Odontology DOI 10.1007/s10266-014-0158-1
ORIGINAL ARTICLE
Cyclophosphamide inhibits root development of molar teeth in growing mice Tomomi Kawakami • Yuko Nakamura Hiroyuki Karibe
•
Received: 27 November 2013 / Accepted: 23 April 2014 Ó The Society of The Nippon Dental University 2014
Abstract Root development of permanent teeth is disturbed in survivors of childhood cancer. Cyclophosphamide (CY) is a cytostatic drug commonly used for chemotherapy in children with cancer. This study aimed to evaluate the effects of CY on the development of molar teeth until the completion of occlusion in young mice, focusing on Hertwig’s epithelial root sheath (HERS). We treated thirty-two 12-day-old ICR mice with CY (100 mg/ kg; 100-CY group), and 36 control mice with saline. At 12, 14, 16, 20, 24, 27, 39, 60, and 76 days of age, the mandibular molars were removed. Soft X-ray radiographs were obtained in lateral projection. The root/crown length (R/ C) ratio of the first molar was calculated. Serial sagittal sections were prepared and histomorphological hematoxylin and eosin (HE) staining and immunohistochemical (cytokeratin) studies were performed. The R/C ratio of the 100-CY group (0.78) was smaller than that of the control group (1.23) at 76 days (p \ 0.05, t test). While all roots developed further after injection, microscopic examination showed that the roots of the first molars that developed in the 100-CY group were shorter than those in the control group. In addition, experimental mice showed apical closure of the roots. By 20 days after injection, the HERS had disappeared from the root surface in the 100-CY group. In conclusion, this study indicates that CY can induce a defect in HERS and cause early loss of HERS. Disruption of the epithelial sheath inhibits normal root formation, and it could cause irreversible short-root development.
T. Kawakami (&) Y. Nakamura H. Karibe Department of Pediatric Dentistry, The Nippon Dental University School of Life Dentistry at Tokyo, 1-9-20, Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan e-mail: [email protected]
Keywords Root development Cyclophosphamide Mice Hertwig’s epithelial root sheath Late effects
Introduction In recent decades, improvements in therapies for childhood cancers have increased the number of survivors. The treatments used for childhood cancer include chemotherapy, irradiation, surgery, stem cell transplantation, and a combination of these modalities, and these treatments are becoming increasingly effective. The survival rates of childhood cancers are reportedly 70 to 75 % in some parts of Europe and North America [1]. The survival rate for children with cancer in Japan is now approximately 80 %, and more than 0.1 % of young adults are survivors of childhood cancer [2]. However, as the survival rate of children with cancer has improved considerably, the late effects of antineoplastic therapy have increased in importance. Concern has been raised about the long-term adverse health effects in several areas including learning, motor function development, fertility, cardiac and renal function, vision, hearing, and the risk of secondary cancers [2–4]. It has been estimated that in Japan, approximately two-thirds of childhood cancer survivors will have at least one late effect [2]. Late effects involving dental disturbances have been reported in a group of patients treated with cancer therapy [5–8]. Adverse effects of cancer and cancer therapy during childhood on dental health have been reported in terms of mineralization disturbances, dental caries, microdontia, hypodontia, root stunting, and taurodontism [6, 7]. For many pediatric cancer patients, since some of the dental sequelae of chemotherapy and radiation are irreversible, oral sequelae and discomfort related to their treatment can
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have long-term and potentially lethal consequences. Some investigators have suggested that children treated with chemotherapy in the early years of their lives exhibit a high incidence of dental defects, and immature teeth are at greater risk of dental abnormalities than fully developed teeth [6, 9]. Dental disturbances can result from chemotherapy, as well as radiation therapy. Chemotherapy in children treated for neuroblastoma can adversely influence tooth development [10]. Experimental studies have shown adverse effects of the frequently used chemotherapeutic agents cyclophosphamide (CY) and doxorubicin on developing teeth [11, 12]. CY is one of the cytostatic drugs present in chemotherapy cocktails that are commonly used to treat childhood cancers. CY targets all cells by cross-linking DNA with its bioactive metabolite, phosphoramide mustard [13]. Though CY is an alkylating agent that is widely used as an antineoplastic drug owing to its capacity to interfere with the division of cancer cells, its toxicity has been ascribed to the fact that it has a similar effect on normal proliferative tissues and cells. While some studies have investigated the effects of CY on tooth formation, mostly rodent incisors have been used. It is difficult to observe these effects until root formation of the apex in this context, because in rodents the incisors grow continuously. Only few studies have investigated the root formation of molars [14, 15], but their observations were made over a limited period, leaving many aspects of the effects of CY unclear. It is well known that tooth root formation is initiated by the development of Hertwig’s epithelial root sheath (HERS). However, relatively little is known about the direct effect of CY on HERS and root development up to completion of the root apex in mouse molars. In the present study, as a first step for clarifying the continuous changes in CY-induced root development up to and after the completion of root development, histopathological examinations were carried out on lower first molars exposed to CY in young mice, focusing on the changes in HERS.
Materials and methods Animals A total of 83 ICR mice (Charles River Japan Inc., Yokohama, Japan) were used. The mice were fed free from their mothers, and allowed free access to standard solid mouse feed and distilled water ad libitum after weaning. All mice were housed under conventional conditions with controlled temperature (24 ± 1 °C), humidity (50 ± 10 %), and light (12-h light/12-h dark).
