Electron microscopic and histochemical studies of the mononuclear odontoclast of the human.код для вставкиСкачать
THE ANATOMICAL RECORD 240:42-51 (1994) Electron Microscopic and Histochemical Studies of the Mononuclear Odontoclast of the Human TAKANORI DOMON, KAZUYUKI SUGAYA, YASUTAKA YAWAKA, MASAKAZU OSANAI, YOSHINORI HANAIZUMI, SHIGERU TAKAHASHI, AND MINORU WAKITA Departments of Oral Anatomy 11 (T.D., Y.H., S.T., M.W.) and Pediatric Dentistry (Y.Y., M.O.), Hokkaido University School of Dentistry, Sapporo, Department of Anatomy 11 (K.S.), Nihon University School of Dentistry at Matsudo, Matsudo, Japan ABSTRACT Background: Osteoclasts and odontoclasts are multinucleated giant cells which resorb hard tissue by the ruffled borders. Recently, the authors reported the presence of a mononuclear osteoclast with a ruffled border in vitro. However, its presence in vivo has not been shown. To demonstrate the presence of a mononuclear odontoclast in humans, the present study used human deciduous teeth. Methods: After fixation and decalcification, tartrate-resistant acid phosphatase (TRACPase) activity was detected with the azo dye method, and then TRACPase-positive cells were observed on resorbing areas of teeth. TRACPase-positive cells could be distinguished from other cells by light microscopy, and the cells for investigation were serially sectioned by alternating semithin and ultrathin sections to observe their ultrastructure and three-dimensional organization. Results: TRACPase activity was detected in both multinucleated odontoclasts and a mononuclear cell from serial sections. By electron microscopy, most of the multinucleated odontoclasts had ruffled borders and clear zones. A mononuclear TRACPase-positive cell with a ruffled border and clear zone was reconstructed three-dimensionally by NIKON COSMOZONE 2SA. The reconstruction showed that this cell had one irregularly shaped nucleus and a wide ring-shaped clear zone and a small ruffled border. Under the ruffled border, this cell formed a small lacuna on the dentin surface. The results suggested that this cell was a mononuclear odontoclast. Conclusions: The present study concludes that cells with ruffled borders and clear zones observed by transmission electron microscopy can be identified as odontoclasts or osteoclasts irrespective of the number of nuclei. 0 1994 Wiley-Liss, Inc. Key words: Osteoclast, Ruffled border, Mononuclear cell, Multinucleation, Electron microscopy, Three-dimensional reconstruction Osteoclasts have been considered multinucleated giant cells which possess special structures called ruff led borders and clear zones observed by transmission electron microscopy (Scott and Pease, 1956; Schenk et al., 1967; King and Holtrop, 1975; Wezeman et al., 1979; Marks and Popoff, 1988). However, many investigators have reported the presence of mononuclear osteoclasts by light microscopy (Garant, 1976; Ries and Gong, 1982; Kaye, 1984; Marshall et al., 1986; Sire et al., 1990; Athanasou et al., 1991; Prallet et al., 19921, by transmission electron microscopy (Baron et al., 1986; Sire et al., 1990), and by scanning electron microscopy (Prallet et al., 1992). Some investigators have also speculated on its theoretical presence as a precursor of osteoclast (Chambers, 1985; Minkin and Shapiro, 1986; Hattersley and Chambers, 1989). 0 1994 WILEY-LISS, INC. With light microscopy, the presence of a ruffled border cannot be demonstrated conclusively. With transmission electron microscopy, a mononuclear cell apparently observed in one section may be multinucleated, and with scanning electron microscopy, the presence of both a ruffled border and single nucleus cannot be precisely demonstrated. Domon and Wakita (1991b) made serial sections of cultured osteoclasts by alternating semithin and ultrathin sections throughout whole cell Received November 19, 1993; accepted March 21, 1994. Address reprint requests to Dr. Takanori Domon, Department of Oral Anatomy 11, Hokkaido University School of Dentistry, Kita 13, Nishi 7, Kita-Ku, Sapporo 060 Japan. MONONUCLEAR ODONTOCLAST WITH RUFFLED BORDER bodies, and