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Cellular Roles in Physiological Root Resorption of Deciduous Teeth in the Cat T. Sasaki, T. Shimizu, C. Watanabe and Y. Hiyoshi J DENT RES 1990 69: 67 DOI: 10.1177/00220345900690011101 The online version of this article can be found at: http://jdr.sagepub.com/content/69/1/67
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Cellular Roles in Physiological Root Resorption of Deciduous Teeth in the Cat T. SASAKI, T. SHIMIZU, C. WATANABE, and Y. HIYOSHI The Second Department of Oral Anatomy, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
This study has attempted to assess the importance of mesenchymal cells, fibroblasts, cementoblasts, and mononuclear phagocytes (i.e., macrophages) in physiological root resorption offeline deciduous teeth. Deciduous incisors of three- to six-month-old kittens undergoing root resorption were investigated by means of electron microscopy. In an early phase of root resorption, the resorption organ consisted of many fibroblasts and relatively few macrophages and odontoclasts, the last with a wide, clear zone and narrow, immature, ruffled border. In the active phase of root resorption, the resorption organ contained many odontoclasts with a well-developed ruffled border and a reduced clear zone, cementoblasts, fibroblasts, macrophages, neutrophils, and many blood vessels. Cementoblasts were present usually on the resorbing dentin surface adjacent to odontoclasts and, in many cases, these cells communicated with each other via gap junctions. Cementoblasts frequently extended broad cell processes with secretion granules and with phagosomes containing collagen fibrils into the dentinal tubules exposed to resorption lacunae. Some macrophages exhibiting a clear zone-like structure also appeared on resorbing dentin surfaces. In the restingphase of root resorption, the dentin surface was covered mostly with cementoblasts resembling bone lining cells. There was an occasional macrophage, but no odontoclasts were observed during this phase. During removal of the periodontal ligament concomitant with root resorption, many fibroblasts phagocytosed mature collagen fibrils, as well as amorphous fluffr material. These results suggest that these mesenchymal cells, as well as odontoclasts, are essential for the cellular removal of dental hard and soft tissues during shedding offeline deciduous teeth. J Dent Res 69(1):67-74, January, 1990
Introduction. In the past two decades, there have been significant advances in elucidation of the cell biology of physiological root resorption of deciduous teeth (Boyde and Lester, 1967; Boyde et al., 1984; Furseth, 1968; Freilich, 1971; Morita et al., 1970; Jones et al., 1984; Ten Cate and Anderson, 1986; Sasaki et al., 1988ab, 1989). These studies have demonstrated that multinucleated giant cells, referred to as odontoclasts (osteoclasts), are the principal mediators of physiological root resorption of deciduous teeth. Although the resorption organ (resorptive tissue) also contains many fibroblasts, mononuclear phagocytes (macrophages), and granular leucocytes (neutrophils) (Furseth, 1968; Ten Cate and Anderson, 1986; Sasaki et al., 1988a, b, 1989), their functional roles in root resorption are not yet known. Ten Cate and Anderson (1986) first examined the cellular removal of dental soft tissues, including periodontal ligament, by fibroblasts during shedding of feline deciduous teeth, and pointed out the existence of two phenotypically different populations of fibroblasts in the region of ligament resorption: one with numerous collagen-containing phagosomes in the cytoplasm, and the other with condensed nucleus and cytoplasm. Moreover, our recent in vitro experiments demonstrated that dentin slices cultured with bone marrow cells were partially phagocytosed by mononuclear phagocytes such as macrophages, as well as being subject to resorption by bone-marrowderived multinucleated cells (Sasaki et al., 1988b). Similar Received for publication April 26, 1989 Accepted for publication September 28, 1989
phagocytosis of mineralized bone particles by macrophages has been reported both in vivo and in vitro (Kahn et al., 1978; Rifkin et al., 1979). Dorey and Bick (1977) further demonstrated the presence of N-acetyl-f3-glucosaminidase, which was related to the hydrolysis of glycosaminoglycans, in the macrophages present in periodontal ligament. These studies suggest that macrophages participate in the total process of bone remodeling and tooth resorption. It seems important, therefore, to clarify how the mesenchymal cells, such as cementoblasts, fibroblasts, and macrophages, are engaged in the removal of dental hard and soft tissues during shedding of deciduous teeth. In the present study, we extend the electron microscopic observations, first made by Ten Cate and Anderson (1986), on feline deciduous teeth undergoing root resorption from an early resorption phase to an active resorption phase just before shedding.
Materials and methods. After an intramuscular injection of ketamine hydrochloride (veterinary Ketalar 50, Sankyo Co., Ltd., Japan), three- to six-month-old kittens were given a transcardiac perfusion for about ten min with a fixative containing 1% glutaraldehyde and 1% formaldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.3). After perfusion, the upper and lower jaws, including deciduous incisors undergoing root resorption, were extracted, placed in fresh fixative for two to four h at 40C, and then demineralized in 10% ethylenediaminetetraacetic acid disodium (pH 7.3) for one to two months at 4TC. After demineralization, longitudinally-cut slices about 0.5 mm thick were prepared. These slices were then post-fixed with 1% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer (pH 7.3) for two h at 40C, block-stained with ethanolated 1% uranyl acetate, dehydrated through a graded ethanol series, and embedded in Epon 812. Thin sections were cut with use of a diamond knife on a Reichert-Jung Ultracut OmU-4 and stained with uranyl acetate and lead citrate. Some thin sections were stained with uranyl acetate and 1% phosphotungstic acid. All sections were examined with a Hitachi HU-12A electron microscope at 75 kV.
Results. Throughout the present study, the processes of root resorption of feline deciduous teeth were classified into three phases: (1) an early phase, with many fibroblasts and occasional macrophages and odontoclasts on dentin surfaces; (2) an active phase, with many well-developed odontoclasts, macrophages, fibroblasts, cementoblasts, and neutrophils; and (3) a resting phase, with cementoblastic lining cells and few macrophages. The root repair by cementum formation reported in human deciduous teeth (Kronfeld, 1932; Westin, 1942; Furseth, 1968) was not observed during the rapid root resorption and shedding of feline deciduous teeth. Early phase of root resorption. -In this phase, the root dentin and cementum were hardly indented, and their resorbing fronts were populated by many cementoblastic lining cells and relatively few multinucleated odontoclasts (Fig. 1A, inset).
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Fig. 1-The resorption organ in an early phase of root resorption. Fig. 1A: A low-magnification electron microscopic view of an odontoclast (OC) resorbing dentin (D) in shallow resorption lacuna and adjacent fibroblasts (FB). Inset is a light micrograph of the resorption organ containing relatively flat odontoclasts (arrows). Figs. 1B and 1C show a broad clear zone (CZ in Fig. 1B) and an immature ruffled border (RB in Fig. 1C) of an odontoclast in Fig. 1A. Fig. 1D shows a macrophage (Mp) and a fibroblast (FB) in resorption organ.