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All animal experiments were approved by the Ethics Committee for Animal Experimentation and conducted in compliance with the Guidelines of the Nippon Dental University, School of Life Dentistry at Tokyo, Section of Biological Sciences, Research Center for Odontology. Study design CY (EndoxanÒ, Shionogi & Co., Ltd., Tokyo, Japan) dissolved in 200 lL of saline was administered to 12-day-old (postnatal; PN12) mice intraperitoneally (i.p.). To investigate the effects of CY, two different CY treatments were designed. To study the dose effects of CY, 15 mice were randomly divided into five groups. Equal numbers of mice (n = 3) were administered i.p. injections of CY at doses of 30, 50, 100, or 200 mg/kg of body weight (indicated as 30-CY, 50-CY, 100-CY, or 200-CY below), at PN12. The control group received injections of an equal volume of saline. The mice were killed at 20 days of age (PN20). The administered doses of CY did not result in the death of any of the mice. In the second experiment, 68 mice were used to investigate the chronological changes in molar development induced by CY, and they were randomly divided into two groups; one group was given a single injection of 100 mg/ kg of CY, and the other group (control) was given saline, on PN12. They were then killed on PN12 (control group only), PN14, PN16, PN20, PN24, PN27, PN39, PN60, and PN76. Indicators of the general condition of the mice in all groups, including body weight and behavior, were monitored once a day throughout the experimental period. Radiographic evaluation On the designated days after drug injection, the mice were anesthetized using sodium pentobarbital (NembutalÒ, Dainihonseiyaku, Tokyo, Japan), and transcardially perfused with 4 % paraformaldehyde. After fixation, the skull was laterally separated along the epicranial midline suture from the parietal bone over the mandible. Soft X-ray radiographs (M-100 W, SOFTEX, Tokyo, Japan) were obtained in lateral projection, under the following conditions: tube voltage, 40 kVp; tube current, 3 mA; acquisition time, 20 s; and focus-subject distance, 25 cm. The acquired film images were scanned and entered into a personal computer. The soft X-ray radiographs were printed on photo paper and compared against the original radiographs to ensure that no distortion had occurred during printing. A previously described method was adapted for the measurement of the distal root length of the first molars [16]. Point ‘‘m’’ was defined as the midpoint of a straight
Odontology
Statistical analysis Results are expressed as mean ± standard deviation (SD). The data were analyzed by a one-way analysis of variance (ANOVA), and when a significant F ratio was identified, groups were compared using Tukey’s post hoc test. Student’s t test was used to compare the differences between groups, and p \ 0.05 was deemed to indicate statistical significance. All statistical analyses were carried out using the statistical package SPSS, version 17.0 J (SPSS, Inc., Tokyo, Japan). Fig. 1 The points used for measuring crown height and root length in the assessment of the root–crown (R/C) ratio. C crown height, R root length, m midpoint, a apical reference line
Results The dose effects of CY on HERS
line connecting the points of intersection between the outer contours of the root and crown in all first molars (Fig. 1). Crown height (C) was the perpendicular line from point m to an occlusal reference line placed to connect the buccal cusps of the first molar. Root length (R) was measured from point m perpendicular to the apical tangent of the distal root, parallel to the occlusal reference line. The R/C ratio of individual tooth was calculated by dividing root length by crown height. Histological evaluation Hematoxylin and eosin staining Histological changes in the periodontal tissues were assessed via hematoxylin and eosin (HE) staining. The mandible of each mouse was dissected and immediately immersed in 4 % paraformaldehyde. Mandibular specimens were decalcified with 10 % EDTA at 4 °C. The specimens were paraffin embedded, and serial sections (4lm thick) were cut on the sagittal plane with a rotary microtome (RM2065, Leica, Wetzlar, Germany). Immunohistochemistry Tissue sections were deparaffinized with xylene, and rehydrated with decreasing concentrations of ethanol. Following rehydration, antigen retrieval was conducted via 0.1 % pepsin treatment for 10 min at 37 °C with 10 mL HCl. The antibodies used were polyclonal rabbit antihuman cytokeratin pan (Progen Biotechnik, Heidelberg, Germany; 1:1000 dilution). Immunoreactivity was visualized using 3,30 -diaminobenzidine (DAB) (SK-4100, Funakoshi, Tokyo, Japan) coloring with the ABC method (ABC Elite kit, Vector Laboratories, Inc., Burlingame, CA). Nuclei were lightly counterstained with 1 % methyl green.
Because HERS is formed at the initiation of tooth root development and plays an important role in determining root shape and size, we examined the effects of various doses of CY on HERS, via immunostaining analysis (Fig. 2). The analysis showed that HERS expressed cytokeratin, which is a marker of epithelium. The HERS in the control group could be seen in the lower first molar germs at PN20, and it still had a double-layered structure. The HERS of experimental groups treated with 30 or 50 mg/kg of CY appeared similar to that of the control group, but the HERS of mice treated with 100 or 200 mg/ kg of CY disappeared in a concentration-dependent manner. Table 1 shows the crown height and the distal root length of the first molars treated with different doses of CY. While the mean crown heights did not change, the mean root lengths decreased in a dose-dependent manner. Furthermore, with regard to comparison of the R/C ratio of the first molars, the R/C ratios decreased in a dosedependent manner, and there were significant differences between the control group and the high-dose CY group (Fig. 2f). These findings indicated that at high doses such as 100 or 200 mg/kg, a single injection of CY had a cytotoxic effect on HERS. The effects of CY on chronological changes A dose of 100 mg/kg of CY was used to investigate chronological CY-induced changes in root formation in lower first molars. The weight change of the two groups up to PN76 is shown in Fig. 3. All mice gained weight at the end of drug administration, though a reduction in the rate of weight gain was observed in the 100-CY group during the first 2 weeks after the CY injection. There were significant differences in weight at PN20 and PN24, but there were no significant differences in weight at any of the other time-points investigated.