observed their ultrastructure by transmission electron microscopy and reconstructed the same cells three-dimensionally from serial sections. As a result, Domon and Wakita (1991b) demonstrated the presence of a mononuclear osteoclast with a ruffled border in vitro. However, this mononuclear osteoclast was merely a cultured cell originating from the mouse, and the presence of mononuclear osteoclasts in vivo has not been shown. There are a number of difficulties in demonstrating the presence of mononuclear osteoclasts in humans. First, it is difficult to obtain intact human bone for examination, and it may be diseased if it can be obtained. Second, bone has many trabeculae with complicated surface contours and it may be impossible to find mononuclear osteoclasts or to make serial sections throughout a complete cell body. To overcome these drawbacks, the present study used human deciduous teeth instead of bone. It is well known that deciduous teeth are resorbed by osteoclast-like cells, odontoclasts (Boyde and Lester, 1967; Furseth, 1968), and that odontoclasts have the same structures as osteoclasts (Furseth, 1968; Yaeger and Kraucunas, 1969; Freilich, 1971; Ohno, 1972; Suzuki, 1974; Nilsen, 1977; Ten Cate and Anderson, 1986; Pierce et al., 1991; Yawaka, 1993). Moreover, deciduous teeth are easier to obtain than human bone because deciduous teeth are naturally shed when the permanent teeth erupt. To identify the odontoclasts on deciduous teeth by light microscopy, the present study used histochemically TRACPase activity, which was considered specific for both odontoclasts (Hasselgren and Stromberg, 1976; Nilsen and Mugnusson, 1979; Yawaka, 1993) and osteoclasts (Hammarstrom et al., 1971; Minkin, 1982). The present study aimed to demonstrate the presence of a mononuclear odontoclast with a ruffled border. 43 room temperature. They were block-stained with 4% uranyl acetate for 30 min, dehydrated in a graded series of ethanol, and then embedded in Epon 812. Specimens were sectioned in the direction perpendicular to the resorbing surface and the cells for investigation were serially sectioned with glass and diamond knives on a Sorval MT-2B ultramicrotome by alternating semithin and ultrathin sections throughout the width of the cells. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (HITACHI H-7000) a t an operating voltage of 75 kV. Three-Dimensional Reconstruction The semithin sections were stained with methylene blue and azure 11, and then photographed by light microscopy. The photographs were enlarged on the printing paper, and the cell surface, dentin surface, nucleus, ruffled border, and clear zone were traced on tracing paper. Based on the outline of the section, these elements were input serially into a personal computer (NEC PC-9801 VM) with the Three-Dimensional Graphic Analytic System, COSMOZONE 2SA (NIKON, Japan). The three-dimensional reconstruction was calculated by computer, and the reconstructed image was photographed from display of computer. RESULTS Histochemistry for TRACPase Activity With a whole-mount light microscope, numerous TRACPase-positive cells with different sizes and shapes appeared dark on the resorbing surfaces in most of the teeth (Figs. 1, 2). We observed TRACPase activity in all specimens and particularly looked for the smallest TRACPase-positive cells, because mononuclear osteoclasts display small contours in vitro (Domon and Wakita, 1991b). After observing the conMATERIALS AND METHOD tours of many TRACPase-positive cells in all speciHistochemistry for TRACPase Activity mens, we selected a small area opposite to the crown Twenty deciduous teeth from 6-10-year-old males (Fig. 2) of one lower right deciduous canine of an and females were used in this study. The teeth were 8-year-old male for further investigation (Fig. 1). extracted under local anesthesia a t Hokkaido UniverTo understand both the ultrastructure and three-disity Dental Hospital (Sapporo, Japan). After extrac- mensional features of TRACPase-positive cells, seven tion, specimens were immediately fixed with 2.5% glu- TRACPase-positive cells of