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Inhibition of Apoptosis in Early Tooth Development Alters Tooth Shape and Size
J.-Y. Kim1,
Y.-G. Cha1,
S.-W. Cho,
E.-J. Kim,
M.-J. Lee,
J.-M. Lee,
J. Cai,
H. Ohshima2 and
H.-S. Jung*
Division in Anatomy and Developmental Biology, Department of Oral Biology, Research Center for Orofacial Hard Tissue Regeneration, Oral Science Research Center, College of Dentistry, Brain Korea 21 Project for Medical Science, Yonsei Center of Biotechnology, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul, 120-752, Korea; and
2 Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Japan
Correspondence: * corresponding author, hsjung{at}yumc.yonsei.ac.kr
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ABSTRACT
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Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.
Key Words: apoptosis • tooth development • tooth shape • tooth size • macrodontia
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INTRODUCTION
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Apoptosis is the mechanism responsible for the physiological elimination of cells and appears to be intrinsically programmed. It has also been identified as an important mechanism in such processes as interdigital cell death (van der Hoeven et al., 1994; Salazar-Ciudad et al., 2003), and has been suggested to play a role in the tooth morphogenesis of mice (Vaahtokari et al., 1996). In particular, apoptosis was first reported to occur in the enamel knot (EK) during tooth development (Vaahtokari et al., 1996). This would begin to silence its function as a signaling center during the late-cap to the early-bell stages (Vaahtokari et al., 1996; Tucker and Sharpe, 1999). It was recently reported that the cessation of cell proliferation in the EK is linked to the expression of the cyclin-dependent kinase inhibitor p21 in mouse molars. This p21 expression is induced by bone morphogenetic protein 4 (BMP-4) in isolated dental epithelium, which is expressed only in the underlying dental mesenchyme at the onset of EK formation (Jernvall et al., 1998). These results support the role of the cyclin-dependent kinase inhibitors as inducible cell differentiation factors in epithelial-mesenchymal interactions (Salazar-Ciudad et al., 2003). Furthermore, the expression of p21 in the EK is followed by Bmp-4 expression, and the differentiated EK cells subsequently undergo extensive apoptosis (Jernvall et al., 1998).
To understand the relationship among tooth morphogenesis, differentiation, and apoptosis in the EK, we performed in vitro tooth cultures using the pancaspase inhibitor z-VAD-fmk, which completely inhibited the apoptosis induced by p21 (Wood and Newcomb, 2000). We examined cellular events such as cell proliferation and apoptosis after the inhibition of apoptosis during tooth development. In addition, we also examined tooth shape and size after 3 wks of transplantation into the kidney capsule of mice. In this study, we hypothesized that apoptosis in the EK would play an important role in regulating tooth shape and size.
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MATERIALS & METHODS
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All experiments were performed in compliance with the guidelines of the Yonsei University College of Dentistry Intramural Animal Use and Care Committee.
Animals
ICR mouse embryos were obtained from time-mated pregnant mice. Embryonic day 0 (E0) was designated as the day that a vaginal plug was confirmed. Embryos at E13.5 were used in this study.
In vitro Organ Culture and Tooth Germ Transplantation into Kidney Capsule
Embryonic mice molar tooth buds were microdissected from the lower jaw at E13.5 by stereo-microscopy in PBS. Tooth buds were cultured in DMEM supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA) and antibiotics by a modified Trowell’s culture method for a designated period (Sarkar and Sharpe, 2000). In the experimental cases, the media were supplemented with 25 µM, 50 µM, and 100 µM z-VAD-fmk [FK-009 z-VAD (OMe)-CH2F O-methylated; Enzyme Systems Products] in DMSO (Sigma, St. Louis, MO, USA) for 2 days, and post-cultured in the control medium for 4, 5, and 7 days, whereas the media without z-VAD-fmk in DMSO were used with the control group. For full tooth development, after in vitro culture for 2 days, at least 20 explants for each concentration of the z-VAD-fmk-treated group and the control group were transplanted into the kidney capsule of syngeneic adult males for 3 wks (Kratochwil et al., 1996).
