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Localization of uncalcified cementum in adult rat molar roots and its relation to physiological tooth movement
Ar~hs WLZ/Biol. Vol. 39. No. 10, pp. X29M2. 1994 Copyright t” 1994 Elsevrer Science Ltd
Pergamon
Prtnted in Great Britain. All riyhrs reserved 0003.9969;94 $7.00 + 0.00
0003-9969(94)00058-l
LOCALIZATION OF UNCALCIFIED CEMENTUM ADULT RAT MOLAR ROOTS AND ITS RELATION PHYSIOLOGICAL TOOTH MOVEMENT MANABU
KAGAYAMA,*
Department
HIROTOSHI AKITA,
of Anatomy,
YASUYUKI
Tohoku University School Sendai 980, Japan
IN TO
SASANO and KOJI KINDAICHI of Dentistry,
Seiryocho
4-1,
(Accepted 6 June 1994) Summary-The study was designed to elucidate the effect of physiological tooth movement on cellular cementum, using the upper molar roots of IO-week-old rats. Paraffin sections stained with haematoxylin and eosin displayed two types of cellular cementum, lightly and darkly staining. The lightly stained was present on the distal half of all molar roots except the mesial root of the first molar. The alveolar bone facing the lightly stained cementum showed resorption lacunae and multinucleated osteoclasts, while the opposite bone surface was lined with osteoblasts. In contact microradiographs of undemineralized ground sections, the X-ray density of the lightly stained cementum was similar to that of the periodontal ligament and pulp, while the X-ray density of the darkly stained cementum was similar to that of alveolar bone. Tetracycline labelling lines were seen at the interface between the two types of cellular cementum as well
as on surfaces of bone and cementum located mesially to the root dentine. The results suggest that the mechanical stress of tooth movement differently affects the alveolar bone and cellular cementum; the bone is resorbed whereas the cementum force of tooth movement. Key words:
uncalcified
cementum,
resists resorption
tooth
and its calcification
movement,
INTRODUCTION
tetracycline
under the compressive
label, adult
rats.
resists resorption during tooth movement is not known. Our study now was designed to elucidate the effect of physiological tooth movement on cellular cementurn.
Cementum is a calcified connective tissue formed on the outer surface of root dentine. It serves as the attachment site of the periodontal ligament, which connects cementum to alveolar bone. Cementum is divided into two types, acellular and cellular; the coronal half of the root surface is covered with acellular and the apical half with cellular cementum (Schroeder, 1986). The cellular cementum of rat molar teeth is formed as posteruptive cementum (Formicola, Krampf and Witte, 1971). Its thickness varies with age and it has been suggested that its formation is continuous throughout life (Zander and Hurzeler, 1958; Louridis, Bazopoulou-Kyrkanidou and Demetriou, 1972). Rat molars are known to drift distally throughout the animal’s life (Kraw and Enlow, 1967). During tooth movement, the alveolar bone displays typical changes, showing resorption lacunae and osteoclasts at the resorptive surface, and active osteoblasts at the forming surface (Kraw and Enlow, 1967; Vignery and Baron, 1980). Although the effect of tooth movement on alveolar bone has been demonstrated, its effect on cementum or on cementogenesis is not known. It has been shown that cementum is more resistant to resorption than bone, and it is for this reason that orthodontic tooth movement is possible (Armitage, 1980). However, the mechanism by which cementum
*To whom correspondence
microradiograph,
is inhibited
MATERIALS
AND METHODS
Two IO-week-old male Wistar rats were used for routine histological examination. Under pentobarbital anaesthesia, animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature. After perfusion, the maxillae were dissected, and immersed in the same fixative for 20 h at 4LC. The specimens were demineralized in 10% EDTA in 0.1 M cacodylate buffer for 4 to 6 weeks at 4’C, dehydrated, and embedded in paraffin via xylene. Serial sections were made horizontally from crown to root apex, stained with haematoxylin and eosin, and observed with a light microscope. Two IO-week-old rats were used for tetracycline labelling and contact microradiographs. The animals were injected with tetracycline hydrochloride (50 mg/kg body wt) 10 days before fixation. After immersion fixation with 4% perfusion and paraformaldehyde in 0.1 M phosphate buffer, the maxillae were dissected, dehydrated, and embedded in Spurr resin. Serial, 200~pm thick sections were cut horizontally, and ground manually to 50pm. Contact microradiographs were made with soft X-ray and
should be addressed. 829
x30
MANAN, KAGAYAMA et trt
spectroscopic safety film type 649-O (Eastman Kodak Co.. U.S.A.). Then, the ground sections were mounted on the slides with 30% glycerine solution. The developed microradiographs and the ground sections were observed and photographed with a light and with a fluorescence microscope microscope, (Olympus. BH2-RFK).