various sizes were selected taraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) in this area (arrows 3-6 in Fig. 2). overnight at 4"C, and then decalcified with 5% EDTA (pH 7.4) for 2 months at 4°C. For the detection of Light Microscopy TRACPase activity, the azo dye method was used (BurThe section a t the site of arrow 3 in Figure 2 showed ston, 1958). Specimens were incubated in 0.1M acetate- two cells on the dentin (Fig. 3). The left cell in Figure buffered medium (pH 5.2) containing naphthol AS-MX 3 corresponded to the small TRACPase-positive cell di(SIGMA, St. Louis, MO) as a substrate, and fast red rectly ahead of the point of arrow 3 in Figure 2, and the violet (SIGMA) as a diazonium chloride, and 10 mM right cell in Figure 3 corresponded the larger cell sodium tartrate (SIGMA) (Minkin, 1982). Incubation immediately beyond it, to the right intoFigure 2. These was carried out at 37°C for 10 min. Specimens for controls were incubated in the medium without a substrate under the same conditions. After the detection of TRACPase activity, specimens were photographed with a light microscope to determine the locations of Abbreviations TRACPase-positive cells on the resorbing surface of the cz clear zone D dentin teeth. Light and Transmission Electron Microscopy Specimens were postfixed with 1%osmium tetroxide in 0.05M sodium cacodylate buffer (pH 7.4) for 3 hr at ER Mt Nu RB V rough-surfacedendoplasmic reticulum mitochondria nucleus ruffled border vacuole 44 T. DOMON ET AL. Fig. 1. Whole mount preparation showing a lower right canine after the detection of TRACPase activity. The lingual (arrowheads) and mesial (M) root surface are resorbed extensively, and there are numerous TRACPase-positive cells on the dentin surface. Enamel was decalcified, and only the dentin of the crown (C) is observed. A small area (brackets) was selected for the observation of a mononuclear odontoclast. Periodontal ligaments (P) are seen on the tooth. x 5. Bar = 2mm. Fig. 2. High magnification showing the bracketed part of Figure 1. There are numerous TRACPase-positive cells on the dentin. The numbered arrows indicate the sites of the observations in Figures 3-6, and two TRACPase-positive cells are indicated at each of the numbered arrows 3, 5, and 6. x 80. Bar = 125 pm. two cells had one nucleus in the cytoplasm and formed lacunae on the dentin surface. Figure 4 showed the TRACPase-positive cell indicated by arrow 4 in Figure 2. The cell had two nuclei in its cytoplasm and fit in the lacuna in this section. The section at the site of arrow 5 in Figure 2 showed two cells on the dentin surface (Fig. 5). The left cell in Figure 5 corresponded to the small TRACPase-positive cell beyond the large TRACPase-positive cell a t the point of arrow 5 in Figure 2. In this section, this cell had one nucleus and many granules in its cytoplasm, and formed a lacuna on the dentin. The right cell in Figure 5 corresponded to the larger cell a t arrow 5. This cell appeared larger and had several nuclei in the cytoplasm. Figure 6 showed two cells corresponding to the two large TRACPase-positive cells to the right of the point of arrow 6 in Figure 2. These two cells were multinucleated giant cells and formed lacunae on the dentin. Observations of the serial sections of these cells allowed us to count precisely the number of nuclei in them. The left cell in Figure 3 had one nucleus (Figs. 7-14), and the right cell had two; the cell in Figure 4 had two nuclei; the left cell in Figure 5 had three nuclei, and the right cell had eight; the left cell in Figure 6 had seven nuclei, and the right cell had five. dentin surface (Fig. 15). The other multinucleated TRACPase-positive cells showed similar ultrastructure. The right cell with two nuclei from the observations of the serial sections in Figure 3 also had a typical ruffled border and clear zone facing the dentin surface. Figures 16-19 showed the serial ultrathin sections of the small TRACPase-positive cell in Figures 3 and 7-14. Figure 16 showed the section 2 pm from the appearance of the cell body. There were two cytoplasmic parts