Tissue Preparation and Immunohistochemistry
The specimens were fixed and embedded in paraffin. Serial, frontal, 5-µm sections were prepared. Sections were stained with hematoxylin-eosin (H-E) for histological observation and prepared for immunohistochemistry. For the cell proliferation assay, mouse monoclonal anti-proliferation cell nuclear antigen (PCNA) antibody (1:200, Neo Markers, CA, USA) was used. Heat treatment in citrate buffer (pH 6.0) was used for PCNA-immunohistochemistry. Apoptosis was detected by the TUNEL (Terminal-transferase-mediated dUTP-biotin NickEnd Labeling) method. An in situ cell death detection kit (Roche Diagnostics GmbH, Germany) was used for the detection of apoptotic cells. Normal serum, instead of the primary antibody, was used as a negative control. For determining the numbers of PCNA- and TUNEL-positive cells in the cultured tooth germs, we selected 15 sections at random from at least 15 specimens. The PCNA- and TUNEL-positive cells were counted, and the data were expressed as the mean ± SD (standard deviation). For statistical analysis, we used the Student’s t test.
Di.I. Microinjection
A lipophilic dye, Di.I. (Molecular Probes, UK; 2 mg/mL in dimethyl formamide), was microinjected as a marker for examination of the migration pattern of tooth bud mesenchymal cells at E13.5, during the early cap stage. In particular, we examined the buccal and lingual sides of tooth germs, to understand the migration patterns of mesenchymal cells contributing to the proper shape and size of the tooth. After the microinjection, tooth germs were cultured for 2 and 4 days by a modified Trowell’s culture method. At least 30 explants were examined. The migration pattern was examined by epi-fluorescence microscopy (Leica, MZ FL III).
Image Processing and Measuring of Teeth
Tooth-transplanted kidney tissue was removed from the host mouse and dissected so that the calcified teeth could be harvested in PBS. Whole teeth were photographed and digitized with a Dimage Scan Multi film scanner (Minolta, Japan). Bright-field and cultured teeth images were also collected with a Spot RT digital camera (Leica, Germany). We used at least 15 teeth explants to examine the shapes and sizes of teeth in each experimental group. Tooth size was measured by means of an eyepiece graticule attached to a binocular microscope. Statistical analysis of these measurements consisted of an independent t test.
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RESULTS
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Tooth Shape was Altered after z-VAD-fmk Treatment in in vitro Organ Culture
At E13.5, after the mice molar tooth buds were microdissected from the mandible, we cultured tooth germs using a modified Trowell’s culture method for 2 and 4 days (Fig. 1A ). The cultured molar tooth germ showed its typical shape after 2 days in both the control and experimental groups (Figs. 1B, 1D). There were no differences in tooth germ shape between the groups. To test the effect of z-VAD-fmk treatment, we post-cultured for 2 days in the control medium. After 4 days of in vitro culture, the size of the tooth germ showed differences between the controls and z-VAD-fmk-treated specimens. In particular, the mesiodistal diameter of the tooth germ in the experimental group was longer than that of the control. After a four-day culture, the control group showed a second molar in the distal part of the first molar (n = 26/28, 92.9%) (Fig. 1C). However, we could detect the second molar in only a few samples (n = 2/35, 5.7%) (Fig. 1E). Compared with the control group, the buccolingual diameter of the tooth germ was not significantly altered after a 100-µM z-VAD-fmk treatment (data not shown).