Odontoclasts exhibited a relatively flat profile and developed an unusually wide clear zone against the resorbing dentin (Figs. 1A and B). Such a wide clear zone was not observed in the odontoclasts found at an active resorption phase (Sasaki et al., 1988a,b). In the cytoplasm proximal to the clear zone, numerous pale vacuoles were accumulated-these were reported as a membrane source of ruffled border in our recent paper (Sasaki et at., 1989) (Figs. 1A and B). Microvillous protrusions forming immature ruffled border-like structures were observed adjacent to the clear zone (Figs. 1A and C). Other structural features of odontoclasts were identical to those described in previous papers (Furseth, 1968; Freilich, 1971; Sasaki et at., 1988a,b, 1989). Around odontoclasts, many fibroblasts and occasional macrophages were distributed roughly in parallel with both the resorbing dentin surface and the enamel organ surface of the successive permanent tooth (Figs. 1A and D). Fibroblasts extended several long slender cell processes in which phagocytosis of collagen fibrils was seldom observed (data not shown). Except for the areas occupied by odontoclasts, the resorbing dentin surface was covered with either fibroblasts, not showing collagen phagocytosis, or cementoblasts, resembling so-called "bone-lining cells" (Miller and Jee, 1987). Active phase of root resorption. -The most prominent feature of the resorption organ in the active phase of root resorption was the presence of many giant odontoclasts of various configurations (Furseth, 1968; Freilich, 1971; Ten Cate and Anderson, 1986; Sasaki et al., 1988a,b, 1989) (Fig. 2A, in-
set). The odontoclasts possessed a well-developed ruffled border against resorbing dentin, but intracellular incorporation of collagen fibrils by odontoclasts was never detected (Figs. 2A and B). Degeneration of collagen fibrils in the dentin surface region overlaid by the odontoclast ruffled border was not observed (Fig. 2B). Adjacent to the odontoclasts, numerous active cementoblast-like cells (hereafter called cementoblasts) were present on resorbing dentin surfaces. Unlike those covering the dentin surface at either an early resorption or a resting phase, cementoblasts in this phase showed a cuboidal and/or a rectangular outline, and were distributed roughly perpendicularly to the dentin surface [Figs. 2A (inset) and 3A]. Adjacent cementoblasts were frequently connected to each other by gap junctions between the cell bodies (Fig. 3A), between cell body and cell process (Fig. 3A, inset at the top left-hand corner), and between the cell processes (Fig. 3A, inset at the top righthand corner). Structurally, these cementoblasts were characterized by well-developed Golgi complexes, secretion granules, many cisternae of rough endoplasmic reticulum (RER), mitochondria, phagosomes, lysosomes, and numerous free polyribosomes. The Golgi complex consisted of five or six saccules with distended cylindrical margins, small coated vesicles, and condensing vacuoles (immature secretion granules) (Figs. 3A and B). Many secretion granules were distributed in the Golgi area and in the cytoplasm close to the dentin surface (Figs. 3A and B). Secretion granules were also observed in the broad cell processes penetrating those dentinal tubules exposed to resorption lacunae (Figs. 3C-E). Abundant newly-
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Fig. 2-The odontoclasts in an active phase of root resorption by phosphotungstic staining. Fig. 2A: The ruffled border region (RB) facing resorbing dentin (D) and adjacent vacuolar region (V) of an odontoclast. Phagocytosis of collagen fibrils is not seen in this odontoclast. Inset is a light micrograph of cementoblasts (CB) adjacent to odontoclast (OC) on resorbing dentin (D). Fig. 2B shows higher magnification of collagen fibrils in resorbing dentin overlaid by the odontoclast ruffled border.
formed thin collagen fibrils were distributed on the resorbing dentin surface overlaid by such cementoblasts (Fig. 3A). Moreover, these cementoblasts seemed to have phagocytosed old collagen fibrils, particularly at the dentin surface and inside the dentinal tubules. Phagocytosis of collagen fibrils through elongated phagocytic vacuoles was especially evident in the cell processes penetrating the dentinal tubules, but rarely observed in the cell bodies (Figs. 3C-E). Abundant fluffy material was accumulated on the dentin surfaces, including dentinal tubules covered with cementoblasts (Figs. 3A and C). In the resorption organ away from the resorbing dentin surface, many fibroblasts resorbed collagen fibrils by way of phagocytic vacuoles. These fibroblasts were, however, devoid of secretion granules (data not shown). Although some macrophages exhibiting the clear zone-like structure were present on the resorbing dentin surface, their interaction with resorbing dentin was uncertain (Fig. 4A). Many macrophages in the resorption organ frequently incorporated red blood cells, neutrophils, and cell debris of unknown origin, but they did not possess a clear zone-like structure (Fig. 4B). Resting phase. -In this phase of root resorption, the relatively smooth dentin surface was covered with flattened cementoblasts resembling "bone-lining cells". Sometimes these cementoblastic lining cells formed small, clear zone-like structures facing the resorbing dentin, and resorbed small dentin particles through the deep membrane invaginations of the clear zone (Fig. 5). Such lining cells possessed a well-developed Golgi apparatus consisting of four or five saccules and small coated vesicles, lysosomes, residual bodies, RER cisternae,
and mitochondria, but were devoid of secretion granules. Coated pits and vesicles were frequently seen in association with the plasma membranes of both the cell bodies and the cell processes of these lining cells. On the resorbing dentin, there was an occasional macrophage, but no active odontoclast was observed. Resorption of periodontal ligament. -During removal of periodontal ligament concomitant with root resorption, fibroblasts showed a flattened profile with several extremely long, slender cell processes (Fig. 6A). The cell body was occupied mainly by a large nucleus, and the narrow perinuclear cytoplasm contained a Golgi apparatus consisting of three or four saccules and related coated vesicles, lysosomes, many RER cisternae, mitochondria, and free ribosomes. Coated vesicles and pits were also visible in association with the plasma membranes. Microfilaments and microtubules ran parallel with the cell long axis. Near the region of ligament resorption, these fibroblasts contained many elongated phagocytic vacuoles in which mature collagen fibrils were found (Fig. 6B). These phagosomes were much more prominent in the cell processes than in the cell bodies. The extracellular matrix of the periodontal ligament consisted of bundles of collagen fibrils and amorphous, fine, fluffy material. The collagen fibril population varied from area to area. In the region of ligament resorption, the extracellular matrix was composed mostly of fluffy material but few collagen fibrils (Figs. 6C and D). Such fluffy material, as well as collagen fibrils, was seen in the phagocytic vacuoles of the fibroblasts. In this region, macrophages frequently appeared among the fibroblasts. The macrophages ex-
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Fig. 3-The cementoblasts on resorbing dentin in an active phase of root resorption. Fig. 3A: Cementoblasts on resorbing dentin contain well-developed Golgi apparatus, abundant RER cisternae, and many secretion granules. They are connected by gap junctions between the cell bodies (arrow in Fig. 3A), between cell body (CB) and cell process (CP) (inset at the top left-hand corner), and between cell processes (inset at the top right-hand corner). Amorphous fluffy material fills the dentinal tubules (DT) and the resorbing dentin surface. Fig. 3B shows a higher-magnification view of the Golgi apparatus (Go) and secretion granules (SG) in a cementoblast. Fig. 3C: A cementoblast (CB) penetrating its broad cell process into a dentinal tubule (DT). Fig. 3D: The cementoblastic cell process marked by a rectangle in Fig. 3C. The process includes many secretion granules (SG) and elongated phagosomes (Ph) containing collagen fibrils. Fig. 3E: A higher-magnification view of collagen fibrils in phagosomes (Ph) in the cementoblastic cell process penetrating a dentinal tubule.
