ARTICLE |
Correspondence to: Antonio Nanci, Université de Montréal, Faculty of Dentistry/Stomatology, PO Box 6128, Station Centre-Ville, Montréal, QC, Canada H3C 3J7. E-mail: Antonio.Nanci@umontreal.ca
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Summary |
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At the beginning of the maturation stage of amelogenesis, ameloblasts deposit a basal lamina (BL) at the interface between their apical surface and maturing enamel. This structure is rich in glycoconjugates and is proposed to exhibit adhesive and/or filtering functions. To clarify its role, we have applied a recently developed surgical window model to locally administer tunicamycin (TM), an antibiotic that interferes with N-glycosylation, in the rat hemimandible using an osmotic minipump. Male Wistar rats were infused with either TM or saline as a control. Lectingold cytochemistry was performed to reveal glycoconjugates in the BL. Immunogold labeling of enamel proteins and albumin was carried out to verify whether depletion of N-linked sugars in the BL affects the content and distribution of endogenous and exogenous proteins in the enamel layer. Under the influence of the drug, the BL became irregular and exhibited alterations in structural organization and composition. The number of Helix pomatia agglutinin binding sites was not significantly affected but their distribution was altered. The labeling density of wheat germ agglutinin over the BL was slightly reduced. Immunoreactivity for enamel proteins showed only a small decrease, but that of albumin, both between ameloblasts and within the enamel layer, increased significantly. No structural alterations were observed in the contralateral incisor and in other sampled tissues and organs. These results demonstrate that it is possible to achieve a localized administration of TM without systemic side effects and lend support to the proposal that the BL represents a specialized structure with filtering functions.
(J Histochem Cytochem 49:165176, 2001)
Key Words: osmotic minipump, tunicamycin, basal lamina, glycoconjugates, enamel proteins, colloidal gold, lectin cytochemistry, immunocytochemistry, incisor, rat
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Introduction |
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During the formative phase of amelogenesis, ameloblasts produce an extracellular matrix that regulates the formation of the extremely long enamel crystals (reviewed in
The BL associated with maturation stage ameloblasts has been proposed to provide the means for their hemidesmosomal attachment to the enamel surface (
To investigate the function of this glycoconjugate-rich BL, we have applied a recently developed experimental model to locally administer tunicamycin (TM), an antibiotic that interferes with glycosylation. The system consists of drilling a "window" in the alveolar bone overlying the apex of the rat incisor and connecting to it an osmotic minipump (
TM inhibits the first step in the lipid carrier-dependent protein glycosylation (dolichol pathway), specifically preventing N-linked glycosylation (
In this study, the effect of TM on enamel maturation was examined at the light and electron microscopic levels. Lectingold cytochemistry was performed to reveal and map the distribution of glycoconjugates. Immunodetection of enamel proteins (EPs) and ALB was carried out to verify whether N-linked sugar depletion in the BL structural components affects the content and distribution of endogenous and exogenous proteins in the enamel layer.
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Materials and Methods |
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Surgical Procedure
Male Wistar rats weighing 100 ± 10 g (Charles River Canada; St-Constant, QC, Canada) were anesthetized with a 0.27-ml IP injection of a 1:1:2 mixture of Hypnorm (fentanyl citrate and fluanisone; Janssen Pharmaceutica, Beerse, Belgium), Versed (midazolam; Hoffmann-LaRoche, Mississauga, ON, Canada), and distilled water. An incision was made through the skin to access the muscle layer, according to an imaginary line joining the auditory meatus and the lip commissure. After separation of the fibers of the masseter muscle and elevation of the periosteum, the bony surface of the ramus was exposed. A slow-speed dental drill equipped with a carbide round burr, size 0.14 (Brassler; Montreal, QC, Canada), was used to create a hole through the alveolar bone under manual saline irrigation (Fig 1A). The hole was drilled approximately 2 mm anterior to the posterior border of the ramus and slightly superior to the bony elevation overlying the apical end of the incisor. Another incision through the skin in the neck area was made to accomodate a 7-day Alzet osmotic minipump (model 2001D; Alza Corporation, Palo Alto, CA). The pump was tunneled into a subcutaneous pouch on the back of the animal and connected to the bony hole using a vinyl tubing and a metal catheter (Fig 1B). The tubing was passed underneath the masseter muscle and through the neck area. Tissue adhesive Indermil (distributed by Sherwood Davis & Geck; St Louis, MO) and bone cement (Zimmer; Warsaw, IN) were used to help immobilize the metal catheter against the bone surface and maintain its tip in the hole. The animals were then sutured and the surgical site was cleaned and disinfected with 70% ethanol. Some rats were administered Temgesic (buprenorphine hydrochloride; Reckitt & Colman, Hull, UK) as an analgesic to control postsurgical pain. Five days after surgery, X-ray radiographs of the rat mandibles were taken to verify that the catheter was well in place (Fig 1C and Fig 1D). Animal procedures and experimental protocols described above were in accordance with guidelines of the Comité de déontologie de l'expérimentation sur les animaux of Université de Montréal.