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Odontology Fig. 2 The effects of different doses of CY on root development of the first molars at PN20. Micrographs of sections immunostained with anti-cytokeratin (a–e). HERS Hertwig’s epithelial root sheath, DP dental pulp, PDL periodontal ligament. Scale bar = 100 lm. Original magnification 920. The R/ C ratio of different doses of CY at PN20 (F). * p \ 0.01, p \ 0.05. Intergroup comparisons were performed using Tukey’s test
Table 1 The crown height and the root length (lm) of the first molars after treatment with different doses of CY PN20 Crown height
Root length
Mean
SD
Mean
SD
Control
1119
71
935
57
30-CY
1034
71
836
97
50-CY 100-CY
1048 1091
120 85
822 694
40 97
200-CY
935
40
453
80
Four days after the injection of 100-CY, the experimental mice began to lose fur. After another 4 days, they had lost all of their fur. However, their fur began to regrow from PN24, and was restored by PN27. The fur of the mice in the control group was unaffected. The effects of CY on the R/C ratios of the first molars The mean crown heights and root lengths are shown in Table 2, and the chronological changes in R/C ratio are shown in Fig. 4. Mean crown height did not change from PN12 to PN76, in either group. However, while mean root length in the 100-CY group did not extend much, there was an increase in the control group. The R/C ratio in the
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Fig. 3 Body weights of experimental and control mice from PN12 to PN76. The results are expressed as mean ± SD. *Statistically significant difference compared with the control (t test, p \ 0.05)
control group increased from PN12 to PN76 (Fig. 4). Conversely, the R/C ratio in the 100-CY group increased slightly up to PN20 and then remained almost unchanged. The R/C ratios in both groups increased significantly. In contrast, there were no significant differences between the mean R/C ratios of the control group and the 100-CY group until PN16. From PN20 to PN76, however, the mean R/ C ratio in the 100-CY group was lower than that of the
Odontology Table 2 The crown height and the root length (lm) of the first molars in the 100-CY group Control
100-CY
Crown height
Root length
Crown height
Root length
Mean
SD
Mean
SD
Mean
SD
Mean
SD
PN12
1105
57
269
28
PN14
1063
28
439
28
1091
54
439
28
PN16
1119
28
646
54
1176
71
609
85
PN20
1119
71
935
57
1091
85
694
97
PN24
1176
54
1162
73
1091
54
737
40
PN27
1162
150
1165
200
992
98
680
93
PN39
935
109
1048
170
963
46
652
73
PN60
949
54
1105
143
1006
73
680
143
PN76
1020
80
1247
122
1077
80
836
54
Fig. 4 The R/C ratio of the first molars. The results are expressed as mean ± SD. *Statistically significant difference compared with the control (t test, p \ 0.05)
control group, and there were significant differences between the two groups during that period. This result indicated that CY had inhibited root formation. Histological evaluation of the first molar after treatment Figure 5 shows the chronological changes of the effects of CY on HERS with anti-cytokeratin antibodies. In the control group, HERS formed a double layer of cells comprised of inner enamel epithelium and outer enamel epithelium that extended in an apical direction at PN14 and became slightly thinner at PN16 (Fig. 5a, b). At PN20, the length of the HERS became progressively shorter (Fig. 5c), and the sheet-like structure of HERS was broken at PN24 (Fig. 5d) and it began to detach from the root surface (Fig. 5e). Conversely, the HERS in the 100-CY group did
not exhibit a double-layered structure at PN14, though a sheet-like structure was discernible (Fig. 5f). HERS began to break at PN16, and had separated from the root (Fig. 5g). HERS had disappeared completely by PN20, but the cytokeratin-positive cells scattered on the root surface were only observed as epithelial rests of Malassez (Fig. 5h–j). The HERS in the experimental group changed shape and was gone by PN16. This state was similar to the HERS of the control group at PN27. From PN39 onwards, no cytokeratin-positive cells resembling the HERS were observed in either group (data not shown). To observe the state of root formation and periodontal tissue, we examined HE-stained sections of the developing root of the mandibular first molars from PN12 to PN76. Fig. 6 shows histological changes in distal root development chronologically, and Fig. 7 shows higher magnification of the apical part of the distal root. At PN12, just before CY administration, root formation had completed furcation, and progressed to approximately half of the root of the first molar, but the apical area was still wide open and the first molar had not yet erupted (data not shown). The distal root length of the first molars clearly extended until PN27 in the control group (Fig. 6a–h). Conversely, while the distal root length of the first molars in the 100-CY group did extend slightly until PN16, at that point it almost ceased (Fig. 6i–p). In both the control and the 100-CY group, at PN14 the first molars had not erupted into the oral cavity, and their apical portions were wide open (Fig. 6a, i). Cervical loop structure was present in both control and experimental groups, but in the 100-CY group the cytoplasm of the odontoblasts had begun to atrophy as compared to the control group (Fig. 7a, b). At PN16, eruption of the first molar had commenced in the control group (Fig. 6b). Conversely, half of each first molar in the 100-CY group was covered with epithelium and had not erupted (Fig. 6j). In the 100-CY group, the cervical loop of the apex could not be discerned and the apical portions observed were smaller as compared to those of the control group. Cementum addition was apparent from PN20 in the 100-CY group, although the root length of the first molar changed little after PN16 (Fig. 7d). In the control group, cementum addition was observed from PN24. In addition, from PN27 until PN76, cementum addition in the 100-CY group increased and was formed not only around the apical area but also the middle part of the root (Fig. 6p, f).
Discussion On the basis of long-term follow-up studies, it has been reported that long-term survivors who received chemotherapy treatment in childhood have an increased risk of
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Odontology Fig. 5 Immunohistochemical staining for cytokeratin in HERS of mandibular first molars at various time-periods in the control group (a–e), and the 100-CY group (f–j). HERS Hertwig’s epithelial root sheath, DP dental pulp, OD odontoblasts, PDL periodontal ligament, ERM epithelial rests of Malassez. Scale bar = 100 lm. Original magnification 940
Fig. 6 Development of the distal roots of mandibular first molars from PN14 to PN76. Hematoxylin and eosin staining of sections showing the distal root of lower first molars in the control group (a–
h), and the 100-CY group (i–p). DP dental pulp, AB alveolar bone, C cementum. Scale bar = 100 lm. Original magnification 94.2
dental disturbances such as short-root tooth and microdontia [6–10, 17–19]. The present study shows that CY inhibits the regular formation of HERS and can cause disturbances to the developing root of the molar. CY is widely used as an antineoplastic drug in childhood cancer therapy, and a correlation between CY and dental disturbances has been reported in a clinical study [18]. To
evaluate the effects of CY on root development until occlusion completion, in this study we used CY alone. An additional reason for conducting the study was that in most clinical reports investigating the effects of cytostatic drugs on developing teeth in humans, several different drugs, sometimes in combination with irradiation, have been used [7, 10, 19]. This has made it difficult to distinguish the
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Odontology Fig. 7 Higher magnification of apical parts of the distal roots at PN14, PN20, and PN76 in the control group (a, c and e), and the 100-CY group (b, d and f). HERS Hertwig’s epithelial root sheath, DP dental pulp, OD odontoblasts, PDL periodontal ligament, C cementum. Scale bar = 100 lm. Original magnifications 940 (a–d), and 920 (e and f)
harmful effects of each component of the treatment on tooth development. Laboratory studies enable us to study the effects of different antineoplastic drugs under controlled conditions. CY is an alkylating agent, accounting for its capacity to interfere with the division of cancer cells. However, the toxicity of CY has been ascribed to a similar effect on normal proliferative tissues [13]. In addition, CY is a non-cell-cycle-specific and dose-dependent cytotoxic chemotherapy agent. The common side effects of chemotherapy include temporary hair loss, nausea, and bone marrow suppression [13]. Though the mice treated with 100 mg/kg of CY lost their hair and temporarily exhibited a slowed rate of body weight gain, these results were presumed to be side effects of the drug. The roots of growing hairs also have high metabolic and mitotic activity. It is not surprising, therefore, that CY interferes markedly with the normal functioning of these roots, as demonstrated in this study. Generally, the treatment of childhood cancer utilizes higher doses of radiation and chemotherapy than that are used to treat adults. In addition, a shorter treatment period tends to be used in children, since children can withstand higher doses over a short period of time (more intensive treatment) than adults, before strong side effects occur.