with many microvilli in this section. In the section 4 pm from the cell surface (Fig. 17), the cellular profiles were larger and there were many mitochondria, rough-surfaced endoplasmic reticulum, and vacuoles in the cytoplasm. The clear zone was seen on the dentin surface, but there was no ruff led border. Seven micrometers from the cell surface (Fig. 181, the cellular profiles were again larger. There was one nucleus in the central area of the cytoplasm and developed Golgi apparatus surrounding it, and a large number of mitochondria, rough-surfaced endoplasmic reticulum, and vacuoles were seen in it. This cell had a ruffled border and clear zone (Figs. 18, 201, and the cytoplasmic processes composing the ruff led border were both finger- and plate-like. On the dentin surface under the ruffled border there was a small lacuna where the collagen fibers of the dentin were disrupted (Fig. 20). Fen micrometers from the cell surface (Fig. 191, the cellular profiles were smaller and no nucleus was observed in the cytoplasm. Only the clear zone was seen on the dentin, and no ruffled border was observed. There was no dentin disruption under the clear zone. Transmission Electron Microscopy The left cell in Figure 6 showed several nuclei with the Golgi apparatus surrounding these, as well as numerous mitochondria, rough-surfaced endoplasmic reticulum, and vacuoles in the cytoplasm (Fig. 15). This cell had a ruffled border and clear zone facing the MONONUCLEAR ODONTOCLAST WITH RUFFLED BORDER Fig. 3-6. Light micrographs showing semithin sections of TRACPase-positive cells sectioned a t arrows 3-6 in Figure 2. Figs. 3 and 4, x 1,100.Figs. 5 and 6, x 1,000,Bar = 10 pm. Fig. 3. Two cells have revealed resorptive lacunae (arrowheads) on the dentin. 45 Fig. 5. A cell with numerous granules and multinucleated cell are related to resorptive lacunae (arrowheads) on the dentin. Fig. 6. Two multinucleated cells are associated with resorptive lacunae (arrowheads) on the dentin. Fig. 4. Rounded cell occupies the resorptive lacuna (arrowheads). Three-DimensionalReconstruction The small TRACPase-positive cell in Figure 3 was reconstructed from the serial sections in Figures 7-14. These reconstructions included the dentin interface with the cellular surface, nucleus, ruffled border, and clear zone. When the cellular and dentin surfaces were displayed together (Fig. 21), the cell showed a rounded gourd-shaped outline with a total width of about fourteen micrometers. When the nucleus was added to the reconstruction (Fig. 22), one irregularly shaped nucleus was observed in the central area of the cytoplasm, and there was a small lacuna on the dentin surface under the cell. The diameter of the lacuna was about 6 Fm. When the clear zone and ruff led border were added (Fig. 23), the clear zone formed a wide ring-shaped structure encircling a small area of ruffled border. The area of the ruffled border was relative to that of lacuna in Figure 22. All elements were displayed together in Figure 24; this cell was shown to possess the one nucleus and a small ruffled border surrounded by a wide clear zone on the dentin surface. This cell overlay a small lacuna. DISCUSSION The present study used the azo dye method to visualize the light microscopic TRACPase-positive cells on the resorbing surface of human deciduous teeth. Most of the TRACPase-positive cells were multinucleated with more than two nuclei, possessing ruffled borders and clear zones. These cells formed lacunae on the dentin surface under the ruffled border. These characteristic features are the same as those of odontoclasts observed by transmission electron microscopy (Furseth, 1968; Yaeger and Kraucunas, 1969; Freilich, 1971; Ohno, 1972; Suzuki, 1974; Nilsen, 1977; Ten Cate and Anderson, 1986; Pierce et al., 1991, Yawaka, 1993), and it is concluded that TRACPase-positive cells on the deciduous teeth are odontoclasts. The precursor of osteoclast or odontoclast has been thought to be the preosteoclast or preodontoclast, re- 46 T. DOMON ET AL. figs.7-14. Serial semithin sections of the left cell in Figure 3 