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Fig. 4-Macrophages in resorption organ in an active phase of root resorption. Fig. 4A: A macrophage (Mp) exhibiting a clear zone-like structure (CZ) on resorbing dentin (D). Fig. 4B: Macrophages in the resorption organ contain varied cell debris (arrows).
hibited an elongated profile and possessed many large electrondense lysosomes with heterogeneous inclusions in the perinuclear cytoplasm. Although deep membrane invaginations containing fluffy material were observed in macrophages, we failed to find phagocytosis of collagen fibrils by macrophages (Fig. 6D). This resorption of the periodontal ligament was a common finding throughout the process of root resorption.
Discussion. The present observations suggest that, in addition to odontoclasts, several other mesenchymal cells such as fibroblasts, cementoblasts, and macrophages are actively involved in cellular removal of dental hard and soft tissues during the shedding of deciduous teeth. We have previously indicated that neutrophils in the resorption organ were related to the degeneration of exhausted odontoclasts (Sasaki et al., 1989). During root resorption of human deciduous teeth, cementoblasts deposit cementum matrix onto resorption lacunae in the phase of tooth repair (Furseth, 1968). However, this study suggests that cementoblasts are engaged in both matrix secretion and resorption during the phase of active root resorption. The findings described here are not restricted to feline deciduous teeth, because the identical phenomenon was recently found in physiological root resorption of human deciduous teeth (unpublished). Earlier biochemical studies have demonstrated that the resorption organ engages in collagenolytic activity (Morita et al., 1970; Woessner and Cahill, 1974), but the cellular origin of
the activity is not yet known. In this regard, it has been reported that collagenase is synthesized and released by osteoblasts in response to parathyroid hormone (PTH) (Sakamoto and Sakamoto, 1982, 1984). Collagenase mediates the extracellular degradation of structural collagen during bone remodeling. Chambers et a. (1985) have suggested that removal of the osteoid layer by collagenase-induced osteoclastic bone resorption in vitro, by permitting osteoclasts to come into contact with resorption-stimulating bone mineral. Recently, the osteoblast has been suspected of being involved in the hormonal control of bone resorption and in the direct activation of osteoclasts by products of osteoblastic hormone action (Rodan and Martin, 1981). In fact, the osteoblast is known to stimulate osteoclastic bone resorption by releasing an unknown soluble factor in response to PTH (McSheehy and Chambers, 1986a,b). Furthermore, the osteoblast stimulates osteoclastic responsiveness to interleukin 1 (IL-1), and thereby increases osteoclastic bone resorption (Heath et al., 1985; Thomson et al., 1986). A recent in vitro study has also indicated that, in the presence of lox,25-dihydroxyvitamin D3 (1,25(OH)2D3), a co-culture of osteoblastic cells with spleen cells induced formation of multinucleated cells which, in turn, produced numerous resorption lacunae on a co-cultured dentin slice (Takahashi et al., 1988). These studies suggest that various hormones, such as IL-1, PTH, PGE2, and 1,25(OH)2D3, act primarily on osteoblasts having receptors for these hormones to stimulate osteoclastic bone resorption. Thus, it is strongly suggested that osteoblasts are required for osteoclast differentiation. In our recent cytochemical study, cementoblasts on resor-
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Fig. 5-Cementoblastic lining cells (LC) on resorbing dentin (D) in a resting phase of root resorption. Inset figure shows a clear zone-like structure (CZ) of a lining cell, in which phagocytosis of small dentin particles is noted (arrows). Fig. 6-Fibroblasts and macrophages in the periodontal ligament in an active phase of root resorption. Fig. 6A: Fibroblasts in the periodontal ligament possess many phagosomes containing collagen fibrils (arrows). Fig. 6B: A higher-magnification view of phagosomes containing collagen fibrils (arrows) in a fibroblast.
bing dentin showed alkaline phosphatase activity, a marker enzyme for the osteoblast (unpublished). These cementoblasts are morphologically identical to osteoblasts (Furseth, 1968, 1969; Jande and Belanger, 1970; present study). It is an important finding that adjacent cementoblasts communicate with each other via gap junctions, as in the osteoblast layer (Doty, 1981). Such gap junctions are well-known to provide electrical coupling between the cells by transcellular transfer of ions and low-molecular-weight metabolites (Pappas, 1975; Lowenstein, 1979). It is our contention that collagenolytic activity in the resorption organ is provided by the cementoblasts. The presence of abundant fluffy material on the dentin surface overlaid by the cementoblasts might be due to their collagenolytic activity. Therefore, if cementoblasts on resorbing dentin are actually identical to osteoblasts, both morphologically and functionally, they might induce surface modification or remodeling of resorbing dentin, and thereby stimulate odontoclast differentiation. In fact, the remodeling (the synthetic and resorptive processes involved in the resorbing of dentin) occurs when active cementoblasts are present, whereas the dentin surface in the resting phase is covered with flattened lining cells. This cementoblastic function might be essential for activation of odontoclastic functioning in tooth resorption. Synchronous resorptive and synthetic activities are a common feature of hard-tissue-forming cells in their active phase:
the odontoblast (Sasaki et al., 1982, 1984), the ameloblast (Kallenbach, 1980; Sasaki, 1984; Nanci et al., 1985), the osteoblast (Sasaki et al., 1985; Takahashi et al., 1986), and perhaps also the cementoblast. In this connection, the repair of resorption lacunae by cementoblasts may reflect the matrix formative activity of the cementoblast in its active phase. During the active resorption phase of feline deciduous teeth, these cementoblasts seemed to be engaged in secretion of matrix substance, but the actual formation of cellular cementum was not observed. This may be due to the rapid shedding of feline deciduous teeth. In human deciduous teeth, cellular cementum was found to be partially formed on the resorption lacunae in the phase of active root resorption (unpublished). Active cementoblasts are not present in an early phase of root resorption or in a resting phase during which odontoclastic resorption does not actively take place. These results suggest that the functional significance of cementoblasts during an active resorption phase is primarily related to activation of odontoclasts. The precise mechanism by which the cementoblast activates the odontoclast has yet to be determined. Phagocytosis of collagen fibrils by these cementoblasts suggests that they are also engaged in removal of collagen fibrils from the resorbing dentin, while odontoclasts are not. Bone resorption is considered to be mediated not only by osteoclasts, but also by two different cell types-fibroblasts and monocyte-derived macrophages (Heersche, 1978). Be-
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Fig. 6C shows the region of ligament resorption, in which the extracellular matrix consists of abundant fluffy material. Fig. 6D shows a macrophage (Mp) and a fibroblast (FB) in the periodontal ligament.
