ARTICLE |
Correspondence to: Antonio Nanci, Département de Stomatologie, Faculté de Médecine Dentaire, Université de Montréal, CP 6128, Station Centre-Ville, Montréal, Québec, Canada H3C 3J7. E-mail: antonio.nanci@umontreal.ca
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Summary |
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The mineral phase in calcified tissues represents an additional factor to be considered during their preservation for ultrastructural analyses. Microwave (MW) irradiation has been shown to facilitate fixative penetration and to improve structural preservation and immunolabeling in a variety of soft tissues. The aim of the present study was to determine whether MW processing could offer similar advantages for hard tissues. Rat hemimandibles were immersed in 4% formaldehyde + 0.1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2, and exposed to MWs for three periods of 5 min at temperatures not exceeding 37C. They were then decalcified in 4.13% EDTA, pH 7.2, for 15 hr, also under MW irradiation. Osmicated and non-osmicated samples were dehydrated in graded concentrations of ethanol and embedded in LR White resin. Sections of incisor, molars, and alveolar bone were processed for postembedding colloidal gold immunolabeling using antibodies against ameloblastin, amelogenin, bone sialoprotein, or osteopontin. Ultrastructural preservation of tissues was in most cases comparable to that obtained by perfusion-fixation, and there was no difference in distribution of labeling with those previously reported for the antibodies used. However, the immunoreactivities obtained were generally more intense, particularly at early stages of tooth formation. Amelogenin was abundant between differentiating ameloblasts and labeling for osteopontin appeared over the Golgi apparatus of odontoblasts after initiation of dentine mineralization. We conclude that MW irradiation represents a simple method that can accelerate the processing of calcified tissues while yielding good structural preservation and antigen retention.
(J Histochem Cytochem 49:10991109, 2001)
Key Words: enamel proteins, noncollagenous matrix proteins, calcified tissues, extracellular matrix, microwave processing, immunocytochemistry
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Introduction |
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The tissue processing protocol for immunocytochemistry is usually a compromise between the quality of structural preservation and the degree of retention of antigenicity. Chemical fixation with aldehydes yields good ultrastructural preservation. However, concentrations normally used in morphological studies frequently interfere with preservation of antigenicity. There are two main approaches for chemical fixation, immersion and perfusion. The first is commonly used but its application for ultrastructural studies is limited because preservation is affected by the rate of penetration of fixative into the tissues (reviewed in
Microwave (MW) processing has been shown to improve tissue preservation during immersion-fixation in a variety of soft tissues (
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Materials and Methods |
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All animal procedures were in accordance with guidelines of the Comité de déontologie de l'expérimentation sur les animaux of Université de Montréal.
Microwave Processing of Tissues
Male Wistar rats (Charles River Canada; St-Constant, PQ, Canada) weighing approximately 100 g were anesthetized with Metofane (methoxyfluorane; Janssen Pharmaceutica, North York, ON, Canada) and decapitated. The hemimandibles were dissected out and quickly immersed in 4% paraformaldehyde + 0.1% glutaraldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. All soft tissues covering the bone were gently removed. In addition, the alveolar bone overlying the buccal aspect at the apical end of the incisor was partially broken and the tip of the incisor was fractured using a bone-cutting forceps. Specimens were immersed in a beaker containing 40 ml of fixative at room temperature (RT), which was subsequently placed in a 20 x 20-cm glass recipient filled with ice and placed in a Pelco 3440 laboratory MW oven (Ted Pella; Redding, CA). The temperature probe of the oven was submersed in the fixative and the specimens were immediately exposed to MW irradiation at a 100% setting for three periods of 5 min with the temperature programmed to a maximum of 37C (
Postembedding Colloidal Gold Immunocytochemistry
Ultrathin sections of osmicated samples were pretreated with an aqueous solution of 5% sodium metaperiodate for 60 min (
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Results |
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Preservation of Tissues
Cells of incisor and molar tooth organs, as well as of alveolar bone, exhibited good ultrastructural preservation (Fig 1 and Fig 2). The plasma membrane was well delineated and the distinctive structural details of organelles were clearly apparent. Mitochondria were not swollen and showed tightly packed cristae. There was no space between cells and the extracellular matrix they produce. In particular, there was no "stippled material" interposed between ameloblasts and enamel, as is usually seen in immersion-fixed teeth (see
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Postembedding Colloidal Gold Immunocytochemistry
As immunolabelings of enamel, dentine, bone and cementum with the antibodies used in the present study have been extensively described for conventionally fixed tissues, only those results that highlight issues pertinent to MW fixation and to the discussion will be presented.