taken, respectively, at 1 , 3 , 4 , 5 , 6 , 8 , 9 , 11, and 12 pm from the cell surface. The serial sections show that the cell has only one nucleus (arrows) in the cytoplasm and that it forms a resorptive lacuna (asterisks) on the dentin. A leukocyte (L) is observed near this cell (Fig. 7). x 1,500. Bar = 7 pm. The mononuclear odontoclast in the present study spectively. It has been reported that the preosteoclasts are mononuclear cells with some of the characteristic showed a rounded outline in three-dimensions. It has features of osteoclasts; however, they do not possess been shown that actively resorbing osteoclasts have any ruffled border conclusively (Scott, 1967; Luk et al., rounded contours and that migrating ones have flat 1974; Rifkin et al., 1980; Ejiri, 1983). Preodontoclasts and irregular shapes in vitro (Domon and Wakita, with TRACPase activity were not observed on the de- 1991a). Although the reconstructed cell was present in ciduous tooth in this study. However, we have observed vivo, the results would allow the mononuclear odontoTRACPase-positive cells originating from long bone of clast in the present study to be classified as a resorbthe mouse on dentine slices in vitro, and reported that ing-type odontoclast. In fact, the three-dimensional reTRACPase activity was detected in both osteoclasts construction showed that this cell had a ring-shaped and preosteoclasts (Domon and Wakita, 1991b). Con- clear zone encircling a small ruffled border, and formed sidering the fact that preosteoclasts also possessed a small lacuna. It has been reported that 80% of human odontoclasts TRACPase activity in vitro, it is possible that mononuclear preodontoclasts may be present among many (Addison, 1978) and 81% of chick osteoclasts (Piper et TRACPase-positive cells on the resorbing areas of al., 1992) have ten or fewer nuclei. In the present study, we also counted the number of nuclei in odontoteeth. The present study showed that one mononuclear clasts and found that all of them had eight or fewer TRACPase-positive cell possessed a typical ruff led bor- nuclei. Addison (1978) and Piper et al. (1992) deliberder and clear zone by transmission electron micros- ately excluded the mononuclear cells from their analcopy. Its ultrastructural features were similar to those yses of the distribution of nuclei because there had of multinucleated odontoclasts, and the three-dimen- been no conclusive evidence of the presence of monosional reconstruction showed that this cell had only one nuclear odontoclasts and osteoclasts. The present and irregularly shaped nucleus. Therefore, the ultrastruc- our recent study (Domon and Wakita, 1991b) have ture and three-dimensional structure suggest that this shown the presence of mononuclear odontoclasts and TRACPase-positive cell is a mononuclear odontoclast. osteoclasts with the ruffled borders. Considering these MONONUCLEAR ODONTOCLASTWITH RUFFLED BORDER 47 Fig. 15. Eleclrun micrograph of the left odontoclast in Figure 6 . The multinucleated odontoclast has a ruffled border (inset)and clear zone on the dentin. There is a Golgi apparatus (Go),many mitochondria, rough-surfaced endoplasmic reticulum, and vacuoles in the cytoplasm. ~3,600,Bar = 3 pm. Inset: x 11,000, Bar = 1 pm. facts, we suggest that most osteoclasts and odontoclasts are the cells with a small number of nuclei, and that these small osteoclasts and odontoclasts play an important role in hard tissue resorption. Hattersley and Chambers (1989) speculated that the osteoclast was initially mononuclear and might remain so, but it generally became multinucleated. The present study showed the presence of mononuclear odontoclasts. This fact suggests that the odontoclast may be initially mononuclear. We were not able to establish whether the mononuclear odontoclast became multinucleated or not. Mononuclear odontoclasts may have two roles, the resorption of the tooth and the recruitment of precursors for multinucleation. The origin of mononuclear odontoclasts also remains unknown. Kolliker (1873) observed multinucleated giant cells in Howship’s lacunae on bones by