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Preparation and Administration of TM
Tunicamycin (Sigma Chemical; St Louis, MO) was first dissolved in 0.01 M sodium hydroxide, pH 9, and then diluted to a final concentration of 10 µg/ml with 0.1 M PBS, pH 7.2 (230 µl of TM solution. The minipumps were connected to a vinyl tubing, also filled with the drug, and soaked in sterile saline at 37C for 13 hr before placement, as described above. Controls consisted of three rats implanted with the same minipumps filled with physiological saline (Sigma).
Tissue Processing
On Day 8 after placement of the minipumps, the animals were anesthetized with an IP injection of 0.25 ml of 20% chloral hydrate (Sigma) and sacrificed by intravascular perfusion through the left ventricle. The vasculature was pre-rinsed with lactated Ringers'solution (Abbott Laboratories; Montreal, QC, Canada) for about 30 sec, followed by perfusion with a fixative solution consisting of 1% glutaraldehyde in 0.1 M sodium phosphate (PB), pH 7.2, for 20 min. Both hemimandibles were dissected out and immersed in the fixative overnight at 4C. They were then washed in 0.1 M PB, pH 7.2, and decalcified either in 4.13% disodium ethylene tetra-acetic acid (EDTA) for 21 days at 4C (
Each tooth segment was oriented for sectioning along its longitudinal axis. One-µm-thick sections were cut with glass knives on a Reichert Jung Ultracut E ultramicrotome and stained with toluidine blue. Thin sections of selected areas were then prepared with a diamond knife and mounted on 200-mesh nickel grids having a carbon-coated Formvar film. The sections were then processed for postembedding lectingold cytochemistry for detection of glycoconjugates (reviewed in
Lectin Cytochemistry
Helix pomatia agglutinin (HPA), specific for N-acetyl-D-galactosamine (GalNAc), and wheat germ agglutinin (WGA; Triticum vulgaris), specific for N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc), were used (
Immunocytochemistry
For immunolabeling of the two main classes of EPs, amelogenin and non-amelogenin (see
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Results |
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Macroscopic Observations and Light Microscopy
Appropriate positioning of the bony window was confirmed on X-rays and by visual inspection at time of dissection (Fig 1C and Fig 1D). Continous exposure of the dental organ to TM for 7 days affected tooth eruption such that the treated incisor was about 0.51 mm shorter than the contralateral one (Fig 2B). None of the rats implanted with saline minipumps showed a notable difference in length between the two incisors (Fig 2A). In some cases, a slight erosion was noted surrounding the hole, probably due to the combined inflammatory effects of the adhesives used.
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Histologically, in early to mid-maturation, there was no significant alteration of the enamel organ (Fig 3A). However, in some regions, groups of odontoblasts appeared to have degenerated (Fig 3B) or to have lost their secretory polarity (Fig 3C). In the latter case, the predentin layer was thickened (Fig 3C). Dentin consistently exhibited a differential metachromatic staining pattern at about half its thickness (Fig 3A and Fig 3B). None of the above cell and matrix alterations was observed in control rats. Duodenum, kidney, liver, and parotid cells of treated and control animals showed no significant structural alterations.
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Ultrastructural Observations
The BL in incisors from hemimandibles infused with saline (Fig 4A and Fig 5A) and from contralateral teeth from both saline- and TM-infused rats showed structural characteristics similar to those previously described in the incisors of normal animals (
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Lectin Cytochemistry
In control rats, the BL showed uniform and intense labeling with HPA (Fig 4A and Fig 5A). At the start of the maturation stage, the labeling over the BL in TM-treated rats appeared weaker than in controls, and was sometimes interrupted (Fig 4B), but for the most part the labeling appeared to have the same density as in controls. In some cases, the labeling normally associated with the BL appeared to diffuse into the overlying enamel matrix (compare Fig 5C and Fig 5A). In regions at which the BL was enlarged, HPA binding sites were found throughout the structure, sometimes showing a concentration at the interface with enamel (Fig 6A).
