ARTICLE |
Correspondence to: Takaaki Aoba, Dept. of Pathology, Nippon Dental University, 1-9-20 Fujimi Chiyoda-ku, Tokyo, Japan 102-8159. E-mail: pathology-ndu@tokyo.ndu.ac.jp
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Summary |
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Osteopontin (OPN) has been proposed to act as a substrate for osteoclast adhesion during bone resorption. The aim of the present study was to examine the presence and distribution of OPN at sites of resorption in traumatized radicular pulp. The upper first molars of 6-week-old male SpragueDawley rats were luxated and then repositioned in the original sockets. The animals were sacrificed by intracardiac perfusion at 10 and 14 days after tooth reimplantation. The teeth were decalcified in EDTA and then processed for embedding in paraffin for histochemistry or LR White resin for immunocytochemistry. Odontoclasts were identified by their multinucleated morphology and expression of tartrate-resistant acid phosphatase (TRAP). Osteopontin was immunolocalized using postembedding colloidal gold labeling with a chicken egg yolk anti-rat OPN antibody. After reimplantation of the teeth, TRAP-positive cells were present along the pulp dentin wall. Osteopontin was not consistently detected at exposed predentin/dentin surfaces. However, gold particles were often found at the margin of resorption lacunae. Labeling was also seen over the Golgi region and cytoplasmic vesicles of odontoclasts and of neutrophils and fibroblast-like cells. The results suggest that accumulation of OPN at the predentin/dentin surface is not a prerequisite for adhesion of odontoclasts to the wall substance and that recruited odontoclasts produce OPN locally to mediate cell and/or matrix events within the resorption lacuna. (J Histochem Cytochem 50:911921, 2002)
Key Words: odontoclast, osteopontin, dentin resorption, traumatized pulp, tooth reimplantation
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Introduction |
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INTERNAL RESORPTION on the coronal and root dentin surrounding the pulp tissue is a rare pathological process in human permanent teeth (
Osteopontin (OPN), a sialic acid-rich phosphorylated glycoprotein, is found in bone and a variety of calcified tissues (reviewed by vß3 integrin (
Although there have been a number of studies on the association of OPN with matrix resorption in bone, cartilage, and external root (e.g., dentin and cementum), little is known about odontoclastic resorption along the predentin/dentin surface in the tooth pulp tissue. The aim of the present study was to elucidate the involvement of OPN in the pathogenesis of odontoclastic dentin resorption. We previously developed an experimental protocol to monitor time-sequential events taking place in the pulp chamber after reimplantation of rat molars (
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Materials and Methods |
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Surgical Procedure
Six-week-old male SpragueDawley rats were used in the study (Charles River; Tokyo, Japan). All surgical procedures were carried out under general anesthesia with an IV injection of sodium pentobarbital (Nembutal, 30 mg/kg body weight; Abbott Lab, North Chicago, IL). The animals were placed in the supine position on a surgical board and the heads were held in a stable position with the aid of a head holder and ear rods. The upper first molars were extracted by luxating the tooth with the aid of a needle holder. The teeth were immediately placed in Hank's buffered solution and verified for signs of root fracture or damage under a stereoscope before repositioning in the original socket. All surgical procedures were carried out under sterile conditions and were completed within 15 min. The experimental protocol was approved by the Animal Care Committee of the Nippon Dental University.
Light Microscopy
The animals were sacrificed at 10 or 14 days after reimplantation of the molars because it had previously been shown that internal radicular resorption showed a high incidence at these time intervals (
Electron Microscopy
Tissue specimens for transmission electron microscope observation were fixed by perfusion with 1% glutaraldehyde in 0.08 M sodium cacodylate buffer (pH 7.4). The dissected maxillae were decalcified in 4.13% EDTA, pH 7.4 (
Immunocytochemistry
Some of the specimens fixed with glutaraldehyde were processed for immunocytochemistry. After demineralization, non-osmicated specimens were embedded in LR White resin (
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Results |
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As the collection consisted of 17 roots of rat first molar (four medial root, six middle roots, and seven distal roots), the selected teeth showing signs of resorption amounted to six roots (two medial roots, three middle roots, and one distal root) in this study. Fig 1 illustrates representative morphological features of resorption lacunae developed along the pulp-side wall of root dentin at 10 and 14 days after tooth reimplantation. The pulp tissue in root canals of reimplanted teeth was severely degenerated or necrotic and was gradually replaced by granulation tissue infiltrated with inflammatory cells such as neutrophils and macrophages. The odontoblasts were damaged and detached from the predentin/dentin surface, but the predentin layer appeared intact. Various stages of resorption by TRAP-positive multinuclear cells were discerned along the exposed root wall. Histological examination of several root samples at 14 days after reimplantation showed the presence of fibroblast-like mononuclear cells in the resorption lacunae and the deposition of new matrix. A large portion of the exposed predentin remained free of resorption activity and was associated with spindle-shaped mononuclear cells. The following phases of resorption were observed: (a) initial contact phase, evidenced by the presence of multinucleated giant cells contact to the predentin; (b) active phase, evidenced by the presence of odontoclasts attached to the exposed dentin; (c) intermittent or resting phase, featured by the detachment or disappearance of TRAP-positive cells from the lacunae; and (d) repairing phase, characterized by the filling of lacunae with newly deposited matrix, on which a layer of cuboidal mononuclear cells was frequently seen.