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Figure 1. Morphological observations of the in vitro cultured teeth of control (A–C) and 100 µM z-VAD-fmk-treated (D,E) groups from 0 to 4 days after dissection under the dissecting microscope. Z-VAD-fmk-treated tooth germs were cultured in a medium containing z-VAD-fmk for 2 days and then cultured in the control medium. (F–H) Di.I. microinjections were performed in the mesenchymal parts of the buccal and lingual lateral sides of a tooth germ, so that we could understand the patterning for tooth formation in the buccolingual axis of the tooth germ. (A) In vitro cultured tooth germ at E13.5, at the onset of culture. (B) In vitro cultured tooth germ showing specific shape. The tooth germ was observed in the distal part of a cultured 1st molar tooth germ. (C) The distal region of the control cultured tooth germ includes the 2nd molar. (D) The size of the tooth germ was not altered after a two-day culture. (E) After 4 days, the shape of the tooth germ in the experimental group was larger than that of the control, especially the length of the mesiodistal diameter, although there were no significant alterations in buccolingual length. (F) At E13.5, the lateral sides of the tooth germ were labeled with Di.I. injection. (G) Di.I.-labeled cells had migrated along the mesiodistal axis and into the tooth germ. (H) After 96 hrs, Di.I.-positive cells were examined along the mesiodistal axis, and some of these cells were recruited into the tooth germ area. An asterisk indicates that the 2nd molar was not formed in the z-VAD-fmk-treated tooth germ after 96 hrs. Mes, mesial; Dis, distal; B, buccal; L, lingual; M1, first molar; M2, second molar. Scale bar: 200 µm.
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To clarify the patterning of mesenchymal cells contributing to the formation of the tooth, we also examined the migration pattern of mesenchymal cells of a tooth germ at E13.5, using Di.I. micro-injections. The Di.I. was micro-injected at each lateral and medial side of the tooth germ (Fig. 1F ). After 2 days, the appearance of cultured molar tooth germ indicated that the distribution of Di.I.-positive cells was near the margin of the tooth germ (Fig. 1G). After 4 days, we observed that the Di.I.-positive cells were recruited in the tooth-germ-forming area, and some of these cells exhibited a migration pattern along the tooth-germ boundary (Fig. 1F). Both the control and experimental groups demonstrated similar patterning after Di.I. microinjections (Figs. 1F-1H).
Histological Evaluation of Tooth Germ after z-VAD-fmk Treatment
In vitro-cultured teeth were histologically examined with H-E staining. After a seven-day culture, we could identify the dental papilla, inner dental epithelium, stellate reticulum, and outer dental epithelium of the bell-stage tooth germ in both the control and experimental groups (Figs. 2A, 2B). In both the control and the z-VAD-fmk-treated specimens, PCNA was localized to the cells located in the inner dental epithelium and dental papilla, and there were no differences between them (Figs. 2C, 2D). However, many apoptotic cells were observed in the control. This apoptosis was detected in the inner dental epithelium and the dental papilla beneath the dental organ (Fig. 2E). Interestingly, there were few positive apoptotic bodies in z-VAD-fmk-treated specimens (Fig. 2F).
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Figure 2. H&E staining (A,B), PCNA immunohistochemistry (C,D), and TUNEL staining (E,F) were observed after the in vitro tooth germs were cultured for 7 days from E13.5. Control group (A,C,E). The z-VAD-fmk-treated group was cultured for 2 days in medium containing the z-VAD-fmk, then post-cultured in the control medium for 5 days (B,D,F). To examine the formation of the calcified tooth, we cultured the tooth germ for 2 days using a modified Trowell’s culture method, then transplanted it into the mouse kidney capsule for 3 wks (G,H). (I) Occlusal view of (G). (J) Occlusal view of (H). (A) Tooth germ is composed of dental papilla (DP), inner dental epithelium (IDE), and outer dental epithelium. (B) The histological structures are similar to those of the control. (C,D) Blue arrows indicate PCNA-positive cells. Both the control and the z-VAD-fmk-treated tooth germs show a similar expression pattern of PCNA. PCNA-positive cells were localized in the inner dental epithelium and dental papilla. (E) Apoptotic bodies appeared with a broad localization pattern in the cultured tooth in the inner dental epithelium and the dental papilla of the control tooth. Red arrows indicate the apoptotic bodies. (F) Few apoptotic bodies were detected after 100-µM z-VAD-fmk treatment. Red arrows indicate the apoptotic bodies. (G) Six cusps were recognized in the control-cultured tooth. The whole shape of the calcified tooth was similar to a rectangular structure. (I) Occlusal view of (G). (H) Tooth shape of the 100-µM z-VAD-fmk-treated tooth germ was altered. The mesiodistal diameter became longer, and the crown height was significantly decreased compared with that of the control. (J) Occlusal view of (H). (I,J) The calcified teeth, both the control and z-VAD-fmk-treated, showed 6 cusps [(B1, B2, B3, and L1, L2, L3), according to the nomenclature of Gaunt (1955, 1961)]. DP, dental papilla; IDE, inner dental epithelium. White arrowheads indicate the mesial direction. Scale bars: A-F, 100 µm; G-J, 200 µm.