Although the 100 mg/kg concentration of CY used in this study is a higher dose than that used in low or standard risk treatment protocols for cancer treatment in children, this concentration is sometimes used in clinical therapy, such as for those in high-risk groups or extremely high-risk groups, depending on the risk or type of cancer [20]. Furthermore, it is known that the total dose of an alkylating agent is associated with its cytotoxicity. In clinical protocols, CY is sometimes administered daily. If the total dose of CY exceeds the amount compatible with normal root formation, there is a possibility of exacerbation of root malformation. Most previous studies on the effects of CY on developing teeth have involved continuously growing adult rat incisors. Those studies have shown that the effects of the drug were restricted to undifferentiated epithelial and mesenchymal cells, and that the extent of disruption of odontogenesis was related to the dose of the drug [11, 21, 22]. This model, using incisors, facilitates the observation of continuous changes in odontogenesis, but it does not incorporate monitoring of the formation of the root apex. The advantage of studying developing mouse molars is that it is possible to examine the effects of the cytostatic agent on crown and root formation, and the effects can be followed for a long period.
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Based on a small number of CY studies performed using rat molars, Na¨sman et al. [14, 15] reported that CY reduced the root length in first and second molars, and reduced the size of the third molar. These changes indicate that the effects of CY are related to the developmental stage of the teeth [14, 15]. The mice in our study were injected with CY at PN12, when the crowns of the first molars were fully formed; the roots of the first molars had already divided into two, and each mesial and distal root had formed. The R/C ratio results in this study suggest that CY inhibits molar root development in a dose-dependent manner, and the roots in the 100-CY group elongated little from PN16 to PN76. Shorter root length in the 100-CY group was also apparent in the HE-stained sections. We surmised that this reduction in root length may be caused by cessation of the downward growth of the epithelial root sheath, and performed immunohistochemical staining to investigate the morphology of HERS. HERS was observed as a two-layer structure of cytokeratinpositive cells at the apical region, at PN12 (data not shown). With regard to chronological observations, the HERS in the 100-CY group had already changed shape by PN14, and subsequently broke up and disappeared early, as compared to the control group. The severity of the disturbances induced by CY was positively associated with dose. Tooth development requires complex interactions between the epithelium of the enamel organ and the mesenchyme of the dental papilla, known as epithelial– mesenchymal interactions. Tooth crown formation precedes root development through epithelial–mesenchymal interactions; then, root development begins under the control of HERS. The role of HERS cells in root formation is widely accepted, including the size, shape, and number of roots [23]. Various growth factors and cytokines regulate the elongation and proliferation of HERS, including fibroblast growth factor-2 [24], sonic hedgehog [25], and insulin-like growth factor [26]. Inhibitory effects of CY on HERS development, which requires active proliferation of cells, would disturb the regulatory signaling involved in root formation. This result could explain why the loss of HERS changes the morphology of roots at an early stage, disrupting their development, such that the tooth root may not be elongated. Not only did the roots of these teeth fail to reach normal lengths, but closing of the apices also occurred. Anton [27] showed that non-dividing columnar odontoblasts and ameloblasts were not affected by 300 mg/kg CY administered as a single injection in the rat incisor. The early closing of the apices in the 100-CY group might have been caused by the formation of odontogenesis by the lessaffected odontoblasts in this area. It may be that V-shaped roots were built up by this continuous odontogenesis, in spite of the cessation of root elongation.
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Interestingly, in the 100-CY group cementum began to be deposited on the root surface immediately after HERS broke up, and the amount of cementum remained greater than that of the control group up to PN76. This was one of the reasons for the low increase in R/C ratio in the 100-CY group. Although the different functions attributed to HERS affect induction and regulation of root formation, the fate of HERS and the precise function of it are debatable [28]. Diekwisch [29] reported that HERS disintegrated prior to any cementum deposition, thus HERS might play a role in regulating cementum deposition. In this study, cementogenesis in the 100-CY group started earlier than it did in the control group. The early rupture and loss of HERS might be involved in the early formation of cementum. However, cementum accumulation after the completion of occlusion may be another possible reason; it may facilitate avoidance of harmful movement resulting from the short V-shaped root. Though the network of HERS and surrounding cells is crucial for tooth root development and the modeling of periodontal tissue, the mechanism of cementogenesis at an early stage observed in this study remains uncertain. Reduction in root size, and deviations from normal root morphology have also been reported in young humans treated with CY for different types of cancer [6, 7, 10, 17, 18]. Several studies have shown that such patients had significantly reduced root sizes in comparison to healthy children [8, 9, 19, 30, 31]. With regard to morphology, arrested root development resulting in short V-shaped roots and apical closure were relatively common in the CYtreated children [18, 32]. It is reasonable to assume that these abnormalities are due to disturbances in cell activity similar to those seen in the mice given CY in the present study; however, there are numerous potential causes of disturbances in the tooth development of long-term survivors of malignant diseases that may alter growth. Further studies should elucidate dose, age, and time-related effects of anticancer treatment on dental development, since many questions still remain. Dental development in humans and mice cannot be directly compared, nevertheless, the present results indicate that administration of CY may explain some of the effects seen in human teeth after CY therapy. Considering that the numbers of long-term survivors of childhood cancer will increase steadily in the future, an oral care program to prevent periodontal disease is important, given the irreversible short tooth roots of these survivors. In conclusion, this study indicates that CY can induce a defect in HERS, and that it causes early loss of HERS. Since HERS plays an important role in tooth root formation and periodontal tissue modeling, disruption of the epithelial sheath inhibits normal root formation, and this may cause irreversible short-root development.