cause neither osteoclasts nor odontoclasts exhibit collagenase activity or phagocytose collagen fibrils exposed at resorbing bone and/or dentin surfaces, these fibrils are believed to be phagocytosed by either fibroblasts or macrophages. Osteoclasts and/or odontoclasts are believed to demineralize the bone and/or dentin surface partially and degrade noncollagenous matrix (Heersche, 1978; Sasaki et al., 1988a,b; present study). A close functional relationship between fibroblasts and osteoclasts was also suggested by electron microscopic observation of the periodontal ligament (Garant, 1976). Our present observation of collagen phagocytosis by cementoblasts on resorbing dentin adjacent to odontoclasts seems to support their
findings. Mononuclear phagocytes (macrophages) seem to have at least two functions in root resorption: phagocytosis, and subsequent digestion, of exhausted and/or degenerated cells in the resorption organ; and the phagocytosis of dentin particles. Although the present study did not show phagocytosis of mineralized dentin by macrophages, we have recently demonstrated that macrophages with a clear zone phagocytose small dentin particles in vitro (Sasaki et al., 1988b). Similar phagocytosis of mineralized bone particles by macrophages has already been indicated in vivo and in vitro (Dorey and Bick, 1977; Kahn et al., 1978; Rifkin et al., 1979; Takahashi et al., 1986). It follows that macrophages found on resorbing dentin may be involved in the phagocytosis of fragments derived from dentin matrix. Phagocytosis of collagen fibrils by macrophages (Parakkal, 1969; Deporter, 1979) was, however, not confirmed in this study. Although similar phagocytosis of dentin particles
was observed in cementoblastic lining cells in a resting phase of resorption, its functional significance is yet to be determined. Removal of periodontal ligament by fibroblasts has been reported repeatedly in previous investigations (Ten Cate, 1972; Ten Cate and Deporter, 1974; Listgarten, 1973; Garant, 1976; Svoboda et al., 1981; Beertsen, 1987). The periodontal ligament is known to undergo rapid turnover of collagen fibrils. Quantitatively, phagocytosis of collagen fibrils by fibroblasts during the normal maintenance of periodontal ligament is much more frequent and faster than in the connective tissues of attached gingiva and skin (Sodek, 1977; Svoboda et a!., 1981). In periodontal ligament, fibroblasts are involved in both resorption and secretion of collagen fibrils, and also show regional variation in cell activity (Garant, 1976). In the region of ligament resorption, the ligament space was mostly filled with amorphous fluffy material, rather than with collagen fibrils. This may be due to extracellular breakdown of collagen fibrils. Both fibroblasts and macrophages may be involved in resorption of fluffy material. Beertsen (1987) recently reported that collagen fibrils in periodontal ligament of teeth made hypofunctional decreased from 50% to 30% of the total population during the experiment. He demonstrated that a substantial loss of collagen occurred within the first few days of hypofunction, and that periodontal ligament fibroblasts reacted very rapidly to functional changes of teeth. The process of collagen loss in hypofunctional teeth might correspond to that for removal of periodontal ligament during shedding of deciduous teeth.
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PARAKKAL, P.F. (1969): Involvement of Macrophages in Collagen Resorption, J Cell Biol 49:345-354. RIFKIN, B.R.; BAKER, R.L.; and COLEMAN, S.J. (1979): An Ultrastructural Study of Macrophage-mediated Resorption of Calcified Tissue, Cell Tissue Res 202:125-132. RODAN, G.A. and MARTIN, T.J. (1981): Role of Osteoblasts in Hormonal Control of Bone Resorption. A Hypothesis, Calcif Tissue Int 33:349-351. SAKAMOTO, S. and SAKAMOTO, M. (1982): Biochemical and Immunohistochemical Studies on Collagenasc in Resorbing Bone in Tissue Culture, J Periodont Res 17:523-526. SAKAMOTO, M. and SAKAMOTO, S. (1984): Immunocytochemical Localization of Collagenasc in Isolated Mouse Bone Cells, Biomed Res 5:29-38. SASAKI, T.; ISHIDA, I.; and HIGASHI, S. (1982): Ultrastructure and Cytochemistry of Old Odontoblasts in Rat Incisors, J Electron Microsc 31:378-388. SASAKI, T. (1984): Tracer, Cytochemical, and Freeze-Fracture Study on the Mechanisms Whereby Secretory Ameloblasts Absorb Exogenous Proteins, Acta Anat 118:23-33. SASAKI, T.; MOTEGI, N.; SUZUKI, H.; WATANABE, C.; TADOKORO, K.; YANAGISAWA, T.; and HIGASHI, S. (1988a): Dentine Resorption Mediated by Odontoclasts in Physiologic Root Resorption of Human Deciduous Teeth, Am JAnat 183:303-315. SASAKI, T.; SHIMIZU, T.; SUZUKI, H.; and WATANABE, C. (1989): Cytodifferentiation and Degeneration of Odontoclasts in Physiologic Root Resorption of Kitten Deciduous Teeth, Acta Anat 135:330-340. SASAKI, T.; TAKAHASHI, N.; WATANABE, C.; SUZUKI, H.; HIGASHI, S.; and SUDA, T. (1988b): Cytodifferentiation of Odontoclasts in Physiologic Root Resorption of Human Deciduous Teeth. In: The Biological Mechanisms of Tooth Eruption and
Root Resorption, Z. Davidovitch, Ed., Birmingham: EBSCO Media, pp. 321-328. SASAKI, T.; TOMINAGA, H.; and HIGASHI, S. (1984): Endocytotic Activity of Kitten Odontoblasts in Early Dentinogenesis. I. Thin Section and Freeze-Fracture Study, JAnat 138:485-492. SASAKI, T.; YAMAGUCHI, A.; HIGASHI, S.; and YOSHIKI, S. (1985): Uptake of Horseradish Peroxidase by Bone Cells During Endochondral Bone Development, Cell Tissue Res 239:547-553. SODEK, J. (1977): A Comparison of the Rates of Synthesis and Turnover of Collagen and Non-Collagen Proteins in Adult Rat Periodontal Tissues and Skin Using a Microassay, Arch Oral Biol 22:635-665. SVOBODA, E.L.A.; SHIGA, A.; and DEPORTER, D.A. (1981): A Stereologic Analysis of Collagen Phagocytosis by Fibroblasts in Three Soft Connective Tissues with Differing Rates of Collagen Turnover, Anat Rec 199:473-480. TAKAHASHI, N.; AKATSU, T.; UDAGAWA, N.; SASAKI, T.; YAMAGUCHI, A.; MOSELEY, J.M.; and SUDA, T. (1988): Ostcoblastic Cells are Involved in Ostcoclast