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Alterations of Tooth Shape and Size in Calcified Teeth
We examined the shape and size of the in vitro-cultured teeth after 3 wks of tooth transplantation into a kidney capsule. Prior to transplantation, the tooth germs at E13.5 were cultured for 48 hrs by a modified Trowell’s method, with various concentrations of z-VAD-fmk. The control teeth showed the proper crown formation, with 6 cusps (Fig. 2D). After 25-µM and 50-µM z-VAD-fmk treatments, the in vitro-cultured teeth showed results similar to those of the control, except for a shorter crown height and a longer mesiodistal diameter than the control (data not shown). Teeth treated with 100-µM z-VAD-fmk showed much wider mesiodistal diameters and shorter crown heights (Figs. 2H, 3C). After tooth transplantation into the kidney capsule for 3 wks, we also examined the histological sections using H-E staining. The observed occurrence of dentin and dentinal tubules in both 50-µM and 100-µM z-VAD-fmk-treated tooth germs was similar to that observed in the control (data not shown). These results suggest that z-VAD-fmk treatment did not affect cytodifferentiation, but instead altered the shapes and sizes of teeth in a concentration-dependent manner (Fig. 3).
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Figure 3. Mesiodistal diameter and crown height of calcified teeth after treatment with various concentrations of z-VAD-fmk. This value is plotted (on a log scale) against the concentration of z-VAD-fmk treatment. (A) The mesiodistal diameter is directly proportional to the concentration of z-VAD-fmk. (B) The crown height is inversely related to the concentration of z-VAD-fmk. (C) The mesiodistal diameter and crown height of calcified teeth were measured under the microscope. Diameter, mesiodistal diameter; height, crown height. (D) Numbers of PCNA- and TUNEL-positive cells. There were no significant differences between the control and z-VAD-fmk-treated groups in PCNA. However, apoptosis was significantly reduced in the z-VAD-fmk-treated group. *P < 0.01.
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DISCUSSION
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Apoptosis in Tooth Morphogenesis
Apoptosis is well-known as a powerful mechanism that controls the proper shapes of organs in embryonic organogenesis, as in the case of digital development with interdigital cell death (Kelley, 1970; Salazar-Ciudad et al., 2003). The developing tooth is a valuable model system for understanding the cellular mechanisms that control organ morphogenesis (Maas and Bei, 1997; Thesleff and Sharpe, 1997; Peterkova et al., 2003). Apoptosis in the EK has been suggested to play a role in the regulation of tooth shape during development in mice (Vaahtokari et al., 1996). A previous report concerning apoptosis of the EK in mice molar teeth described the expression of the cyclin-dependent kinase inhibitor p21 (Jernvall et al., 1998). The EK cells express p21 after Bmp-4, and subsequently undergo extensive apoptosis (Jernvall et al., 1998). The EK plays a pivotal role in the regulation of tooth shape through the unequal growth of the dental epithelium resulting from p21-related apoptosis (Jernvall et al., 2000). A previous report also showed the effects of inhibiting apoptosis during tooth development (Coin et al., 2000). These investigators focused on the formation of the cusp structure after inhibiting apoptosis. However, they did not examine the shapes and sizes of calcified teeth using an in vitro culture method. Based on this previous study, we decided to examine the alterations in calcified teeth and to evaluate the histological changes, including cell proliferation and apoptosis, after inhibiting apoptosis at the early cap stage.