RESULTS
Two types of cellular cementum, lightly and darkly staining, were identified in paraffin sections stained with haematoxylin and eosin (Fig. I). Both types appeared at random depending on the position of each section. With the use of serial sections, however.
Fig. I. Light micrograph of a parafftn section showing representative appearances of cellular cementum of rat molar tooth. Both the lightly and darkly stained cementum can be identified clearly. In this distopalatal root of first molar tooth, the lightly stained cementum is seen on distopalatal half of the root, whereas the darkly stained is seen on the opposite half. The arrow indicates the mesiodistal direction. x 750 Fig. 2. A light micrograph of a ground section showing the cellular cementum of the distopalatal root of first molar root. Note that part of the cementum is transparent and its localization is similar to that of the lightly stained cementum in Fig. 1. The other part shows lacunae and canaliculi of cementocytes. The same ground section was used for Figs 2. 3 and 4. The arrow indicates the mesiodistal direction. x 750 Fig. 3. A contact microradiograph of the same ground section as in Figs 2 and 4. Note that the X-ray density of the lightly stained cementum is similar to that of the periodontal ligament and pulp, whereas that of the darkly stained cementum is similar to that of alveolar bone. x750 Fig. 4. Tetracycline labelling lines of the same ground sectton as in Figs 2 and 3. The lines are seen at the surfaces of cementum and bone. and the interface between the two types of cementum (arrow head). Note that the bone surface facing the periodontal ligament is positive at one side and negative at the other, suggesting physiological tooth movement. x 750
Uncdlcified
cementum
it was found that the lightly stained cementum was present on all molar tooth roots except the largest mesial root of the first molar tooth. The lightly stained cementum was localized at specific positions on molar roots; most was seen on the distal half or on the distobuccal half of the buccal roots or the distopalatal half of the palatal roots. The lightly stained cementum appeared thickest at the middle of the apical half of the roots, and decreased in thickness in sections cut more cervically or apically. The cementurn surrounding the root apex was darkly stained. The surface of alveolar bone facing the lightly stained cementum displayed resorption lacunae and multinucleated osteoclasts, while the opposite bone surface showed active osteoblasts (data not shown). There was no resorptive activity at the surface of two types of cellular cementurn, although resorption lacunae were often seen on the coronal half of the root surface. The lightly stained cementum appeared to be uncalcified when ground sections were examined by contact microradiography or tetracycline labelling (Figs 3 and 4). In the light micrographs of the ground sections, the lightly stained cementum appeared transparent and structureless (Fig. 2). In the contact microradiographs, the X-ray density of the lightly stained cementum was similar to that of the periodontal ligament and pulp, and that of darkly stained cementum (calcified cementurn) similar to alveolar bone (Fig. 3). The specific position of the uncalcified cementum was identical to that of the lightly stained cementum in paraffin sections. Labelling lines of tetracycline were seen at the interface between the two types of cementum (Fig. 4, arrow heads), as well as at the bone and cementum surfaces that faced mesially to the root. but not at those facing distally to the root.