Odontology Acknowledgments This work was supported by Grants-in-Aid for Scientific Research (C) 20592416 and (C) 23593048 from the Japan Society for the Promotion of Science, Tokyo, Japan. Conflict of interest related to this study.
The authors report no conflicts of interest
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15. Na¨sman M, Hultenby K, Forsberg CM. A scanning electron microscopy study of disturbances in the developing rat molar induced by cyclophosphamide. Acta Odontol Scand. 1997;55:186–91. 16. Lind V. Short root anomaly. Scand J Dent Res. 1972;80:85–93. 17. Pedersen LB, Clausen N, Schroder H, Schmidt M, Poulsen S. Microdontia and hypodontia of premolars and permanent molars in childhood cancer survivors after chemotherapy. Int J Paediatr Dent. 2012;22:239–43. 18. Hsieh SG, Hibbert S, Shaw P, Ahern V, Arora M. Association of cyclophosphamide use with dental developmental defects and salivary gland dysfunction in recipients of childhood antineoplastic therapy. Cancer. 2011;117:2219–27. 19. Ho¨ltta P, Hovi L, Saarinen-Pihkala UM, Peltola J, Alaluusua S. Disturbed root development of permanent teeth after pediatric stem cell transplantation. Dental root development after SCT. Cancer. 2005;103:1484–93. 20. Ishida Y, Maeda M, Urayama KY, Kiyotani C, Aoki Y, Kato Y, Goto S, Sakaguchi S, Sugita K, Tokuyama M, Nakadate N, Ishii E, Tsuchida M, Ohara A, The QOLcoTCsCSG. Secondary cancers among children with acute lymphoblastic leukaemia treated by the Tokyo Children’s Cancer Study Group protocols: a retrospective cohort study. Br J Haematol. 2014;164:101–12. 21. Adatia AK. The effects of cyclophosphamide on odontogenesis in the rat. Arch Oral Biol. 1975;20:141–4. 22. Adatia AK. Cytotoxicity of cyclophosphamide in the rat incisor. Br J Cancer. 1975;32:208–18. 23. Ten Cate AR. The role of epithelium in the development, structure and function of the tissues of tooth support. Oral Dis. 1996;2:55–62. 24. Madan AK, Kramer B. Immunolocalization of fibroblast growth factor-2 (FGF-2) in the developing root and supporting structures of the murine tooth. J Mol Histol. 2005;36:171–8. 25. Nakatomi M, Morita I, Eto K, Ota MS. Sonic hedgehog signaling is important in tooth root development. J Dent Res. 2006;85:427–31. 26. Fujiwara N, Tabata MJ, Endoh M, Ishizeki K, Nawa T. Insulinlike growth factor-I stimulates cell proliferation in the outer layer of Hertwig’s epithelial root sheath and elongation of the tooth root in mouse molars in vitro. Cell Tissue Res. 2005;320:69–75. 27. Anton E. Ultrastructural study of the effect of cyclophosphamide on the growth area of incisor teeth of DBA/2 and C57BL/6 mice. Int J Exp Pathol. 1996;77:83–8. 28. Thomas HF. Root formation. Int J Dev Biol. 1995;39:231–7. 29. Diekwisch TG. The developmental biology of cementum. Int J Dev Biol. 2001;45:695–706. 30. Na¨sman M, Forsberg CM, Dahllo¨f G. Long-term dental development in children after treatment for malignant disease. Eur J Orthod. 1997;19:151–9. 31. Avsar A, Elli M, Darka O, Pinarli G. Long-term effects of chemotherapy on caries formation, dental development, and salivary factors in childhood cancer survivors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;104:781–9. 32. Kaste SC, Goodman P, Leisenring W, Stovall M, Hayashi RJ, Yeazel M, Beiraghi S, Hudson MM, Sklar CA, Robison LL, Baker KS. Impact of radiation and chemotherapy on risk of dental abnormalities: a report from the Childhood Cancer Survivor Study. Cancer. 2009;115:5817–27.
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ORIGINAL ARTICLE
Cyclophosphamide inhibits root development of molar teeth in growing mice Tomomi Kawakami • Yuko Nakamura Hiroyuki Karibe
•
Received: 27 November 2013 / Accepted: 23 April 2014 Ó The Society of The Nippon Dental University 2014
Abstract Root development of permanent teeth is disturbed in survivors of childhood cancer. Cyclophosphamide (CY) is a cytostatic drug commonly used for chemotherapy in children with cancer. This study aimed to evaluate the effects of CY on the development of molar teeth until the completion of occlusion in young mice, focusing on Hertwig’s epithelial root sheath (HERS). We treated thirty-two 12-day-old ICR mice with CY (100 mg/ kg; 100-CY group), and 36 control mice with saline. At 12, 14, 16, 20, 24, 27, 39, 60, and 76 days of age, the mandibular molars were removed. Soft X-ray radiographs were obtained in lateral projection. The root/crown length (R/ C) ratio of the first molar was calculated. Serial sagittal sections were prepared and histomorphological hematoxylin and eosin (HE) staining and immunohistochemical (cytokeratin) studies were performed. The R/C ratio of the 100-CY group (0.78) was smaller than that of the control group (1.23) at 76 days (p \ 0.05, t test). While all roots developed further after injection, microscopic examination showed that the roots of the first molars that developed in the 100-CY group were shorter than those in the control group. In addition, experimental mice showed apical closure of the roots. By 20 days after injection, the HERS had disappeared from the root surface in the 100-CY group. In conclusion, this study indicates that CY can induce a defect in HERS and cause early loss of HERS. Disruption of the epithelial sheath inhibits normal root formation, and it could cause irreversible short-root development.