light microscopy, and called these multinucleated cells “Ostoklasten,” or osteoclasts. After this report, multinucleated cells seen on bone by light microscopy have generally been thought to be osteoclasts. Scott and Pease (1956) were the first to observe osteoclasts by transmission electron microscopy. They reported that bone was resorbed by special structures of osteoclasts, and called these the ruffled borders. Therefore, the cells with ruffled borders observed by transmission electron microscopy have been considered to be osteoclasts. Besides multinucleation and the presence of a ruffled border, many characteristics of osteoclasts have been proposed: the presence of a ruffled border and clear zone (Dudley and Spiro, 1961; Schenk et al., 1967; Kallio et al., 1971; King and Holtrop, 1975; Wezeman et al., 1979; Marks and Popoff, 1988), TRACPase activity (Hammarstrom et al., 1971; Minkin, 1982; Baron et al., 1984; Andersson et al., 1986; Glowacki and Cox, 1986; Helfrich et al., 1989; Takahashi et al., 1988; Udagawa et al., 19901, calcitonin receptors (Warshawsky et al., 1980; Chambers and Magnus, 1982; Arnett and Dempster, 1987; Hattersley and Chambers, 1989), carbonic anhydrase (Gay and Mueller, 1974; Anderson et al., 19821, ATPase (Baron et al., 19851, vitronectin receptors (Davies et al., 19891, and cellular surface antigens different from those of macrophage-monocyte lineage cells (Horton et al., 1985; Nijweide et al., 1985; Athanasou et al., 1991; Collin-Osdoby et al., 1991). The nomenclature of osteoclast and odontoclast applies generally, based on the hard tissue substrate which is resorbed; osteoclasts resorb bone and odontoclasts resorb tooth. Previously, the multinucleated giant cells resorbing deciduous teeth had been also called osteoclasts by light microscopy (Kronfeld, 19321, and Furseth (1968) was the first to observe them by transmission electron microscopy, and has referred to them as odontoclasts. Accordingly, it has been also known that odontoclasts are multinucleated giant cells which have the same characteristic features as osteoclasts: the presence of the ruffled border and clear zone (FurSeth, 1968; Yaeger and Kraucunas, 1969: Freilich, 1971; Ohno, 1972; Suzuki, 1974; Nilsen, 1977; Ten Cate and Anderson, 1986; Pierce et al., 1991; Yawaka, Figs. 16-1 9. Electron micrographs showing serial ultrathin sections of the mononuclear odontoclast in Figures 3 taken, respectively, at 2, 4, 7, and 10 pm from the cell surface. X 3,000. Bar = 4 pm. Fig. 16. Two areas of the cytoplasm are observed in this site. There is a leukocyte (L) near the dentin. Fig. 17. Cytoplasmic areas are larger, showing many mitochondria, rough-surfaced endoplasmic reticulum, and vacuoles. Only the clear zone is seen next to the dentin. Fig. 18. The cell has one nucleus, and the ruffled border and clear zone are seen at the dentin surface. There is a small lacuna (asterisk) under the ruffled border. Most of the rough-surfaced endoplasmic reticulum occurs between the nucleus and the Golgi apparatus (Go). Many mitochondria and vacuoles are observed in the cytoplasm. Fig. 19. There is no nucleus present in this section, and only the clear zone is seen next to the dentin. Many mitochondria, roughsurfaced endoplasmic reticulum, and vacuoles are present in the cytoplasm. MONONUCLEARODONTOCLASTWITHRUFFLEDBORDER 49 Fig. 20. Electron micrograph showing a part of Figure 18 a t a higher magnification. The ruffled border surrounded by the clear zone is seen against the dentin surface. Under the ruffled border, there is a small lacuna (asterisk), and the collagen fibers composing the dentin are disrupted. x 12,000. Bar = 1 pm. 1993) and TRACP activity (Hasselgren and Stromberg, 1976; Nilsen and Mugnusson, 1979; Yawaka, 1993). Therefore, osteoclasts and odontoclasts would be called "elastic" cells for hard tissue resorption. In fact, osteoclasts can also resorb enamel and dentin which are components of tooth in vitro (Jones et al., 1984). Although many characteristics of osteoclasts and odontoclasts have been proposed, the conclusive criterion for identification of