DISCUSSION
We clearly demonstrate two types of cellular cementum, lightly and darkly stained, in paraffin sections. and uncalcified and calcified in ground sections at a specific position on the rat molar roots. Although there have been repeated studies of the development, growth, and calcification of both acellular and cellular cementum (Schroeder, 1986), no one has, to the best of our knowledge, described a typical uncalcified or lightly stained cementum, even in adult rats (Paynter and Pudy, 1958). The differcnces in the results obtained from the previous and present studies may be due to the different methods of observation. Most studies on cementum have used sagittal sections in order to observe the coronal and apical cementum within one section, and do not analyse serial sections. In sagittal sections, the uncalcified cementum may be cut tangentially because of its specific localization and would not be easily identifiable as a normal structure. We found that observation of serial sections was required to confirm the presence of the lightly stained cementum in all of the molar roots except the mesial. root of the first molar, because that cementum appeared at random depending on the position of the section within each root.
in rat molar
teeth
831
The cellular cementum of the rat molar is formed as posteruptive cementum at the apical half of the root (Formicola et al., 1971). Its thickness varies with age and it is probably formed continuously throughout life (Zander and Hurzeler, 1958; Louridis e/ (11.. 1972). Most of the tetracycline labelling lines that mark the calcification front at the time of injection are reportedly parallel (Formicola ef al., 1971; Owens, 1975), which suggests that calcification of cellular cementum progresses from crown to root apex. A previous study using the tetracycline labelling method (Owens, 1975) demonstrated a V-shaped labelhng line at the apical root of the dog premolar tooth, and suggested that cementum formation spreads from the apical region towards the crown rather than from the crown to the root apex. Because the uncalcified cementum that we observed appeared mainly in the middle of the apical half of the root and was not seen at the apical end, and because the tetracycline line was seen between the calcified and the uncalcified cementum in horizontal sections. we suppose that a V-shaped or similar tetracycline line would be present in sagittal ground sections of rat molar roots as in the dog premolar roots (Owens, 1975). In other words, uncalcified cementum may occur in other animals, although the nature of cementurn differs greatly among species (Schroeder. 1986. 1992). Rat molars are known to drift distally (Kraw and Enlow, 1967). During tooth movement, one side of the alveolar bone is resorbed. and bone is formed at the opposite side. Orthodontic forces can alter the remodelling activities of alveolar bone, which supports the concept that the alveolar bone is resorbed by the compressive force and formed by the tensional force of mechanical stress. Although the effect of tooth movement on alveolar bone has been demonstrated, its effect on cementum is unknown. Because alveolar bone is resorbed at the distal side of the roots and is supposedly under the compressive force of tooth movement, the occurrence of the uncalcified or the lightly stained cementum on the distal half of the roots may relate to that force. This suggests differing effects of mechanical stress on alveolar bone and cementum; the resorption of the bone and not resorption but inhibition of calcification of cementurn. This may be related to the relative resistance of cementum to mechanical stress during tooth movement. It will be of great interest to examine the cellular or molecular mechanisms of this phenomenon. technical assistance of Mr M. Mr Y. Mikami is gratefully acknowledged. This supported in part- by grants-in-aid (50030100. from the Ministry of Science. Education and Japan.
Acknowledgement.s-The
Eguchi and work was 71020400) Culture of
REFERENCES
Armitage
G. C. (1980) Cementum. In Orhan’s Oral HISEmbryology (Ed. Bhaskar S. N.). 9th edn. Chap. 6. p. 199. The C. V. Mosby Co., London. Formicola A. J., Krampf J. 1. and Witte E. G. (1971) Cementogenesis in developing rat molars. J. Peritxfoont. 42, 766773. tology
and
X32
MANABC KAUAYAMA 01 crl
Krdw A. G. and Enlow D. M. (1967) Continuous attachment of the periodontal membrane. Am. J. Anal. 120, 1333148. Louridis 0.. Bazopoulou-Kyrkanidou E. and Demetriou N. (1972) Age effect upon cementum width of albino rat: a histometric study. J. Periodon/. 43, 533-536. Owens P. D. A. (1975) Patterns of mineralization indicated by tetracycline labelling in the premolar teeth of dogs. Archs oral Biol. 20, 709-712. Paynter K. J. and Pudy G. (1958) A study of the structure, chemical nature and development of cementum in the rat. Anat. Rec. 131, 233-251.
Schroeder H. E. (1986) The Periodon/ium. Springer-Verlag, Berlin. Schroeder H. E. (I 992) Biological problems of regenerative cementogenesis: synthesis and attachment of collagenous matrices on growing and established root surfaces. In/. Ret. Cw~l. 142, I-59. Vignery A. and Baron R. (1980) Dynamic histomorphometry of alveolar bone remodeling in the adult rat. Anat. Rec. I%, 191-200. Zander H. A. and Hurzeler B. (1958) Continuous cementum apposition. J. dent. Rus. 37, 1035-1044.