T. Kawakami (&) Y. Nakamura H. Karibe Department of Pediatric Dentistry, The Nippon Dental University School of Life Dentistry at Tokyo, 1-9-20, Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan e-mail: [email protected]
Keywords Root development Cyclophosphamide Mice Hertwig’s epithelial root sheath Late effects
Introduction In recent decades, improvements in therapies for childhood cancers have increased the number of survivors. The treatments used for childhood cancer include chemotherapy, irradiation, surgery, stem cell transplantation, and a combination of these modalities, and these treatments are becoming increasingly effective. The survival rates of childhood cancers are reportedly 70 to 75 % in some parts of Europe and North America [1]. The survival rate for children with cancer in Japan is now approximately 80 %, and more than 0.1 % of young adults are survivors of childhood cancer [2]. However, as the survival rate of children with cancer has improved considerably, the late effects of antineoplastic therapy have increased in importance. Concern has been raised about the long-term adverse health effects in several areas including learning, motor function development, fertility, cardiac and renal function, vision, hearing, and the risk of secondary cancers [2–4]. It has been estimated that in Japan, approximately two-thirds of childhood cancer survivors will have at least one late effect [2]. Late effects involving dental disturbances have been reported in a group of patients treated with cancer therapy [5–8]. Adverse effects of cancer and cancer therapy during childhood on dental health have been reported in terms of mineralization disturbances, dental caries, microdontia, hypodontia, root stunting, and taurodontism [6, 7]. For many pediatric cancer patients, since some of the dental sequelae of chemotherapy and radiation are irreversible, oral sequelae and discomfort related to their treatment can
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have long-term and potentially lethal consequences. Some investigators have suggested that children treated with chemotherapy in the early years of their lives exhibit a high incidence of dental defects, and immature teeth are at greater risk of dental abnormalities than fully developed teeth [6, 9]. Dental disturbances can result from chemotherapy, as well as radiation therapy. Chemotherapy in children treated for neuroblastoma can adversely influence tooth development [10]. Experimental studies have shown adverse effects of the frequently used chemotherapeutic agents cyclophosphamide (CY) and doxorubicin on developing teeth [11, 12]. CY is one of the cytostatic drugs present in chemotherapy cocktails that are commonly used to treat childhood cancers. CY targets all cells by cross-linking DNA with its bioactive metabolite, phosphoramide mustard [13]. Though CY is an alkylating agent that is widely used as an antineoplastic drug owing to its capacity to interfere with the division of cancer cells, its toxicity has been ascribed to the fact that it has a similar effect on normal proliferative tissues and cells. While some studies have investigated the effects of CY on tooth formation, mostly rodent incisors have been used. It is difficult to observe these effects until root formation of the apex in this context, because in rodents the incisors grow continuously. Only few studies have investigated the root formation of molars [14, 15], but their observations were made over a limited period, leaving many aspects of the effects of CY unclear. It is well known that tooth root formation is initiated by the development of Hertwig’s epithelial root sheath (HERS). However, relatively little is known about the direct effect of CY on HERS and root development up to completion of the root apex in mouse molars. In the present study, as a first step for clarifying the continuous changes in CY-induced root development up to and after the completion of root development, histopathological examinations were carried out on lower first molars exposed to CY in young mice, focusing on the changes in HERS.
Materials and methods Animals A total of 83 ICR mice (Charles River Japan Inc., Yokohama, Japan) were used. The mice were fed free from their mothers, and allowed free access to standard solid mouse feed and distilled water ad libitum after weaning. All mice were housed under conventional conditions with controlled temperature (24 ± 1 °C), humidity (50 ± 10 %), and light (12-h light/12-h dark).
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All animal experiments were approved by the Ethics Committee for Animal Experimentation and conducted in compliance with the Guidelines of the Nippon Dental University, School of Life Dentistry at Tokyo, Section of Biological Sciences, Research Center for Odontology. Study design CY (EndoxanÒ, Shionogi & Co., Ltd., Tokyo, Japan) dissolved in 200 lL of saline was administered to 12-day-old (postnatal; PN12) mice intraperitoneally (i.p.). To investigate the effects of CY, two different CY treatments were designed. To study the dose effects of CY, 15 mice were randomly divided into five groups. Equal numbers of mice (n = 3) were administered i.p. injections of CY at doses of 30, 50, 100, or 200 mg/kg of body weight (indicated as 30-CY, 50-CY, 100-CY, or 200-CY below), at PN12. The control group received injections of an equal volume of saline. The mice were killed at 20 days of age (PN20). The administered doses of CY did not result in the death of any of the mice. In the second experiment, 68 mice were used to investigate the chronological changes in molar development induced by CY, and they were randomly divided into two groups; one group was given a single injection of 100 mg/ kg of CY, and the other group (control) was given saline, on PN12. They were then killed on PN12 (control group only), PN14, PN16, PN20, PN24, PN27, PN39, PN60, and PN76. Indicators of the general condition of the mice in all groups, including body weight and behavior, were monitored once a day throughout the experimental period. Radiographic evaluation On the designated days after drug injection, the mice were anesthetized using sodium pentobarbital (NembutalÒ, Dainihonseiyaku, Tokyo, Japan), and transcardially perfused with 4 % paraformaldehyde. After fixation, the skull was laterally separated along the epicranial midline suture from the parietal bone over the mandible. Soft X-ray radiographs (M-100 W, SOFTEX, Tokyo, Japan) were obtained in lateral projection, under the following conditions: tube voltage, 40 kVp; tube current, 3 mA; acquisition time, 20 s; and focus-subject distance, 25 cm. The acquired film images were scanned and entered into a personal computer. The soft X-ray radiographs were printed on photo paper and compared against the original radiographs to ensure that no distortion had occurred during printing. A previously described method was adapted for the measurement of the distal root length of the first molars [16]. Point ‘‘m’’ was defined as the midpoint of a straight