Formation, Endocrinology 123:2600-2602. TAKAHASHI, T.; SO, S.; WANG, D.; TAKAHASHI, K.; KURIHARA, N.; and KUMEGAWA, M. (1986): Phagocytosis of Different Matrix Components by Different Cell Types at Bone-forming Sites in Cultured Mouse Calvariae, Cell Tissue Res 245:9-17. TEN CATE, A.R. (1972): Morphological Studies of Fibrocytes in Connective Tissue Undergoing Rapid Remodelling, JAnat 112:401414. TEN CATE, A.R. and ANDERSON, R.D. (1986): An Ultrastructural Study of Tooth Resorption in the Kitten, J Dent Res 65:10871093. TEN CATE, A.R. and DEPORTER, D.A. (1974): The Role of the Fibroblast in Collagen Turnover in the Functioning Periodontal Ligament of the Mouse, Arch Oral Biol 19:339-340. THOMSON, B.M.; SAKLAVALA, J.; and CHAMBERS, T.J. (1986): Osteoblasts Mediate Interleukin 1 Stimulation of Bone Resorption by Rat Osteoclasts, J Exp Med 164:104-112. WESTIN, G. (1942): Uber Zahndurchbruch und Zahnwechsel, Z Mikrosk Anat Forsch 51:393-471. WOESSNER, J.F. and CAHILL, D.R. (1974): Collagen Breakdown in Relation to Tooth Eruption and Resorption in the Dog, Arch Oral Biol 19:1195-1201.
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Cellular Roles in Physiological Root Resorption of Deciduous Teeth in the Cat T. Sasaki, T. Shimizu, C. Watanabe and Y. Hiyoshi J DENT RES 1990 69: 67 DOI: 10.1177/00220345900690011101 The online version of this article can be found at: http://jdr.sagepub.com/content/69/1/67
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Cellular Roles in Physiological Root Resorption of Deciduous Teeth in the Cat T. SASAKI, T. SHIMIZU, C. WATANABE, and Y. HIYOSHI The Second Department of Oral Anatomy, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
This study has attempted to assess the importance of mesenchymal cells, fibroblasts, cementoblasts, and mononuclear phagocytes (i.e., macrophages) in physiological root resorption offeline deciduous teeth. Deciduous incisors of three- to six-month-old kittens undergoing root resorption were investigated by means of electron microscopy. In an early phase of root resorption, the resorption organ consisted of many fibroblasts and relatively few macrophages and odontoclasts, the last with a wide, clear zone and narrow, immature, ruffled border. In the active phase of root resorption, the resorption organ contained many odontoclasts with a well-developed ruffled border and a reduced clear zone, cementoblasts, fibroblasts, macrophages, neutrophils, and many blood vessels. Cementoblasts were present usually on the resorbing dentin surface adjacent to odontoclasts and, in many cases, these cells communicated with each other via gap junctions. Cementoblasts frequently extended broad cell processes with secretion granules and with phagosomes containing collagen fibrils into the dentinal tubules exposed to resorption lacunae. Some macrophages exhibiting a clear zone-like structure also appeared on resorbing dentin surfaces. In the restingphase of root resorption, the dentin surface was covered mostly with cementoblasts resembling bone lining cells. There was an occasional macrophage, but no odontoclasts were observed during this phase. During removal of the periodontal ligament concomitant with root resorption, many fibroblasts phagocytosed mature collagen fibrils, as well as amorphous fluffr material. These results suggest that these mesenchymal cells, as well as odontoclasts, are essential for the cellular removal of dental hard and soft tissues during shedding offeline deciduous teeth. J Dent Res 69(1):67-74, January, 1990
Introduction. In the past two decades, there have been significant advances in elucidation of the cell biology of physiological root resorption of deciduous teeth (Boyde and Lester, 1967; Boyde et al., 1984; Furseth, 1968; Freilich, 1971; Morita et al., 1970; Jones et al., 1984; Ten Cate and Anderson, 1986; Sasaki et al., 1988ab, 1989). These studies have demonstrated that multinucleated giant cells, referred to as odontoclasts (osteoclasts), are the principal mediators of physiological root resorption of deciduous teeth. Although the resorption organ (resorptive tissue) also contains many fibroblasts, mononuclear phagocytes (macrophages), and granular leucocytes (neutrophils) (Furseth, 1968; Ten Cate and Anderson, 1986; Sasaki et al., 1988a, b, 1989), their functional roles in root resorption are not yet known. Ten Cate and Anderson (1986) first examined the cellular removal of dental soft tissues, including periodontal ligament, by fibroblasts during shedding of feline deciduous teeth, and pointed out the existence of two phenotypically different populations of fibroblasts in the region of ligament resorption: one with numerous collagen-containing phagosomes in the cytoplasm, and the other with condensed nucleus and cytoplasm. Moreover, our recent in vitro experiments demonstrated that dentin slices cultured with bone marrow cells were partially phagocytosed by mononuclear phagocytes such as macrophages, as well as being subject to resorption by bone-marrowderived multinucleated cells (Sasaki et al., 1988b). Similar Received for publication April 26, 1989 Accepted for publication September 28, 1989
phagocytosis of mineralized bone particles by macrophages has been reported both in vivo and in vitro (Kahn et al., 1978; Rifkin et al., 1979). Dorey and Bick (1977) further demonstrated the presence of N-acetyl-f3-glucosaminidase, which was related to the hydrolysis of glycosaminoglycans, in the macrophages present in periodontal ligament. These studies suggest that macrophages participate in the total process of bone remodeling and tooth resorption. It seems important, therefore, to clarify how the mesenchymal cells, such as cementoblasts, fibroblasts, and macrophages, are engaged in the removal of dental hard and soft tissues during shedding of deciduous teeth. In the present study, we extend the electron microscopic observations, first made by Ten Cate and Anderson (1986), on feline deciduous teeth undergoing root resorption from an early resorption phase to an active resorption phase just before shedding.