a cell as an osteoclast or odontoclast has been considered the presence of a ruffled border and clear zone observed by transmission electron microscopy (Scott and Pease, 1956; Schenk et al., 1967). With this criterion, many investigators have identified cells with ruffled borders and clear zones as osteoclasts or odontoclasts, and in the present study we identified the mononuclear cell with these features as a mononuclear odontoclast. However, osteoclasts and odontoclasts have been generally considered to be multinucleated giant cells, and not mononuclear cells (Kolliker, 1873). The significance of the multinucleation of osteoclasts and odontoclasts must be better understood before it is possible to identify the mononuclear cell with a ruff led border as an odontoclast. Based on light microscopic observations, multinucleation has been proposed as one criterion for osteoclast identification (Kolliker, 1873). The presence of a ruffled border, the other criterion, has been based on transmission electron microscopic observation (Scott and Pease, 1956). Therefore, considering the conditions of observation, these two criteria are quite different. If osteoclasts and odontoclasts must always satisfy both criteria, the mononuclear cell with a ruff led border in this study cannot be classified as either. Hattersley and Chambers (1989) suggested that multinuclearity was an unreliable marker for the osteoclastic phenotype in culture. Prallet et al. (1992) also speculated that multinucleation might not be necessary for full expression of the osteoclast function in bone resorption. Moreover, multinucleated cells observed by light microscopy are not always osteoclasts or odontoclasts. It is known that multinucleated giant cells encircle bone particles in vivo (Popoff and Marks, 1986). Macrophages have similar structures to those of osteoclasts, and the clear zones are seen in both macrophages and osteoclasts; however, macrophages lack a ruffled border at the interface between the cell and the hard tissue substrate (Kahn et al., 1978; Rifkin et al., 1979; Popoff and Marks, 1986). Therefore, the conclusive criterion to identify a cell as an osteoclast or odontoclast is the presence of a ruffled border and clear zone observed by transmission electron microscopy. Our recent study (Domon and Wakita, 1991b) and the present study have demonstrated the presence of a mononuclear cell with a ruffled border and clear zone both in vitro and in vivo. Therefore, we conclude that cells with ruffled borders and clear zones observed by transmission electron microscopy can be identified as osteoclasts or odontoclasts, irrespective of the number of nuclei. However, this conclusion does not apply to mononuclear cells observed by light microscopy, because a t this level it is not possible to conclusively demonstrate the presence of a ruffled border. A further 50 T. DOMON ET AL. Figs. 21-24. Models showing the three-dimensional structure of the mononuclear odontoclast. These images are displayed as looking obliquely to the resorbing surface. The odontoclast (Ocl) shows a rounded gourd-shaped outline on the dentin (Fig. 21). When the cell surface is displayed as semitransparent (Figs.22-24), one irregularly shaped nucleus is seen in the central area of the cytoplasm, and a lacuna (La) can be seen on the dentin under the cell (Fig. 22). A wide ring-shaped clear zone and small ruffled border are present on the dentin (Fig. 23). Displaying all structures together allows a n understanding of the three-dimensional arrangement (Fig. 24). x 2,000. Bar = 5 pm. evaluation of multinucleation as a criterion of both osteoclasts and odontoclasts is needed. Arnett, T.R., and D.W. Dempster 1987 A comparative study of disaggregated chick and rat osteoclasts in vitro; effects of calcitonin and prostaglandin. Endocrinology, 120.602-608. Athanasou, N.A., B. Puddle, J . Quinn, and C.G. Woods 1991 Use of monoclonal antibodies to recognize osteoclasts in routinely processed biopsy specimens. J . Clin. Pathol., 44.664-666. Baron, R., L. Neff, D. 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