ooo3-9%9/91 $3.00 + 0.00 Copyright 0 1991 Pqamon Press plc

Archs oral Bid. Vol. 36, No. 9, pp. 693-696, 1991 Rioted in Great Britain. All rights reserved

SHORT COMMUNICATION AN E,LECTRON MICROSCOPIC STUDY OF DENTAL PLAQUE OF THE RAT INCISOR P. D. NOVAES,’0. P. ALMEIDA,’R. G. JAEGER’and V. S. ITO’ Department of Oral Pathology, ‘Faculty of Odontology of Piracicaba and *Faculty of Odontology of Slo Paulo, Brazil (Accepted 13 March 1991) Summary-Because rat incisors continuously erupt they provide an opportunity for the study of dental plaque at all stages of its development. The youngest plaque would be visible at the gingival margin of the tooth as it erupts, and the older plaque higher on the tooth. The ultrastructural features of these plaques were studied by transmission electron microscopy. Cocci and short rods colonized the cementum surface, forming a monolayer. The plaque had a maximum thickness of about 40 pm, with the inner third rich in fibrillar matrix and the organisms forming microcolonies perpendicular to the tooth surface. Cells were haphazardly distributed in a loose matrix on the surface of the plaque. In the area of plaque disorganization the cementum was covered by isolated groups of bacteria and the matrix had holes in it. The rat mandilbular incisor may provide a unique model for study of how plaque on cementum is initially formed, matures and finally is degraded. Key words: plaque, rat, incisor, ultrastructure.

The morphological aspects of colonization and organization of plaque on the enamel and cementum in vitro and in vivo have been described many times (Theilade and Theilade, 1970; Listgarten, 1976; Brecx et al., 1981; Ten Nape1 et al., 1985; Nyvad and Fejerskov, 1987a,b; Carassi, Santarelli and Abati, 1989). None of these studies, however, describes the sequential colonization, organization and destruction of dental plaque simultaneously on the same surface. On the continuously erupting rat incisor dental plaque can be examined at all stages of its development. In order therefore to elucidate more fully the features of plaque formation we have now studied the morphology of plaque on rat incisor cementurn by transmission electron microscopy. Eight mandibular incisors of four normal Wistar rats weighing 200-220 g were used. The animals were killed by cervical dislocation and their mandibles fixed in 2.5% buffered glutaraldehyde. The teeth were dissected out at the gingival margin, demineralized in EDTA according to Warshawsky and Moore (1967), and postfixed in 1% OsO.,. The incisor was cut in a series of segments from the gingival margin to the in&al edge and embedded in Araldite. Thick sections (0.5 pm) were stained with 1% toluidine blue and ultrathin sections with uranyl acetate and lead citrate. Observations were made in a Zeiss EM10 of the plaque formed over the cementurn covering the distal aspect of the incisor. On the distal surface the cementum was covered by a uniform layer of plaque except at the incisal edge. The distance from the gingival margin to the initial appearance of a monolayer of coccoid and rodshaped micro-organisms was about 100 pm, and this was quickly followed by the organization of multilay-

ers of 4-5 cells loosely aggregated with sparse intercellular material (Fig. 1). The amorphous material initially deposited on the cementum surface was heterogeneous, granular, and contained irregular vacuolar membranous structures. When the bacterial population increased, some contained septa indicative of cell division, and the matrix acquired a dense fibrillar structure, with fibrils parallel to the long axis of the organisms and perpendicular to the tooth surface (Fig. 2). The mature plaque extended as a continuous layer along the cementurn surface with a relatively even thickness of about 40 pm (Fig. 3). The types of organisms as well as the density and structure of the matrix were variable. Close to the cementum the matrix was dense and fibrillar, with cocco-bacillary types predominating, forming micro-colonies in a palisade-like arrangement perpendicular to the tooth surface, with many cells in division (Fig. 4). Bacteria or fibrils of the matrix were never observed penetrating into the cementurn. In the central parts of the plaque the bacterial density was variable, with some regions formed by dense aggregates of filaments, cocci and rods, while others had ample spaces among the bacteria, containing a loose, granular substance. In the superficial layers the microbiota was more variable, with different types of bacteria randomly disposed in a matrix of very low electron-density. Some bacteria were not attached to the plaque, apparently leaving or tenuously adhering to its surface (Fig. 5). On the incisal third of the incisor the oldest plaque was suffering destruction, with aggregates of microorganisms in a sparse matrix dispersed haphazardly over the cementum. As plaque became progressively

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thinner the cementum was again covered by a discontinuous monolayer of bacteria, then by a thin acellular matrix and finally was denuded and exposed to the mouth (Fig. 6). The isolated bacteria were only partly surrounded by matrix, indicating that they were not firmly attached to it. The colonization, organization and degradation of plaque can be conveniently studied on the rat incisor, although due to the relatively rapid rate of eruption, periods of study are limited. Also, as a consequence of the continuous growth the plaque is supragingival only, and adjacent to the gingival margin a few bacteria are seen close to the cementurn. The pellicle initially deposited on the cementurn was granular and with the presence of bacteria became dense and fibrillar, as described by Nyvad and Fejerskov (1987a,b). The structure of the fibrillar matrix was similar to the mutan produced by streptococci and it is known that these micro-organisms, during the early phases of plaque formation, produce plentiful extracellular polysaccharide (Minah and Loesche, 1977). The cementum was initially colonized mainly by cocco-bacillary forms, as described by various investigators (Theilade and Theilade, 1970; Brecx et al., 1981; Nyvad and Fejerskov, 1987a,b; Carassi, Santarelli and Abati, 1989). The monolayer abruptly turned into a denser, even plaque with a maximum thickness of about 4Opm, implying that local factors do not allow continuous growth. On the inner third the growth of plaque was largely by cell proliferation as it was formed by microcolonies of septate bacteria perpendicular to the root surface (Listgarten, Mayo and Tremblay, 1975). In the superficial layer the matrix was loose, bacteria did not form micro-colonies, and on the actual surface organisms seemed to float, as if adhering to or leaving the plaque. Bacterial interactions as described by Jones (1971, 1972) with ‘corn cob’ formations were not observed, probably because of the relatively short maturation time of this plaque. The destruction of the plaque was abrupt, with bacteria forming small, irregular aggregates or isolated cells partly covering the cementum. The alveolar structure of the matrix during this disorganization can be explained by the dislodgement of bacteria, as each alveolus corresponds in size and shape to micro-