Pergamon
Prtnted in Great Britain. All riyhrs reserved 0003.9969;94 $7.00 + 0.00
0003-9969(94)00058-l
LOCALIZATION OF UNCALCIFIED CEMENTUM ADULT RAT MOLAR ROOTS AND ITS RELATION PHYSIOLOGICAL TOOTH MOVEMENT MANABU
KAGAYAMA,*
Department
HIROTOSHI AKITA,
of Anatomy,
YASUYUKI
Tohoku University School Sendai 980, Japan
IN TO
SASANO and KOJI KINDAICHI of Dentistry,
Seiryocho
4-1,
(Accepted 6 June 1994) Summary-The study was designed to elucidate the effect of physiological tooth movement on cellular cementum, using the upper molar roots of IO-week-old rats. Paraffin sections stained with haematoxylin and eosin displayed two types of cellular cementum, lightly and darkly staining. The lightly stained was present on the distal half of all molar roots except the mesial root of the first molar. The alveolar bone facing the lightly stained cementum showed resorption lacunae and multinucleated osteoclasts, while the opposite bone surface was lined with osteoblasts. In contact microradiographs of undemineralized ground sections, the X-ray density of the lightly stained cementum was similar to that of the periodontal ligament and pulp, while the X-ray density of the darkly stained cementum was similar to that of alveolar bone. Tetracycline labelling lines were seen at the interface between the two types of cellular cementum as well
as on surfaces of bone and cementum located mesially to the root dentine. The results suggest that the mechanical stress of tooth movement differently affects the alveolar bone and cellular cementum; the bone is resorbed whereas the cementum force of tooth movement. Key words:
uncalcified
cementum,
resists resorption
tooth
and its calcification
movement,
INTRODUCTION
tetracycline
under the compressive
label, adult
rats.
resists resorption during tooth movement is not known. Our study now was designed to elucidate the effect of physiological tooth movement on cellular cementurn.
Cementum is a calcified connective tissue formed on the outer surface of root dentine. It serves as the attachment site of the periodontal ligament, which connects cementum to alveolar bone. Cementum is divided into two types, acellular and cellular; the coronal half of the root surface is covered with acellular and the apical half with cellular cementum (Schroeder, 1986). The cellular cementum of rat molar teeth is formed as posteruptive cementum (Formicola, Krampf and Witte, 1971). Its thickness varies with age and it has been suggested that its formation is continuous throughout life (Zander and Hurzeler, 1958; Louridis, Bazopoulou-Kyrkanidou and Demetriou, 1972). Rat molars are known to drift distally throughout the animal’s life (Kraw and Enlow, 1967). During tooth movement, the alveolar bone displays typical changes, showing resorption lacunae and osteoclasts at the resorptive surface, and active osteoblasts at the forming surface (Kraw and Enlow, 1967; Vignery and Baron, 1980). Although the effect of tooth movement on alveolar bone has been demonstrated, its effect on cementum or on cementogenesis is not known. It has been shown that cementum is more resistant to resorption than bone, and it is for this reason that orthodontic tooth movement is possible (Armitage, 1980). However, the mechanism by which cementum
*To whom correspondence
microradiograph,
is inhibited
MATERIALS
AND METHODS
Two IO-week-old male Wistar rats were used for routine histological examination. Under pentobarbital anaesthesia, animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature. After perfusion, the maxillae were dissected, and immersed in the same fixative for 20 h at 4LC. The specimens were demineralized in 10% EDTA in 0.1 M cacodylate buffer for 4 to 6 weeks at 4’C, dehydrated, and embedded in paraffin via xylene. Serial sections were made horizontally from crown to root apex, stained with haematoxylin and eosin, and observed with a light microscope. Two IO-week-old rats were used for tetracycline labelling and contact microradiographs. The animals were injected with tetracycline hydrochloride (50 mg/kg body wt) 10 days before fixation. After immersion fixation with 4% perfusion and paraformaldehyde in 0.1 M phosphate buffer, the maxillae were dissected, dehydrated, and embedded in Spurr resin. Serial, 200~pm thick sections were cut horizontally, and ground manually to 50pm. Contact microradiographs were made with soft X-ray and
should be addressed. 829
x30
MANAN, KAGAYAMA et trt
spectroscopic safety film type 649-O (Eastman Kodak Co.. U.S.A.). Then, the ground sections were mounted on the slides with 30% glycerine solution. The developed microradiographs and the ground sections were observed and photographed with a light and with a fluorescence microscope microscope, (Olympus. BH2-RFK).
