RAPID COMMUNICATION |
Correspondence to: Antonio Nanci, Lab. for Electron Microscopy, Faculty of Dentistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montréal, QC, Canada H3C 3J7.
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Summary |
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After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins. (J Histochem Cytochem 46:135142, 1998)
Key Words: immunocytochemistry, lectin cytochemistry, amelogenin, ameloblastin, bone sialoprotein, osteopontin, collagen, tooth, cementum, epithelium
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Introduction |
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The dentin of mammalian teeth is overlaid by two distinct mineralized tissues. Enamel covers the dentin of the crown and does not contain collagen, whereas cementum, a collagen-based mineralized tissue, forms along the root. Enamel is formed by ameloblasts that differentiate from the inner enamel epithelium (IEE). The origin of cementoblasts, however, is less clear. It is widely believed that cementoblasts differentiate from dental follicle cells, which derive from the cranial neural crest (reviewed in
A knowledge of the nature and the origin of the first deposited matrix constituents along root dentin is of importance to elucidate the function of HERS in cementogenesis. In some animals, this matrix is believed to be formed in the presence of epithelial cells (
Other studies have shown that, at a time coincident with the initiation of cementogenesis, the root surface stains for bone sialoprotein (BSP) and osteopontin (OPN) (
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Materials and Methods |
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Tissue Preparation
Wistar rats (Charles River; St.-Constant, QC, Canada) at 1, 3, 5, 11, and 14 days after birth were used in this study. A minimum of three rats per interval were analyzed. The animals were anesthetized with chloral hydrate (0.4 mg/g body weight) and perfused for 30 sec through the left ventricle with lactated Ringer's solution (Abbott Laboratories; Montréal, QC, Canada), followed by fixative consisting of 1% glutaraldehyde in 0.08 M sodium cacodylate buffer containing 0.05% calcium chloride (pH 7.3). The lower and upper jaws were dissected and immersed in the same fixative for an additional 12 hr at 4C. Most specimens were decalcified at 4C in 4.13% disodium ethylenediaminetetraacetic acid (EDTA) for 14 days (
Immunocytochemistry
The high-resolution protein Agold technique (reviewed in
Sections of osmicated tissues embedded in Epon were first floated on a drop of saturated solution of sodium metaperiodate for 15 min and washed with distilled water. The epoxy sections were then incubated for 3 hr on a drop of egg yolk chicken anti-rat AMEL antibody diluted 1:150 with PBS (
After incubations with primary or secondary antibody, the grids were rinsed with PBS, floated on PBS1% ovalbumin for 10 min, and incubated with protein Agold complexes prepared using gold particles of approximately 8 or 12 nm in diameter (
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Results |
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Antibodies to AMEL and AMBN labeled the enamel matrix, and those against BSP and OPN the cementum proper and bone. The labeling pattern obtained with the various anti-AMEL antibodies used did not show any significant differences. As revealed in adjacent sections, gold particle labeling for BSP and OPN occupied the same matrix regions. Control incubations resulted in few, randomly distributed gold particles over the tissue section.
Enamel-free Area
The EFA matrix was lined throughout crown development by inner enamel epithelial (IEE) cells, as indicated by the presence of tonofilaments and (hemi)desmosomes (Figure 1A, Figure 1B, Figure 2A, and Figure 2B). Occasionally, irregular globular masses of organic matrix (Figure 1B and Figure 2A) containing barely recognizable collagen fibrils (Figure 2A) extended between the epithelial cells in 14-day-old rats. In younger animals (Days 15), on the other hand, only uncalcified collagen fibrils were visible among the epithelial cells (Figure 2B). The EFA matrix was immunoreactive for AMEL (Figure 1A), AMBN (Figure 1B and Figure 2B), BSP (not shown), and OPN (Figure 2A).
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Root Surface
In 14-day-old animals, HERS covered the apical end of the forming root, and its fragmentation started slightly apical to the level where mineralization of the predentin matrix initiated (not shown). The remaining portion of the root was lined by epithelial cells. Between these epithelial cells and the root dentin, a thin layer of collagen-deficient cementum gradually formed, as revealed by a steady increase in thickness in the cervical direction. In the region of the cementoenamel junction, enamel matrix extended for a short distance between root dentin and cementum. This matrix labeled for AMBN (Figure 3 and Figure 4A) and AMEL (Figure 4B), but the labeling for AMBN extended slightly more apically. Beyond this narrow region near the cementoenamel junction, immunoreactivity for AMEL (Figure 5A) and AMBN (Figure 5B) was no longer detectable along the root despite the consistent presence of epithelial cells (Figure 5AC). The initially deposited layer of cementum consisted mainly of an electron-dense, granular organic matrix that labeled for BSP (not shown) and OPN (Figure 5C). Collagen fibrils were scarce in cementum but abundant adjacent to it (i.e., in precementum), where they formed bundles (as seen in cross-sectional profiles) in bay-shaped concavities formed by the epithelial cells (Figure 5AC).
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Discussion |
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Previous studies in rodent molars have demonstrated that root-lining cells express mRNA for BSP and OPN (
HERS cells have been proposed to secrete enamel-related proteins before dental follicle-derived cementoblasts form cementum proper (
The matrix at the EFA in rat molars also comprises cementum-related proteins (
The participation of epithelial cells in cementum formation in rat molars must be transient, because only cells with mesenchymal characteristics are present along forming cementum at more advanced stages of root development. It is conceivable that, after the deposition of the initial cementum layer by HERS cells, dental follicle-derived cementoblasts take over the subsequent formation of cementum. On the other hand, it is also possible that not all epithelial cells withdraw from the root surface and that these cells undergo epithelialmesenchymal transformation to give rise to cementoblasts (
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Acknowledgments |
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Supported by the Medical Research Council of Canada through an operating grant (AN) and a fellowship (DDB).
We are grateful to Ms Sylvia Zalzal for preparing the protein Agold complexes used for immunolabeling.
Received for publication September 23, 1997; accepted October 9, 1997.
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