Odontology
Statistical analysis Results are expressed as mean ± standard deviation (SD). The data were analyzed by a one-way analysis of variance (ANOVA), and when a significant F ratio was identified, groups were compared using Tukey’s post hoc test. Student’s t test was used to compare the differences between groups, and p \ 0.05 was deemed to indicate statistical significance. All statistical analyses were carried out using the statistical package SPSS, version 17.0 J (SPSS, Inc., Tokyo, Japan). Fig. 1 The points used for measuring crown height and root length in the assessment of the root–crown (R/C) ratio. C crown height, R root length, m midpoint, a apical reference line
Results The dose effects of CY on HERS
line connecting the points of intersection between the outer contours of the root and crown in all first molars (Fig. 1). Crown height (C) was the perpendicular line from point m to an occlusal reference line placed to connect the buccal cusps of the first molar. Root length (R) was measured from point m perpendicular to the apical tangent of the distal root, parallel to the occlusal reference line. The R/C ratio of individual tooth was calculated by dividing root length by crown height. Histological evaluation Hematoxylin and eosin staining Histological changes in the periodontal tissues were assessed via hematoxylin and eosin (HE) staining. The mandible of each mouse was dissected and immediately immersed in 4 % paraformaldehyde. Mandibular specimens were decalcified with 10 % EDTA at 4 °C. The specimens were paraffin embedded, and serial sections (4lm thick) were cut on the sagittal plane with a rotary microtome (RM2065, Leica, Wetzlar, Germany). Immunohistochemistry Tissue sections were deparaffinized with xylene, and rehydrated with decreasing concentrations of ethanol. Following rehydration, antigen retrieval was conducted via 0.1 % pepsin treatment for 10 min at 37 °C with 10 mL HCl. The antibodies used were polyclonal rabbit antihuman cytokeratin pan (Progen Biotechnik, Heidelberg, Germany; 1:1000 dilution). Immunoreactivity was visualized using 3,30 -diaminobenzidine (DAB) (SK-4100, Funakoshi, Tokyo, Japan) coloring with the ABC method (ABC Elite kit, Vector Laboratories, Inc., Burlingame, CA). Nuclei were lightly counterstained with 1 % methyl green.
Because HERS is formed at the initiation of tooth root development and plays an important role in determining root shape and size, we examined the effects of various doses of CY on HERS, via immunostaining analysis (Fig. 2). The analysis showed that HERS expressed cytokeratin, which is a marker of epithelium. The HERS in the control group could be seen in the lower first molar germs at PN20, and it still had a double-layered structure. The HERS of experimental groups treated with 30 or 50 mg/kg of CY appeared similar to that of the control group, but the HERS of mice treated with 100 or 200 mg/ kg of CY disappeared in a concentration-dependent manner. Table 1 shows the crown height and the distal root length of the first molars treated with different doses of CY. While the mean crown heights did not change, the mean root lengths decreased in a dose-dependent manner. Furthermore, with regard to comparison of the R/C ratio of the first molars, the R/C ratios decreased in a dosedependent manner, and there were significant differences between the control group and the high-dose CY group (Fig. 2f). These findings indicated that at high doses such as 100 or 200 mg/kg, a single injection of CY had a cytotoxic effect on HERS. The effects of CY on chronological changes A dose of 100 mg/kg of CY was used to investigate chronological CY-induced changes in root formation in lower first molars. The weight change of the two groups up to PN76 is shown in Fig. 3. All mice gained weight at the end of drug administration, though a reduction in the rate of weight gain was observed in the 100-CY group during the first 2 weeks after the CY injection. There were significant differences in weight at PN20 and PN24, but there were no significant differences in weight at any of the other time-points investigated.
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Odontology Fig. 2 The effects of different doses of CY on root development of the first molars at PN20. Micrographs of sections immunostained with anti-cytokeratin (a–e). HERS Hertwig’s epithelial root sheath, DP dental pulp, PDL periodontal ligament. Scale bar = 100 lm. Original magnification 920. The R/ C ratio of different doses of CY at PN20 (F). * p \ 0.01, p \ 0.05. Intergroup comparisons were performed using Tukey’s test
Table 1 The crown height and the root length (lm) of the first molars after treatment with different doses of CY PN20 Crown height
Root length
Mean
SD
Mean
SD
Control
1119
71
935
57
30-CY
1034
71
836
97
50-CY 100-CY
1048 1091
120 85
822 694
40 97
200-CY
935
40
453
80
Four days after the injection of 100-CY, the experimental mice began to lose fur. After another 4 days, they had lost all of their fur. However, their fur began to regrow from PN24, and was restored by PN27. The fur of the mice in the control group was unaffected. The effects of CY on the R/C ratios of the first molars The mean crown heights and root lengths are shown in Table 2, and the chronological changes in R/C ratio are shown in Fig. 4. Mean crown height did not change from PN12 to PN76, in either group. However, while mean root length in the 100-CY group did not extend much, there was an increase in the control group. The R/C ratio in the
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Fig. 3 Body weights of experimental and control mice from PN12 to PN76. The results are expressed as mean ± SD. *Statistically significant difference compared with the control (t test, p \ 0.05)
control group increased from PN12 to PN76 (Fig. 4). Conversely, the R/C ratio in the 100-CY group increased slightly up to PN20 and then remained almost unchanged. The R/C ratios in both groups increased significantly. In contrast, there were no significant differences between the mean R/C ratios of the control group and the 100-CY group until PN16. From PN20 to PN76, however, the mean R/ C ratio in the 100-CY group was lower than that of the
Odontology Table 2 The crown height and the root length (lm) of the first molars in the 100-CY group Control
100-CY
Crown height
Root length
Crown height
Root length
Mean
SD
Mean
SD
Mean
SD
Mean
SD
PN12
1105
57
269
28
PN14
1063
28
439
28
1091
54
439
28
PN16
1119
28
646
54
1176
71
609
85
PN20
1119
71
935
57
1091
85
694
97
PN24
1176
54
1162
73
1091
54
737
40
PN27
1162
150
1165
200
992
98
680
93
PN39
935
109
1048
170
963
46
652
73
PN60
949
54
1105
143
1006
73
680
143
PN76
1020
80
1247
122
1077
80
836
54
Fig. 4 The R/C ratio of the first molars. The results are expressed as mean ± SD. *Statistically significant difference compared with the control (t test, p \ 0.05)