RESULTS
Two types of cellular cementum, lightly and darkly staining, were identified in paraffin sections stained with haematoxylin and eosin (Fig. I). Both types appeared at random depending on the position of each section. With the use of serial sections, however.
Fig. I. Light micrograph of a parafftn section showing representative appearances of cellular cementum of rat molar tooth. Both the lightly and darkly stained cementum can be identified clearly. In this distopalatal root of first molar tooth, the lightly stained cementum is seen on distopalatal half of the root, whereas the darkly stained is seen on the opposite half. The arrow indicates the mesiodistal direction. x 750 Fig. 2. A light micrograph of a ground section showing the cellular cementum of the distopalatal root of first molar root. Note that part of the cementum is transparent and its localization is similar to that of the lightly stained cementum in Fig. 1. The other part shows lacunae and canaliculi of cementocytes. The same ground section was used for Figs 2. 3 and 4. The arrow indicates the mesiodistal direction. x 750 Fig. 3. A contact microradiograph of the same ground section as in Figs 2 and 4. Note that the X-ray density of the lightly stained cementum is similar to that of the periodontal ligament and pulp, whereas that of the darkly stained cementum is similar to that of alveolar bone. x750 Fig. 4. Tetracycline labelling lines of the same ground sectton as in Figs 2 and 3. The lines are seen at the surfaces of cementum and bone. and the interface between the two types of cementum (arrow head). Note that the bone surface facing the periodontal ligament is positive at one side and negative at the other, suggesting physiological tooth movement. x 750
Uncdlcified
cementum
it was found that the lightly stained cementum was present on all molar tooth roots except the largest mesial root of the first molar tooth. The lightly stained cementum was localized at specific positions on molar roots; most was seen on the distal half or on the distobuccal half of the buccal roots or the distopalatal half of the palatal roots. The lightly stained cementum appeared thickest at the middle of the apical half of the roots, and decreased in thickness in sections cut more cervically or apically. The cementurn surrounding the root apex was darkly stained. The surface of alveolar bone facing the lightly stained cementum displayed resorption lacunae and multinucleated osteoclasts, while the opposite bone surface showed active osteoblasts (data not shown). There was no resorptive activity at the surface of two types of cellular cementurn, although resorption lacunae were often seen on the coronal half of the root surface. The lightly stained cementum appeared to be uncalcified when ground sections were examined by contact microradiography or tetracycline labelling (Figs 3 and 4). In the light micrographs of the ground sections, the lightly stained cementum appeared transparent and structureless (Fig. 2). In the contact microradiographs, the X-ray density of the lightly stained cementum was similar to that of the periodontal ligament and pulp, and that of darkly stained cementum (calcified cementurn) similar to alveolar bone (Fig. 3). The specific position of the uncalcified cementum was identical to that of the lightly stained cementum in paraffin sections. Labelling lines of tetracycline were seen at the interface between the two types of cementum (Fig. 4, arrow heads), as well as at the bone and cementum surfaces that faced mesially to the root. but not at those facing distally to the root.
DISCUSSION
We clearly demonstrate two types of cellular cementum, lightly and darkly stained, in paraffin sections. and uncalcified and calcified in ground sections at a specific position on the rat molar roots. Although there have been repeated studies of the development, growth, and calcification of both acellular and cellular cementum (Schroeder, 1986), no one has, to the best of our knowledge, described a typical uncalcified or lightly stained cementum, even in adult rats (Paynter and Pudy, 1958). The differcnces in the results obtained from the previous and present studies may be due to the different methods of observation. Most studies on cementum have used sagittal sections in order to observe the coronal and apical cementum within one section, and do not analyse serial sections. In sagittal sections, the uncalcified cementum may be cut tangentially because of its specific localization and would not be easily identifiable as a normal structure. We found that observation of serial sections was required to confirm the presence of the lightly stained cementum in all of the molar roots except the mesial. root of the first molar, because that cementum appeared at random depending on the position of the section within each root.