In this study, we examined the contribution of apoptosis at the early cap stage to the regulation of tooth shape and size development, by using kidney transplantation. Three weeks after tooth germ transplantation into the kidney, we examined the sizes and shapes of the calcified teeth. The results suggested that tooth shape could be altered through the inhibition of apoptosis with z-VAD-fmk in a concentration-dependent manner at the early cap stage (Fig. 3). The crown height and mesiodistal diameter were changed after treatment with 100 µM of z-VAD-fmk: The mesiodistal distance was increased, and the crown height was decreased. Interestingly, there were no disturbances in the buccolingual lengths for various concentrations of z-VAD-fmk (data not shown). Three-dimensional examinations, such as a reconstruction method and micro CT, will help us understand the alterations of the volume of calcified teeth, and such examinations are planned for the near future.
These results suggest that there are unknown mechanisms that regulate the buccolingual diameter during molar tooth development. As a possible candidate for the mechanism that regulates tooth shape and size, apoptosis of the primary EK could influence mesiodistal diameter and crown height. This possibility is supported by several observations: First, spatial occupation, which should be deleted by apoptosis in normal development, still existed in the tooth-forming area after the inhibition of apoptosis. Second, relative to the specific patterning for tooth formation, dental follicle cells migrated from the lateral sides of a tooth germ to the center part of a tooth germ at the early cap stage, as confirmed by Di.I. microinjection. Third, the direction of apoptosis in the tooth germ at E14 showed that it initiated from the distal to the mesial direction (Jernvall et al., 1998). From our results, the determination of mesiodistal diameter and crown height is governed by cellular apoptosis of the primary EK in molar development. Judging from the evidence in this study, we hypothesize that tooth anomalies such as macrodontia could result from the failure of apoptosis in the EK during early tooth development (Fig. 4).
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Figure 4. Schematic diagram of the putative mechanism of macrodontia. Disruption of apoptosis would result in macrodontia.
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Apoptosis, One Candidate Factor for Macrodontia
Macrodontia is a rare dental anomaly. This term has been used to describe dental gigantism, and may be made in reference to a large proportion of the dentition (true generalized), the entire dentition (relative generalized), or to a single tooth (isolated macrodontia) (Shafer et al., 1974). Isolated macrodontia may be the simple enlargement of all tooth structures, or it may be associated with morphological anomalies (Groper, 1987). After blocking apoptosis by z-VAD-fmk treatment, we observed morphogenesis of mice molar teeth similar to that in human macrodontia, except that there was no alteration in the buccolingual diameter. Human macrodontia, like Ekman-Westborg-Julin and Schinzel-Giedion syndromes, shows increases in both mesiodistal and buccolingual diameters. Interestingly, crown height was decreased in both human macrodontia and the z-VAD-fmk-treated mouse teeth. Although a previous report suggested that reduced crown height in human macrodontia (Dugmore, 2001) results from the lack of space to erupt, we could not exclude the possible involvement of apoptosis. Inhibition of apoptosis during early tooth development would cause such a problem with eruption, since the size of the tooth would be larger than that of the normal tooth. Recently, many reports of bioengineered teeth have showed some difficulties in the regulation of size and shape (Miura et al., 2003; Duailibi et al., 2004; Ohazama et al., 2004). This apoptosis-related mechanism, which was suggested in a previous report, could be an important modulator in controlling the shapes and sizes of bioengineered teeth (Peterkova et al., 2003).
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ACKNOWLEDGMENTS
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This work was supported by grant No. R13-2003-13 from the Korea Science & Engineering Foundation.
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FOOTNOTES
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1 authors contributing equally to this work 
Received for publication May 17, 2005.
Revision received February 7, 2006.
Accepted for publication February 21, 2006.
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Journal of Dental Research, Vol. 85, No. 6,
530-535 (2006)
DOI: 10.1177/154405910608500610
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ABSTRACT
INTRODUCTION