented for normal animals (
In ameloblasts of hypocalcemic rats, the organelles involved in protein synthesis and secretion as well as multivesicular bodies were consistently labeled for ameloblastin (Fig 4B). The intracellular reactivity for amelogenin was variable and was lower than in controls, particularly for multivesicular bodies, whose frequency and density of labeling were both reduced (Fig 4A). Labeling for ameloblastin and amelogenin was heterogeneously distributed throughout the maturing enamel layer (Fig 7 and Fig 9). There were regions of intense immunoreactivity alternating with weakly-labeled ones (Fig 7 and Fig 9). Immunolabeling for albumin was clearly more intense than in control rats and also exhibited a heterogeneous distribution (Fig 11A). The accumulations of organic matrix in the superficial portion of the enamel layer were variably immunoreactive (Fig 9A9C), whereas the content of the cyst-like structures labeled for all three proteins (Fig 10B, Fig 10C, and Fig 11B). Large patches of organic matrix were sometimes found along the basolateral surface of ameloblasts and among cells of the papillary layer. These were intensely immunoreactivity for amelogenin but showed very little or no labeling for ameloblastin (Fig 12).
Control incubations resulted in few gold particles, randomly distributed throughout the entire tissue section (data not shown).
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Discussion |
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This report shows that diet-induced hypocalcemia directly or indirectly results in major alterations of both the enamel organ and the enamel. The results are consistent with previous studies on experimentally induced chronic hypocalcemia suggesting that low calcium levels, rather than phosphate levels, cause enamel hypoplasia (
The organic matrix of enamel comprises two classes of proteins, amelogenins and nonamelogenins. Amelogenins, the dominant component of developing enamel, undergo bulk enzymatic breakdown and removal during enamel formation to accommodate the growth in width and thickness of enamel crystals (reviewed in
Ameloblasts do not synthesize and secrete albumin (
The detection of mRNA signal for amelogenin and ameloblastin in the cells forming cyst-like structures and clusters in the enamel organ of calcium-deficient rats strongly suggests that they are of ameloblastic origin. Indeed, the matrix within the cysts comprises both amelogenin and ameloblastin and mineralizes. One possible mechanism for their formation is an interference with the cell cycle program of ameloblasts. Calcium is known to regulate the cell cycle (
Among the various enamel proteins, only nonamelogenins are believed to show an important degree of glycosylation (reviewed in
In early maturation stage ameloblasts from normal rat incisors, elements of the endosomal/lysosomal system, such as multivesicular bodies, are, in general, intensely immunoreactive for amelogenin and ameloblastin (see
In conclusion, chronic hypocalcemia affects the physiological events that regulate enamel maturation. The enamel organ normally provides a "sealed" and controlled microenvironment for these events. Its disruption therefore represents a major cause for the impairment of mineralization. Protein processing and removal are slowed and the enamel layer is actually flooded by exogenous proteins such as albumin, thereby limiting crystal growth. This situation differs from what happens in bone during chronic hypocalcemia, in which calcium availability may have an important and direct effect on mineral deposition (see
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Acknowledgments |
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Supported by grants from the Medical Research Council of Canada and the Ministry of University and Scientific and Technological Research (MURST) of Italy. P. Mocetti was the recipient of a fellowship from the Québec Ministry of Education.
We are grateful to Dr R. Lepage [Pavillon St-Luc, Centre Hospitallier de l'Université de Montréal (CHUM) Montreal, QC] for carrying out the biochemical assays of blood samples, to Dr Paul H. Krebsbach (School of Dentistry, University of Michigan, Ann Arbor, MI) for providing the anti-ameloblastin antibody, to Micheline Fortin for diligent technical and photographic assistance, and to Sylvia Zalzal for assistance with the supervison of the experimentation.
Received for publication October 30, 1999; accepted March 15, 2000.
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