organisms that initially colonized the tooth surface. This aspect of plaque disorganization has not been previously described. The mechanisms involved in the destruction of the plaque are unknown; the simple mechanical action of chewing would seem to be the most likely, although other local factors may be relevant. Our preliminary observations are that xerostomic rats form less plaque on the incisor than normal. In fact the nutrients of plaque come mainly from saliva (Carlsson, 1980), and Quirynen and van Steenberghe (1989) described, in humans, a smaller formation of plaque during the night, which could be related to the decreased salivary flux. Further studies are needed to elucidate the possible mechanisms. We show, for the first time, the formation, maturation and degradation of plaque on the same tooth surface. The unique characteristics of this plaque also provide a model that can contribute to better understanding of the mechanisms involved in plaque organization. REFERENCES

Brecx M., Ronstrom A., Theilade J. and Attstrom R. (1981)

Early formation of dental plaque on plastic films. 2. Electron microscopic observations. J. periodont. Res. 16, 213-227. Carassi A., Santarelli G. and Abati S. (1989) Early plaque colonization on human cementum. J. c/in. Periodont. 16, 265-267.

Carlsson T. (1980) Symbiosis between host and microorganisms in the oral cavity. Stand. J. Infect. Dis. 24, 74-78.

Jones S. J. (1971) Natural plaque on tooth surfaces: A scanning electron microscope study. Apex, J. Univ. Coil. Hosp. dent. Sot. 5, 95-98.

Jones S. J. (1972) A special relationship between spherical and filamentous microorganisms in mature human dental Dlauue. Archs oral Biol. 17. 613416. Listgaiten M. A. (1976) Str&ture of surface coating on teeth. A review. J. Periodont. 47, 139-147. Listgarten M. A., Mayo H. E. and Tremblay R. (1975) Development of dental plaque on epoxy resin crowns in man. A light and electron microscopic study. J. Periodont. 46, l&25.

Minah G. E. and Loesche W. T. (1977) Sucrose metabolism by prominent members of the flora isolated from cariogenie and non-cariogenic dental plaques. Infect. Immun. 17, 55-61.

Plate 1 Fig. 1. Early colonization of the cementum (C) by cocco-bacillary micro-organisms showing the region where the monolayer abruptly forms a plaque of about 7pm thickness. TEM- x 2000 Fig. 2. Dental plaque formed by 3-5 layers of bacteria. The matrix is fibrillar especially in the proximity of the cementum (star). TEM- x 5000 Fig. 3. Dental plaque fully developed on the cementum surface of the rat incisor, with an even thickness of about 4Opm. The density and types of bacteria are variable, as well as the amount of intercellular material. The cells form colonies perpendicular to the tooth surface, except at the external third. TEM- x 1000 Fig. 4. Inner part of bacterial deposits on cementurn with micro-colonies perpendicular to the root surface, surrounded by a fibrillar intercellular matrix. Many cells have septa indicative of cell division. TEM- x 5000 Fig. 5. Superficial layer of plaque showing bacteria haphazardly distributed in a matrix of very low electron-density. On the very surface the arrow heads point out two cells apparently leaving or adhering to the plaque. Many small vesicles are seen on the surface of the plaque. TEM- x 5000. Fig. 6. Cementum covered by matrix in disorganization containing membranous vacuoles (arrows) and some bacteria on the surface. TEM- x 5000

Dental plaque of the rat incisor

Plate 1

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Nyvad B. and Fejerskov 0. (1987a) Scanning electron microscopy of early microbial colonization of human enamel and root surfaces in viva. Stand. J. dent. Res. 95,287-296. Nyvad B. and Fejerskov 0. (1987b) Transmission electron microscopic of early microbial colonization of human enamel and root surfaces in vivo. Stand. J. dent. Res. 95, 297-307. Quirynen M. and van Steenberghe D. (1989) Is early plaque growth rate constant with time? J. clin. Periodont. 16, 278-283.

Ten Nape1 J. H., Theilade J., Matsson L. and Attstrom R. (1985) Ultrastructure of developing subgingival plaque in beagle dogs. J. clin. Periodont. 12, 507-524. Theilade E. and Theilade J. (1970) Bacteriological and ultrastructural studies of developing dental plaque. In Dental Plaque (Ed. McHugh W. D.), pp. 27-48. Livingstone, Edinburgh. Warshawsky H. and Moore G. (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J. Htitochem. Cytochem. 15, 542-549.

An electron microscopic study of dental plaque of the rat incisor.

Because rat incisors continuously erupt they provide an opportunity for the study of dental plaque at all stages of its development. The youngest plaq...
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