Materials and methods. After an intramuscular injection of ketamine hydrochloride (veterinary Ketalar 50, Sankyo Co., Ltd., Japan), three- to six-month-old kittens were given a transcardiac perfusion for about ten min with a fixative containing 1% glutaraldehyde and 1% formaldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.3). After perfusion, the upper and lower jaws, including deciduous incisors undergoing root resorption, were extracted, placed in fresh fixative for two to four h at 40C, and then demineralized in 10% ethylenediaminetetraacetic acid disodium (pH 7.3) for one to two months at 4TC. After demineralization, longitudinally-cut slices about 0.5 mm thick were prepared. These slices were then post-fixed with 1% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer (pH 7.3) for two h at 40C, block-stained with ethanolated 1% uranyl acetate, dehydrated through a graded ethanol series, and embedded in Epon 812. Thin sections were cut with use of a diamond knife on a Reichert-Jung Ultracut OmU-4 and stained with uranyl acetate and lead citrate. Some thin sections were stained with uranyl acetate and 1% phosphotungstic acid. All sections were examined with a Hitachi HU-12A electron microscope at 75 kV.
Results. Throughout the present study, the processes of root resorption of feline deciduous teeth were classified into three phases: (1) an early phase, with many fibroblasts and occasional macrophages and odontoclasts on dentin surfaces; (2) an active phase, with many well-developed odontoclasts, macrophages, fibroblasts, cementoblasts, and neutrophils; and (3) a resting phase, with cementoblastic lining cells and few macrophages. The root repair by cementum formation reported in human deciduous teeth (Kronfeld, 1932; Westin, 1942; Furseth, 1968) was not observed during the rapid root resorption and shedding of feline deciduous teeth. Early phase of root resorption. -In this phase, the root dentin and cementum were hardly indented, and their resorbing fronts were populated by many cementoblastic lining cells and relatively few multinucleated odontoclasts (Fig. 1A, inset).
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Fig. 1-The resorption organ in an early phase of root resorption. Fig. 1A: A low-magnification electron microscopic view of an odontoclast (OC) resorbing dentin (D) in shallow resorption lacuna and adjacent fibroblasts (FB). Inset is a light micrograph of the resorption organ containing relatively flat odontoclasts (arrows). Figs. 1B and 1C show a broad clear zone (CZ in Fig. 1B) and an immature ruffled border (RB in Fig. 1C) of an odontoclast in Fig. 1A. Fig. 1D shows a macrophage (Mp) and a fibroblast (FB) in resorption organ.
Odontoclasts exhibited a relatively flat profile and developed an unusually wide clear zone against the resorbing dentin (Figs. 1A and B). Such a wide clear zone was not observed in the odontoclasts found at an active resorption phase (Sasaki et al., 1988a,b). In the cytoplasm proximal to the clear zone, numerous pale vacuoles were accumulated-these were reported as a membrane source of ruffled border in our recent paper (Sasaki et at., 1989) (Figs. 1A and B). Microvillous protrusions forming immature ruffled border-like structures were observed adjacent to the clear zone (Figs. 1A and C). Other structural features of odontoclasts were identical to those described in previous papers (Furseth, 1968; Freilich, 1971; Sasaki et at., 1988a,b, 1989). Around odontoclasts, many fibroblasts and occasional macrophages were distributed roughly in parallel with both the resorbing dentin surface and the enamel organ surface of the successive permanent tooth (Figs. 1A and D). Fibroblasts extended several long slender cell processes in which phagocytosis of collagen fibrils was seldom observed (data not shown). Except for the areas occupied by odontoclasts, the resorbing dentin surface was covered with either fibroblasts, not showing collagen phagocytosis, or cementoblasts, resembling so-called "bone-lining cells" (Miller and Jee, 1987). Active phase of root resorption. -The most prominent feature of the resorption organ in the active phase of root resorption was the presence of many giant odontoclasts of various configurations (Furseth, 1968; Freilich, 1971; Ten Cate and Anderson, 1986; Sasaki et al., 1988a,b, 1989) (Fig. 2A, in-
set). The odontoclasts possessed a well-developed ruffled border against resorbing dentin, but intracellular incorporation of collagen fibrils by odontoclasts was never detected (Figs. 2A and B). Degeneration of collagen fibrils in the dentin surface region overlaid by the odontoclast ruffled border was not observed (Fig. 2B). Adjacent to the odontoclasts, numerous active cementoblast-like cells (hereafter called cementoblasts) were present on resorbing dentin surfaces. Unlike those covering the dentin surface at either an early resorption or a resting phase, cementoblasts in this phase showed a cuboidal and/or a rectangular outline, and were distributed roughly perpendicularly to the dentin surface [Figs. 2A (inset) and 3A]. Adjacent cementoblasts were frequently connected to each other by gap junctions between the cell bodies (Fig. 3A), between cell body and cell process (Fig. 3A, inset at the top left-hand corner), and between the cell processes (Fig. 3A, inset at the top righthand corner). Structurally, these cementoblasts were characterized by well-developed Golgi complexes, secretion granules, many cisternae of rough endoplasmic reticulum (RER), mitochondria, phagosomes, lysosomes, and numerous free polyribosomes. The Golgi complex consisted of five or six saccules with distended cylindrical margins, small coated vesicles, and condensing vacuoles (immature secretion granules) (Figs. 3A and B). Many secretion granules were distributed in the Golgi area and in the cytoplasm close to the dentin surface (Figs. 3A and B). Secretion granules were also observed in the broad cell processes penetrating those dentinal tubules exposed to resorption lacunae (Figs. 3C-E). Abundant newly-
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Fig. 2-The odontoclasts in an active phase of root resorption by phosphotungstic staining. Fig. 2A: The ruffled border region (RB) facing resorbing dentin (D) and adjacent vacuolar region (V) of an odontoclast. Phagocytosis of collagen fibrils is not seen in this odontoclast. Inset is a light micrograph of cementoblasts (CB) adjacent to odontoclast (OC) on resorbing dentin (D). Fig. 2B shows higher magnification of collagen fibrils in resorbing dentin overlaid by the odontoclast ruffled border.