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Quantitatively, at 10 days after treatment resorption lacunae were classified as one initial contact phase and seven active phases. At this time intermittent phase and repair phase lacunae were not yet observed. At 14 days after treatment, resorption lacunae were classified in six active phases, four intermittent phases and seven repair phases. At sites of initial contact the odontoblasts covered the root surface, multinucleated giant cells extended wide cytoplasmic processes through the dentin, and the body of the cell remained on the predentin surface. Abundant mitochondria, small vesicles, and rough endoplasmic reticulum characterized these odontoclasts. Initiation of ruffled border formation was observed within the area of the clear zone-like structure. Concomitant with these cell surface alterations, many vesicles appeared in the cell processes. There was no significant immunolabeling for OPN along the ruffled border-like and sealing zone-like areas of odontoclast (Fig 2B and Fig 2C), and over the predentin and dentin matrix.
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Sites showing active resorption exhibited cells on the root surface with ultrastructural characteristics of clastic cells (Fig 3A). They were multinucleated large cells, containing many mitochondria and several Golgi structures with variously sized vesicles and vacuoles in their cytoplasm. The mature odontoclasts had prominent cell polarity, especially at the resorbing surface. A sealing zone with scant cytoplasmic organelles was found at the periphery of the cell (Fig 3B), and a well-developed ruffled border (Fig 3C) was found. Extracellularly, there was diffuse labeling for OPN near the ruffled border area (Fig 3D), but labeling was more tightly distributed outside of the sealing zone (Fig 3E). Gold particles were also found over the Golgi areas at perinuclear regions of odontoclasts (Fig 3E, inset). In some cases, neutrophils frequently seemed to co-migrate and closely contacted active odontoclasts inside OPN-coated lacunae (Fig 4A and Fig 4B). There was immunoreactivity over cytoplasmic vacuoles in neutrophils and along fine fibrils apposed to the lacunae wall.
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After odontoclasts detached from the resorption lacuna, the exposed dentin surface frequently showed intense metachromatic staining (Fig 1B). It consisted of an irregular dentin edge, an electron-dense line, and a layer of amorphous material, which on occasion was covered with an electron-dense line (Fig 5A). Short and irregular collagen fibers were present within the lacunae. These fibrils were oriented approximately at the surface of the lacunae. Fibroblast-like mononuclear cells were seen close to the resorbed dentin surface. OPN labeling was present over the old root matrix but dentin beyond the superficial region was not immunoreactive. The electron-dense line observed at the dentin surface was intensely labeled for OPN. When present, the amorphous material between old root matrix and the electron-dense line was very weakly or not at all immunoreactive.
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After the damaged odontoblasts detached from the predentin, fibroblast-like cells without evidence of clastic resorption covered a large portion of the exposed predentin wall. Mononuclear cells close to the denuded dentin frequently established contacts with its surface. Immunostaining of the corresponding region revealed that underneath fibroblastic cells, gold particles were present over the dentin matrix, particularly at its mineralized surface in association with degraded collagen fibrils. In some areas an electron-dense line, which was labeled for OPN, was observed between the dentin matrix and degraded organic matrix. Gold particles were also found over cytoplasmic organelles of various types of infiltrating mononuclear cells, including fibroblastic cells (Fig 6).
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Discussion |
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On the basis of the histological observations in the current and previous studies (
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Osteoclasts express vß3 integrin (
9ß1 integrin after exposure to thrombin cleavage (
9ß1 integrin is highly expressed on neutrophils (
Osteopontin-enriched electron-dense lines (i.e., cement line, lamina limitans) were not consistently detected at sites where odontoclasts contacted the exposed dentin wall. When present, OPN showed a diffuse distribution throughout the exposed dentin surface. The infrequent presence of OPN immunoreactivity suggests that the major mechanism for odontoclast adhesion onto dentin is not mediated by this non-collagenous protein. Osteoclasts have been shown to interact with uncalcified bone surface in the absence of a cement line (2ß1 integrin and denatured collagen via
vß3 integrin (
vß3 ligands with lower affinities, e.g., bone sialoprotein, but the weaker interactions may result in less efficient osteoclast function (
As regards the origin of OPN that coats the unresorbed exposed dentin wall, it may be produced locally by mononuclear cells (fibroblasts, macrophages, neutrophils) and free odontoclasts. Circulating molecules may also contribute to the formation of surface coatings. In this context, it has been demonstrated that exogenously administered OPN is incorporated into cement lines and newly formed bone (
In conclusion, the present results indicate that the internal root surface of rat molars can be used as an experimental model for investigating the recruitment and activation of odontoclasts. The cue to development of odontoclastic dentin resorption is the replacement of the innate pulp tissue with granulation tissue. The initial attachment of odontoclasts to the predentin wall does not necessarily need preconditioning with OPN and appears to be mediated by alternative recognition mechanisms.
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Acknowledgments |
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Supported by grants-in-aid for Scientific Research (12671866) from the Ministry of Education, Science and Culture of Japan (YS) and the Canadian Institutes of Health Research (AN).
We are grateful to Dr K. Sato for her great help in preparation of the manuscript and illustrations, Sylvia Zalzal for assistance with immunolabeling, and Micheline Fortin for technical assistance.
Received for publication January 9, 2002; accepted January 30, 2002.
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