,;, Periodontal Res, 10: 191-196

Scanning electron microscopic study of human gingival epithelial cells on the surface of teeth and glass slides REINALDO SAGLIE, JAN R, JOHANSEN AND TORE TOLLEFSEN

Department of Periodontology, Dental Faculty, University of Oslo, Oslo, Norway Scanning electron microscopy was used for a comparative study of gingival epithelial-)ike cells grown on glass slides atid epithelial remnants on extracted teeth. The micrographs showed that the shape, surface morphology, edges, intercellular contacts, light areas, and fibrous-like products of the cells were quite similar. The method facilitated identification of epithelial cell remnants in the gingival pocket area of extracted teeth, (Received for publication April 7, 1975; accepted April 28,1975) Introduction

Material and Methods

The presence of epithelial attachment remnants in the gingival pocket area on the surface of extracted teeth has been reported (Bass 1946, Waerhaug 1952, Saglie, Johansen & Flotra 1974, Saglie, Johansen & Tollefsen 1975), Other structures have also been shown to be present, such as adherent bacterial plaque and calculus, cuticuiar structures, collagen fibers and blood cells. Information of gingival epithelial cell morphology in the light microscope and in the transmission electron microscope is available (Listgarten 1972), However, information about the three-dimensional morphology of gingival epithelial cells as seen in the scanning electron microscope is scarce. The purpose of this investigation was to describe gingival epithelial cells remaining on the surface of extracted teeth in the area of the plaquefree zone (Saglie et al, 1975), For comparison, the morphologic characteristics of cultivated epithelial-like cells were studied.

Celt Culture Monolayers of gingival epithelial-like cells (Smulow & Glickman 1966) were cultivated in Carrel bottles in minimum essential medium (Eagle 1959) with Earie's sails. Catalogue No, F-11 (Gibco, New York) and supplemented with 10 % foetal calf serum. The cells were trypsinized and transferred in medium to petri dishes containing 4 glass cover slips. The petri dishes were incubated at 37°C in a gas phase containing 5 % CO, in air. After about 48 hours, the cells had formed a monolayer on the cover slips. At this time the culture medium was removed and the cells washed for 2 min in a sodium hypochloride solution. The cells were then fixed in 3 % glutaraldehyde in Sorensen's buffer at pH 7,4 for 48 hours at a temperature of 20° C, They were postfixed for 4 hours in 2 % osmium tetroxide and dried according to the critical point method of Anderson (1951), The cells were then coated

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with a thin layer of carbon and gold and examined in a Jeol 50A scanning electron micrascope (SEM operated at 10 to 20 kV. The Teeth Epithelial cells remaining on the surface of the teeth were studied on 6 teeth with chronic periodontifis, (Pocket depths ranging from 3 to 5 mm,) After extraction, the teeth were rinsed in tap water to remove all loose debris, fixed, dehydrated and coated as described above. Results

Gingival Epitheiial-like Ceil Culture The cells showed a wide variation in shape and size. Some were round (Figs, 1, 2), others flattened (Fig, 5) and other polygonally shaped (Fig. 1), Several cells appeared to be in mitosis (Figs, 1, 2), The surfaces were completely (Fig, 4) or partially (Fig, 2) covered by blebs ranging in size from 0,2 to 1 p,m, Microvilli (small single extensions on the cell surface) having a uniform diameter of about 0.1 (,im and variable lengths, generally 1,5 jim, were found (Fig. 3), Fiiopodja (single surface extensions from 2 to 5 |im, with the same diameter as the microvilli) extending from the surface of the cells to the substrate were also seen (Fig, 2) together with light areas associated with the edges of the cells (Fig, 1), Fibrous-like cell products, resembling a mesh, were also observed (Fig, 6), The borders of the cells were folded or straight and usually irregular when in contact with adjacent cells (Figs, 1, 5),

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Gingival Epithelial Celis Remaining on Extracted Teeth In general, the observations almost paralleled those of cultivated cells. The shape (Figs, 7, 8, 13) and surface morphology (Figs, 9, 10, 13) correlated well with those of cultivated cells, but the size varied in both cell lines, fn two specimens cells in a stage indicating mitosis were seen in the apical part of the epithelial remnants (Figs, 7, 8), Light areas, cellular edges, together with intercellular connections were similar to those found in the cultivated cells (Figs, 7, 11), Small amounts of fibrous-like material were often seen (Fig, 12), Discussion