formed thin collagen fibrils were distributed on the resorbing dentin surface overlaid by such cementoblasts (Fig. 3A). Moreover, these cementoblasts seemed to have phagocytosed old collagen fibrils, particularly at the dentin surface and inside the dentinal tubules. Phagocytosis of collagen fibrils through elongated phagocytic vacuoles was especially evident in the cell processes penetrating the dentinal tubules, but rarely observed in the cell bodies (Figs. 3C-E). Abundant fluffy material was accumulated on the dentin surfaces, including dentinal tubules covered with cementoblasts (Figs. 3A and C). In the resorption organ away from the resorbing dentin surface, many fibroblasts resorbed collagen fibrils by way of phagocytic vacuoles. These fibroblasts were, however, devoid of secretion granules (data not shown). Although some macrophages exhibiting the clear zone-like structure were present on the resorbing dentin surface, their interaction with resorbing dentin was uncertain (Fig. 4A). Many macrophages in the resorption organ frequently incorporated red blood cells, neutrophils, and cell debris of unknown origin, but they did not possess a clear zone-like structure (Fig. 4B). Resting phase. -In this phase of root resorption, the relatively smooth dentin surface was covered with flattened cementoblasts resembling "bone-lining cells". Sometimes these cementoblastic lining cells formed small, clear zone-like structures facing the resorbing dentin, and resorbed small dentin particles through the deep membrane invaginations of the clear zone (Fig. 5). Such lining cells possessed a well-developed Golgi apparatus consisting of four or five saccules and small coated vesicles, lysosomes, residual bodies, RER cisternae,
and mitochondria, but were devoid of secretion granules. Coated pits and vesicles were frequently seen in association with the plasma membranes of both the cell bodies and the cell processes of these lining cells. On the resorbing dentin, there was an occasional macrophage, but no active odontoclast was observed. Resorption of periodontal ligament. -During removal of periodontal ligament concomitant with root resorption, fibroblasts showed a flattened profile with several extremely long, slender cell processes (Fig. 6A). The cell body was occupied mainly by a large nucleus, and the narrow perinuclear cytoplasm contained a Golgi apparatus consisting of three or four saccules and related coated vesicles, lysosomes, many RER cisternae, mitochondria, and free ribosomes. Coated vesicles and pits were also visible in association with the plasma membranes. Microfilaments and microtubules ran parallel with the cell long axis. Near the region of ligament resorption, these fibroblasts contained many elongated phagocytic vacuoles in which mature collagen fibrils were found (Fig. 6B). These phagosomes were much more prominent in the cell processes than in the cell bodies. The extracellular matrix of the periodontal ligament consisted of bundles of collagen fibrils and amorphous, fine, fluffy material. The collagen fibril population varied from area to area. In the region of ligament resorption, the extracellular matrix was composed mostly of fluffy material but few collagen fibrils (Figs. 6C and D). Such fluffy material, as well as collagen fibrils, was seen in the phagocytic vacuoles of the fibroblasts. In this region, macrophages frequently appeared among the fibroblasts. The macrophages ex-
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Fig. 3-The cementoblasts on resorbing dentin in an active phase of root resorption. Fig. 3A: Cementoblasts on resorbing dentin contain well-developed Golgi apparatus, abundant RER cisternae, and many secretion granules. They are connected by gap junctions between the cell bodies (arrow in Fig. 3A), between cell body (CB) and cell process (CP) (inset at the top left-hand corner), and between cell processes (inset at the top right-hand corner). Amorphous fluffy material fills the dentinal tubules (DT) and the resorbing dentin surface. Fig. 3B shows a higher-magnification view of the Golgi apparatus (Go) and secretion granules (SG) in a cementoblast. Fig. 3C: A cementoblast (CB) penetrating its broad cell process into a dentinal tubule (DT). Fig. 3D: The cementoblastic cell process marked by a rectangle in Fig. 3C. The process includes many secretion granules (SG) and elongated phagosomes (Ph) containing collagen fibrils. Fig. 3E: A higher-magnification view of collagen fibrils in phagosomes (Ph) in the cementoblastic cell process penetrating a dentinal tubule.
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Fig. 4-Macrophages in resorption organ in an active phase of root resorption. Fig. 4A: A macrophage (Mp) exhibiting a clear zone-like structure (CZ) on resorbing dentin (D). Fig. 4B: Macrophages in the resorption organ contain varied cell debris (arrows).
hibited an elongated profile and possessed many large electrondense lysosomes with heterogeneous inclusions in the perinuclear cytoplasm. Although deep membrane invaginations containing fluffy material were observed in macrophages, we failed to find phagocytosis of collagen fibrils by macrophages (Fig. 6D). This resorption of the periodontal ligament was a common finding throughout the process of root resorption.
Discussion. The present observations suggest that, in addition to odontoclasts, several other mesenchymal cells such as fibroblasts, cementoblasts, and macrophages are actively involved in cellular removal of dental hard and soft tissues during the shedding of deciduous teeth. We have previously indicated that neutrophils in the resorption organ were related to the degeneration of exhausted odontoclasts (Sasaki et al., 1989). During root resorption of human deciduous teeth, cementoblasts deposit cementum matrix onto resorption lacunae in the phase of tooth repair (Furseth, 1968). However, this study suggests that cementoblasts are engaged in both matrix secretion and resorption during the phase of active root resorption. The findings described here are not restricted to feline deciduous teeth, because the identical phenomenon was recently found in physiological root resorption of human deciduous teeth (unpublished). Earlier biochemical studies have demonstrated that the resorption organ engages in collagenolytic activity (Morita et al., 1970; Woessner and Cahill, 1974), but the cellular origin of
the activity is not yet known. In this regard, it has been reported that collagenase is synthesized and released by osteoblasts in response to parathyroid hormone (PTH) (Sakamoto and Sakamoto, 1982, 1984). Collagenase mediates the extracellular degradation of structural collagen during bone remodeling. Chambers et a. (1985) have suggested that removal of the osteoid layer by collagenase-induced osteoclastic bone resorption in vitro, by permitting osteoclasts to come into contact with resorption-stimulating bone mineral. Recently, the osteoblast has been suspected of being involved in the hormonal control of bone resorption and in the direct activation of osteoclasts by products of osteoblastic hormone action (Rodan and Martin, 1981). In fact, the osteoblast is known to stimulate osteoclastic bone resorption by releasing an unknown soluble factor in response to PTH (McSheehy and Chambers, 1986a,b). Furthermore, the osteoblast stimulates osteoclastic responsiveness to interleukin 1 (IL-1), and thereby increases osteoclastic bone resorption (Heath et al., 1985; Thomson et al., 1986). A recent in vitro study has also indicated that, in the presence of lox,25-dihydroxyvitamin D3 (1,25(OH)2D3), a co-culture of osteoblastic cells with spleen cells induced formation of multinucleated cells which, in turn, produced numerous resorption lacunae on a co-cultured dentin slice (Takahashi et al., 1988). These studies suggest that various hormones, such as IL-1, PTH, PGE2, and 1,25(OH)2D3, act primarily on osteoblasts having receptors for these hormones to stimulate osteoclastic bone resorption. Thus, it is strongly suggested that osteoblasts are required for osteoclast differentiation. In our recent cytochemical study, cementoblasts on resor-
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Fig. 5-Cementoblastic lining cells (LC) on resorbing dentin (D) in a resting phase of root resorption. Inset figure shows a clear zone-like structure (CZ) of a lining cell, in which phagocytosis of small dentin particles is noted (arrows). Fig. 6-Fibroblasts and macrophages in the periodontal ligament in an active phase of root resorption. Fig. 6A: Fibroblasts in the periodontal ligament possess many phagosomes containing collagen fibrils (arrows). Fig. 6B: A higher-magnification view of phagosomes containing collagen fibrils (arrows) in a fibroblast.