Labeling for amelogenin was detected between differentiating ameloblasts before they fully reversed polarity. At this early time point, there were some gold particles in the unmineralized mantle dentine associated with either the basement membrane at the apical surface of ameloblasts or nearby patches of organic matrix (Fig 3A). At a slightly later time, labeled secretory granules were found at the distal pole of ameloblasts. The patches in the forming dentine matrix were larger and more intensely immunoreactive. These were frequently associated with odontoblast cell processes and occasionally with matrix vesicles (Fig 3B). When the basement membrane was no longer present, abundant patches of matrix, intensely immunoreactive for amelogenin, were present among the superficial collagen fibrils (Fig 4A). The ameloblasts associated with these patches exhibited immunoreactivity over the Golgi apparatus, secretory granules, and multivesicular bodies (Fig 4B and Fig 4C). Differentiating ameloblasts showed immunoreactivity for ameloblastin, but there was no readily apparent labeling over the unmineralized mantle dentine. During the secretory stage, the Golgi apparatus, secretory granules, and enamel matrix were intensely labeled (Fig 5A and Fig 5B). Gold particles over enamel were concentrated at growth sites (Fig 5A). No significant labeling for either ameloblastin or amelogenin was observed in differentiating odontoblasts.
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In regions of bone development, labeling for bone sialoprotein (Fig 6) and for osteopontin was observed in close relation to mineralization foci. Cement lines in bone as well as interfibrilar matrix in both bone and cementum were also immunoreactive for both noncollagenous proteins (Fig 7A, Fig 7B, and Fig 8). The lamina limitans outlining osteocyte lacunae and canaliculi lodging their processes occasionally showed labeling for osteopontin (not usually observed with this antibody). Distinctively, at early stages of tooth development, the Golgi apparatus of odontoblasts was labeled for osteopontin (Fig 9D9F). The density of labeling over this organelle increased from the time when mineral crystals appeared in matrix vesicles to when a uniform layer of mantle dentine was evident (Fig 9A9C). However, no significant labeling was observed over dentine matrix at any stage of dentinogenesis.
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Discussion |
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The present study shows that immersion-fixation with MW irradiation can be applied to the immunocytochemical characterization of calcified tissues. The procedure is simple, yields ultrastructural preservation comparable to that obtained with perfusion-fixation, and retains antigenicity of the various noncollagenous matrix proteins examined. Although the exact mechanism of the MW effects is still not fully defined, it has been postulated that irradiation induces oscillation of water molecules (
Decalcification of large mineralized samples, e.g., as human teeth, requires exposure to EDTA for long periods of time, introducing alterations in their ultrastructure. Although irradiation with MWs has been previously used to shorten decalcification time (
After establishing that MW irradiation yielded good ultrastructural preservation, our aim was to determine its impact on the immunodetection of major noncollagenous matrix proteins present in calcified tissues. In general, the immunoreactivity obtained was at least as intense as in previous reports (see references below) and, in most cases, it appeared stronger. This is consistent with the findings that MW irradiation can be used for antigen retrieval (reviewed in
Enamel Proteins
It has long been believed that initiation of dentinogenesis precedes amelogenesis. However, a number of immunocytochemical studies have shown that differentiating ameloblasts already secrete amelogenin during the presecretory stage of amelogenesis (discussed in
Noncollagenous Proteins of Collagen-based Tissues
A number of studies using a variety of approaches, including immunohistochemistry (
Bone sialoprotein and osteopontin are conspicuously present on patches of interfibrillar matrix and over cement lines in bone (reviewed in
In summary, MW-processed calcified tissues show good ultrastructural preservation and no difference in distribution of labeling of representative noncollagenous matrix proteins compared to published studies with conventional tissue-processing methods. In addition, immunoreactivity with all the antibodies used was in general stronger and additional sites of labeling were revealed with the anti-osteopontin antibody, supporting the antigen retrieval potential of MW irradiation. We conclude that this approach offers an advantageous alternative for the processing of calcified tissues, in which the mineral phase may complicate the penetration of reagents such as fixatives and decalcification agents.
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Acknowledgments |
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V. E. AranaChavez was the recipient of a research fellowship (99/11187-5) from FAPESP, Brazil. This work was supported by an operating grant from Canadian Institutes of Health Research (CIHR).
We thank Sylvia Zalzal for help with immunocytochemical procedures and for preparing the protein Agold complexes and Micheline Fortin for ultrathin sectioning. We are grateful to Dr L. W. Fisher (National Institutes of Health, Bethesda, MD) for supplying the LF-87 antibody and to Dr P. H. Krebsbach (University of Michigan, Detroit, MI) for the ameloblastin antibody.
Received for publication December 14, 2000; accepted March 21, 2001.
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