In both control and TM-treated rats, labeling with WGA over the BL was weak and was difficult to distinguish from that over the adjacent enamel (Fig 5C and Fig 5D). In TM-treated teeth, regions of altered BL did not show any accumulation of gold particles near the cell surface (Fig 6B). Qualitative estimation suggested that the overall WGA labeling over the BL and enamel was somewhat weaker in TM-treated than in saline-infused animals (Fig 5C and Fig 5D).
Immunolocalization of Enamel Proteins and Albumin
All three anti-EP antibodies showed slightly weaker immunoreactivity over enamel from TM-treated incisors compared to saline-infused teeth.
Both anti-amelogenin antibodies used labeled the enamel matrix. However, they yielded different distribution patterns. Anti-AMELy showed an intense reaction throughout the enamel layer (Fig 7A), whereas labeling with anti-AMEL M179y was more intense near the enamel surface (Fig 7B). In TM-treated incisors, in regions at which the BL appeared widened, immunoreactivity for amelogenin started farther away from the cell surface (Fig 8). In general, labeling for AMBN over enamel was very weak and in treated rats was almost absent in the region where rods are visible (
In both control and TM-treated rats, ALB was immunodetected in the intercellular space along the basolateral surface of ameloblasts. However, in treated animals, accumulation of ALB between cells was more common and abundant (compare Fig 9B and Fig 9A, and Fig 10B and Fig 10A). There were very few or no gold particles over enamel in control rats, but there was weak and conspicuous labeling at the dentinoenamel junction (Fig 11A and Fig 11B). The number of gold particles over maturing enamel was significantly higher in TM-treated incisors, including at the dentinoenamel junction (Fig 8B, Fig 11C and Fig 11D). Labeling for ALB over enamel was patchy and more intense around regions where the BL was absent or significantly altered.
Control Incubations
Labeling was significantly reduced when sections were incubated with HPA or WGA in the presence of their respective competing saccharides, or when they were incubated with anti-HPA or anti-WGA only, followed by protein Agold. In the case of incubations for EPs and ALB, controls exhibited abolition of the selective labeling observed and the presence of few, randomly distributed gold particles throughout the tissue sections.
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Discussion |
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This study demonstrates that it is possible to achieve a localized administration of TM in the rat hemimandible without systemic side effects. Infusion of this antibiotic through a bony window using an osmotic minipump over a 7-day period causes alterations in both the structure and composition of the BL interposed between ameloblasts and maturing enamel.
Noncollagenous matrix proteins in mineralized tissues, such as bone sialoprotein (BSP) and osteopontin (OPN), are highly glycosylated (reviewed in
In the present study, lectingold cytochemistry was applied to examine compositional alterations of the BL induced by inhibition of N-glycosylation. Under the influence of TM, the BL becomes irregular and/or is replaced by an interfacial region poor in EPs but rich in glycoconjugates. The presence of a large number of HPA binding sites throughout this region suggests that it represents a widened BL. The density of HPA binding sites over the BL did not significantly differ in treated and control animals, indicating that this lectin recognizes glycoconjugates whose concentration is not affected by TM. On the other hand, the density of labeling of WGA appeared to decrease over both the BL and the enamel in TM-treated incisors. This apparent reduction is consistent with the expectation that TM should prevent the incorporation of N-linked sugars, such as GlcNac.
A number of enzymes are involved in the processing and degradation of EPs (reviewed in
The immunodetection of large amounts of ALB in the enamel matrix of TM-treated rats indicates that there has been an abnormal influx of this plasma protein after its administration. Ameloblasts do not synthesize and secrete ALB (
In conclusion, the rat hemimandible bony window model is well suited for studying locally the effect of drugs such as TM. This antibiotic affects both the structure and composition of the BL interposed between maturation stage ameloblasts and enamel. This structure has been proposed to have adhesive and/or filtering functions. No significant detachment of the enamel organ was observed after 7 days of TM administration, but there was an abnormal presence of ALB in maturing enamel. Although not excluding a potential adhesive role, the data therefore lend support to the proposal that this BL is a specialized structure exhibiting filtering functions.
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Acknowledgments |
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Supported by a grant from the Canadian Institute of Health Research.
We thank M. Fortin for general technical assistance and Dr H. Verreault for helping during the surgical procedures.
Received for publication August 10, 2000; accepted August 23, 2000.
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