bing dentin showed alkaline phosphatase activity, a marker enzyme for the osteoblast (unpublished). These cementoblasts are morphologically identical to osteoblasts (Furseth, 1968, 1969; Jande and Belanger, 1970; present study). It is an important finding that adjacent cementoblasts communicate with each other via gap junctions, as in the osteoblast layer (Doty, 1981). Such gap junctions are well-known to provide electrical coupling between the cells by transcellular transfer of ions and low-molecular-weight metabolites (Pappas, 1975; Lowenstein, 1979). It is our contention that collagenolytic activity in the resorption organ is provided by the cementoblasts. The presence of abundant fluffy material on the dentin surface overlaid by the cementoblasts might be due to their collagenolytic activity. Therefore, if cementoblasts on resorbing dentin are actually identical to osteoblasts, both morphologically and functionally, they might induce surface modification or remodeling of resorbing dentin, and thereby stimulate odontoclast differentiation. In fact, the remodeling (the synthetic and resorptive processes involved in the resorbing of dentin) occurs when active cementoblasts are present, whereas the dentin surface in the resting phase is covered with flattened lining cells. This cementoblastic function might be essential for activation of odontoclastic functioning in tooth resorption. Synchronous resorptive and synthetic activities are a common feature of hard-tissue-forming cells in their active phase:
the odontoblast (Sasaki et al., 1982, 1984), the ameloblast (Kallenbach, 1980; Sasaki, 1984; Nanci et al., 1985), the osteoblast (Sasaki et al., 1985; Takahashi et al., 1986), and perhaps also the cementoblast. In this connection, the repair of resorption lacunae by cementoblasts may reflect the matrix formative activity of the cementoblast in its active phase. During the active resorption phase of feline deciduous teeth, these cementoblasts seemed to be engaged in secretion of matrix substance, but the actual formation of cellular cementum was not observed. This may be due to the rapid shedding of feline deciduous teeth. In human deciduous teeth, cellular cementum was found to be partially formed on the resorption lacunae in the phase of active root resorption (unpublished). Active cementoblasts are not present in an early phase of root resorption or in a resting phase during which odontoclastic resorption does not actively take place. These results suggest that the functional significance of cementoblasts during an active resorption phase is primarily related to activation of odontoclasts. The precise mechanism by which the cementoblast activates the odontoclast has yet to be determined. Phagocytosis of collagen fibrils by these cementoblasts suggests that they are also engaged in removal of collagen fibrils from the resorbing dentin, while odontoclasts are not. Bone resorption is considered to be mediated not only by osteoclasts, but also by two different cell types-fibroblasts and monocyte-derived macrophages (Heersche, 1978). Be-
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Fig. 6C shows the region of ligament resorption, in which the extracellular matrix consists of abundant fluffy material. Fig. 6D shows a macrophage (Mp) and a fibroblast (FB) in the periodontal ligament.
cause neither osteoclasts nor odontoclasts exhibit collagenase activity or phagocytose collagen fibrils exposed at resorbing bone and/or dentin surfaces, these fibrils are believed to be phagocytosed by either fibroblasts or macrophages. Osteoclasts and/or odontoclasts are believed to demineralize the bone and/or dentin surface partially and degrade noncollagenous matrix (Heersche, 1978; Sasaki et al., 1988a,b; present study). A close functional relationship between fibroblasts and osteoclasts was also suggested by electron microscopic observation of the periodontal ligament (Garant, 1976). Our present observation of collagen phagocytosis by cementoblasts on resorbing dentin adjacent to odontoclasts seems to support their
findings. Mononuclear phagocytes (macrophages) seem to have at least two functions in root resorption: phagocytosis, and subsequent digestion, of exhausted and/or degenerated cells in the resorption organ; and the phagocytosis of dentin particles. Although the present study did not show phagocytosis of mineralized dentin by macrophages, we have recently demonstrated that macrophages with a clear zone phagocytose small dentin particles in vitro (Sasaki et al., 1988b). Similar phagocytosis of mineralized bone particles by macrophages has already been indicated in vivo and in vitro (Dorey and Bick, 1977; Kahn et al., 1978; Rifkin et al., 1979; Takahashi et al., 1986). It follows that macrophages found on resorbing dentin may be involved in the phagocytosis of fragments derived from dentin matrix. Phagocytosis of collagen fibrils by macrophages (Parakkal, 1969; Deporter, 1979) was, however, not confirmed in this study. Although similar phagocytosis of dentin particles
was observed in cementoblastic lining cells in a resting phase of resorption, its functional significance is yet to be determined. Removal of periodontal ligament by fibroblasts has been reported repeatedly in previous investigations (Ten Cate, 1972; Ten Cate and Deporter, 1974; Listgarten, 1973; Garant, 1976; Svoboda et al., 1981; Beertsen, 1987). The periodontal ligament is known to undergo rapid turnover of collagen fibrils. Quantitatively, phagocytosis of collagen fibrils by fibroblasts during the normal maintenance of periodontal ligament is much more frequent and faster than in the connective tissues of attached gingiva and skin (Sodek, 1977; Svoboda et a!., 1981). In periodontal ligament, fibroblasts are involved in both resorption and secretion of collagen fibrils, and also show regional variation in cell activity (Garant, 1976). In the region of ligament resorption, the ligament space was mostly filled with amorphous fluffy material, rather than with collagen fibrils. This may be due to extracellular breakdown of collagen fibrils. Both fibroblasts and macrophages may be involved in resorption of fluffy material. Beertsen (1987) recently reported that collagen fibrils in periodontal ligament of teeth made hypofunctional decreased from 50% to 30% of the total population during the experiment. He demonstrated that a substantial loss of collagen occurred within the first few days of hypofunction, and that periodontal ligament fibroblasts reacted very rapidly to functional changes of teeth. The process of collagen loss in hypofunctional teeth might correspond to that for removal of periodontal ligament during shedding of deciduous teeth.
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J Dent Res January 1990
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