in rat molar
teeth
831
The cellular cementum of the rat molar is formed as posteruptive cementum at the apical half of the root (Formicola et al., 1971). Its thickness varies with age and it is probably formed continuously throughout life (Zander and Hurzeler, 1958; Louridis e/ (11.. 1972). Most of the tetracycline labelling lines that mark the calcification front at the time of injection are reportedly parallel (Formicola ef al., 1971; Owens, 1975), which suggests that calcification of cellular cementum progresses from crown to root apex. A previous study using the tetracycline labelling method (Owens, 1975) demonstrated a V-shaped labelhng line at the apical root of the dog premolar tooth, and suggested that cementum formation spreads from the apical region towards the crown rather than from the crown to the root apex. Because the uncalcified cementum that we observed appeared mainly in the middle of the apical half of the root and was not seen at the apical end, and because the tetracycline line was seen between the calcified and the uncalcified cementum in horizontal sections. we suppose that a V-shaped or similar tetracycline line would be present in sagittal ground sections of rat molar roots as in the dog premolar roots (Owens, 1975). In other words, uncalcified cementum may occur in other animals, although the nature of cementurn differs greatly among species (Schroeder. 1986. 1992). Rat molars are known to drift distally (Kraw and Enlow, 1967). During tooth movement, one side of the alveolar bone is resorbed. and bone is formed at the opposite side. Orthodontic forces can alter the remodelling activities of alveolar bone, which supports the concept that the alveolar bone is resorbed by the compressive force and formed by the tensional force of mechanical stress. Although the effect of tooth movement on alveolar bone has been demonstrated, its effect on cementum is unknown. Because alveolar bone is resorbed at the distal side of the roots and is supposedly under the compressive force of tooth movement, the occurrence of the uncalcified or the lightly stained cementum on the distal half of the roots may relate to that force. This suggests differing effects of mechanical stress on alveolar bone and cementum; the resorption of the bone and not resorption but inhibition of calcification of cementurn. This may be related to the relative resistance of cementum to mechanical stress during tooth movement. It will be of great interest to examine the cellular or molecular mechanisms of this phenomenon. technical assistance of Mr M. Mr Y. Mikami is gratefully acknowledged. This supported in part- by grants-in-aid (50030100. from the Ministry of Science. Education and Japan.
Acknowledgement.s-The
Eguchi and work was 71020400) Culture of
REFERENCES
Armitage
G. C. (1980) Cementum. In Orhan’s Oral HISEmbryology (Ed. Bhaskar S. N.). 9th edn. Chap. 6. p. 199. The C. V. Mosby Co., London. Formicola A. J., Krampf J. 1. and Witte E. G. (1971) Cementogenesis in developing rat molars. J. Peritxfoont. 42, 766773. tology
and
X32
MANABC KAUAYAMA 01 crl
Krdw A. G. and Enlow D. M. (1967) Continuous attachment of the periodontal membrane. Am. J. Anal. 120, 1333148. Louridis 0.. Bazopoulou-Kyrkanidou E. and Demetriou N. (1972) Age effect upon cementum width of albino rat: a histometric study. J. Periodon/. 43, 533-536. Owens P. D. A. (1975) Patterns of mineralization indicated by tetracycline labelling in the premolar teeth of dogs. Archs oral Biol. 20, 709-712. Paynter K. J. and Pudy G. (1958) A study of the structure, chemical nature and development of cementum in the rat. Anat. Rec. 131, 233-251.
Schroeder H. E. (1986) The Periodon/ium. Springer-Verlag, Berlin. Schroeder H. E. (I 992) Biological problems of regenerative cementogenesis: synthesis and attachment of collagenous matrices on growing and established root surfaces. In/. Ret. Cw~l. 142, I-59. Vignery A. and Baron R. (1980) Dynamic histomorphometry of alveolar bone remodeling in the adult rat. Anat. Rec. I%, 191-200. Zander H. A. and Hurzeler B. (1958) Continuous cementum apposition. J. dent. Rus. 37, 1035-1044.