SEM study of morphologic characteristics of epithelial cells in culture may aid in the identification of epithelial cells remaining on the surface of the teeth, A clear correlation between the shape and surface feature of cultivated gingival epithelial-like cells and cells remaining on the teeth was seen. The substrate on which the cells grow and the ecological conditions probably influence their size and appearance. According to Porter, Prescott & Frye (1973), the surface and overall morphology of cells change as they progress through their life cycle. This may explain the many different shapes and morphologic variations of the cells observed in the present study. The precise function of microprocesses on the surface of the cells is not yet fully understood, but they are probably implicated

Fig, 1, Gingival epitheliai-lii^e cells in cuiture. The ceils are polygonally siitped with cieariy demarcated ceiluiar boundaries. The white arrow is pointihg at celis in the rouoded stage, compatible with mitcsis. X 5D0. Fig, 2. Higiiier magnification c l the ceils indicated by the arrow m fig. 1. The ceiis are attached to the substrate by iong fiiopodia. The surface of the celis is partialiy ccvered by biebs. x 5000, 'Fig. 3, Part of the surface of a ceil in ouiture covered mostiy by micreviiiL x 10,000. Fig, 4, Part of a cultivated celi. The surface is compieteiy covered by biebs. x 10,000. Fig, 5, interceiluiar connections between ceiis in cuiture. x 10,000. Fig, 6, Fi^brous-iiice materiai from the surface of cuitivated ceHs, x 5250:

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Fig. 13. Gingival epitheita! cells attached to the surface of a tooth, demonstrating different characteristics: a round oeli in the center is surrounded by flattened ceiis; ic — interce^ular connection; m — microviiii; b = blebs; f = fiiopodia. x 4000.

in the function of locomotion and of cellular attachment contacts between cells (Ambrose 1967). Accordingly, Hodges. & Muir (1972) stated that the apparently greater frequency and overall distribution of microprocesses on the surface of epitheliaUike cells suggests a highly dynamic cell surface which may reflect an inherent property of cells specialized for absorption or resorption. Microprocesses, such as blebs or microviiii, may be mistaken for clusters of coccal or filamentous microorganisms. However, the width of the microviiii is 0.1 um (Porter et a!. 1973). The average width of elongated microorganisms studied in the SEM is approximately 0.6 ^m (Boyde & Williams 1971, Saglie et al. 1974). Moreover, coccal microorganisms have been reported to be covered and connected by a light material probably produced by the cocci (Carlsson & Sundstrom 1968,

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Schroeder & de Soever 1970, Saglie et al. 1974) which masked the shape of these microorganisms. The blebs on the surface of the cells in the present study were clearly demarcated with well-defined edges. These features seem to exclude the misinterpietation between blebs and cocci. In the region of the apical extent of the junctionai epithelium, especially adjacent to the zone of the partially lyzed fibers (Saglie et al. 1974), the fibrous-like products of gingival epitheliai cells may be mistaken for disrupted periodontal fibrils or fibrin. This material may correspond to the extraneous cell surface coat pointed out by Schroeder (1968) using tratismission electron microscopy. Detached and collapsed blebs may resemble erythrocytes, but consideration of the size will rule out this interpretation. In the zone of the epithelial attachment remnants, isolated cells such as erythrocytes and polymorphonuclear leukocytes may be present. In the SEM the intercellular contacts between the epithelial cells attached to the tooth surface support the interpretation that the cells described in this study are of epithelial origin. Furthermore, the flattened appearance together with the surface morphology described add clues to distinguish between isolated occasionally found cells aod true epithelial ceil remnants (Rubin & Everhart 1973). Epithelial cells in a rounded stage, compatible with mitosis (Porter et al. -1973, Paweletz & Schroeter 1974), were found in the most apical part of the junctionai epithelium (Figs. 7,, 8). This observation suggests that the germinative cells in the most apical

Fag. 7. Epitheliai ceils attached to the surface of a tooth. The white arrow is pointing at ceiis in the rounded stage, compatibie with rr^itosis. x 500, Fig. B. Higher magnificatmn of the ceiis indicated by the arrow in Fig. 7. The ceiis to the ieft show biebs on their surfaces, x 4500. Fig. 9. Part of an epitheiiai ceil attached to a tooth. The surface is covered mostiy by microvilEL x 10,000. Fig. 10. Part of another ceil covered predominantiy by blebs of different size, x 10,000. Fig. 11. interceiiuiar connections between gingfval epitheiiai ceiis from the surface of a tooth. X 10,000. Fig. 12. Fibrous-iii

Scanning electron microscopic study of human gingival epithelial cells on the surface of teeth and glass slides.

,;, Periodontal Res, 10: 191-196 Scanning electron microscopic study of human gingival epithelial cells on the surface of teeth and glass slides REIN...
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