control group, and there were significant differences between the two groups during that period. This result indicated that CY had inhibited root formation. Histological evaluation of the first molar after treatment Figure 5 shows the chronological changes of the effects of CY on HERS with anti-cytokeratin antibodies. In the control group, HERS formed a double layer of cells comprised of inner enamel epithelium and outer enamel epithelium that extended in an apical direction at PN14 and became slightly thinner at PN16 (Fig. 5a, b). At PN20, the length of the HERS became progressively shorter (Fig. 5c), and the sheet-like structure of HERS was broken at PN24 (Fig. 5d) and it began to detach from the root surface (Fig. 5e). Conversely, the HERS in the 100-CY group did
not exhibit a double-layered structure at PN14, though a sheet-like structure was discernible (Fig. 5f). HERS began to break at PN16, and had separated from the root (Fig. 5g). HERS had disappeared completely by PN20, but the cytokeratin-positive cells scattered on the root surface were only observed as epithelial rests of Malassez (Fig. 5h–j). The HERS in the experimental group changed shape and was gone by PN16. This state was similar to the HERS of the control group at PN27. From PN39 onwards, no cytokeratin-positive cells resembling the HERS were observed in either group (data not shown). To observe the state of root formation and periodontal tissue, we examined HE-stained sections of the developing root of the mandibular first molars from PN12 to PN76. Fig. 6 shows histological changes in distal root development chronologically, and Fig. 7 shows higher magnification of the apical part of the distal root. At PN12, just before CY administration, root formation had completed furcation, and progressed to approximately half of the root of the first molar, but the apical area was still wide open and the first molar had not yet erupted (data not shown). The distal root length of the first molars clearly extended until PN27 in the control group (Fig. 6a–h). Conversely, while the distal root length of the first molars in the 100-CY group did extend slightly until PN16, at that point it almost ceased (Fig. 6i–p). In both the control and the 100-CY group, at PN14 the first molars had not erupted into the oral cavity, and their apical portions were wide open (Fig. 6a, i). Cervical loop structure was present in both control and experimental groups, but in the 100-CY group the cytoplasm of the odontoblasts had begun to atrophy as compared to the control group (Fig. 7a, b). At PN16, eruption of the first molar had commenced in the control group (Fig. 6b). Conversely, half of each first molar in the 100-CY group was covered with epithelium and had not erupted (Fig. 6j). In the 100-CY group, the cervical loop of the apex could not be discerned and the apical portions observed were smaller as compared to those of the control group. Cementum addition was apparent from PN20 in the 100-CY group, although the root length of the first molar changed little after PN16 (Fig. 7d). In the control group, cementum addition was observed from PN24. In addition, from PN27 until PN76, cementum addition in the 100-CY group increased and was formed not only around the apical area but also the middle part of the root (Fig. 6p, f).
Discussion On the basis of long-term follow-up studies, it has been reported that long-term survivors who received chemotherapy treatment in childhood have an increased risk of
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Odontology Fig. 5 Immunohistochemical staining for cytokeratin in HERS of mandibular first molars at various time-periods in the control group (a–e), and the 100-CY group (f–j). HERS Hertwig’s epithelial root sheath, DP dental pulp, OD odontoblasts, PDL periodontal ligament, ERM epithelial rests of Malassez. Scale bar = 100 lm. Original magnification 940
Fig. 6 Development of the distal roots of mandibular first molars from PN14 to PN76. Hematoxylin and eosin staining of sections showing the distal root of lower first molars in the control group (a–
h), and the 100-CY group (i–p). DP dental pulp, AB alveolar bone, C cementum. Scale bar = 100 lm. Original magnification 94.2
dental disturbances such as short-root tooth and microdontia [6–10, 17–19]. The present study shows that CY inhibits the regular formation of HERS and can cause disturbances to the developing root of the molar. CY is widely used as an antineoplastic drug in childhood cancer therapy, and a correlation between CY and dental disturbances has been reported in a clinical study [18]. To
evaluate the effects of CY on root development until occlusion completion, in this study we used CY alone. An additional reason for conducting the study was that in most clinical reports investigating the effects of cytostatic drugs on developing teeth in humans, several different drugs, sometimes in combination with irradiation, have been used [7, 10, 19]. This has made it difficult to distinguish the
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Odontology Fig. 7 Higher magnification of apical parts of the distal roots at PN14, PN20, and PN76 in the control group (a, c and e), and the 100-CY group (b, d and f). HERS Hertwig’s epithelial root sheath, DP dental pulp, OD odontoblasts, PDL periodontal ligament, C cementum. Scale bar = 100 lm. Original magnifications 940 (a–d), and 920 (e and f)
harmful effects of each component of the treatment on tooth development. Laboratory studies enable us to study the effects of different antineoplastic drugs under controlled conditions. CY is an alkylating agent, accounting for its capacity to interfere with the division of cancer cells. However, the toxicity of CY has been ascribed to a similar effect on normal proliferative tissues [13]. In addition, CY is a non-cell-cycle-specific and dose-dependent cytotoxic chemotherapy agent. The common side effects of chemotherapy include temporary hair loss, nausea, and bone marrow suppression [13]. Though the mice treated with 100 mg/kg of CY lost their hair and temporarily exhibited a slowed rate of body weight gain, these results were presumed to be side effects of the drug. The roots of growing hairs also have high metabolic and mitotic activity. It is not surprising, therefore, that CY interferes markedly with the normal functioning of these roots, as demonstrated in this study. Generally, the treatment of childhood cancer utilizes higher doses of radiation and chemotherapy than that are used to treat adults. In addition, a shorter treatment period tends to be used in children, since children can withstand higher doses over a short period of time (more intensive treatment) than adults, before strong side effects occur.
Although the 100 mg/kg concentration of CY used in this study is a higher dose than that used in low or standard risk treatment protocols for cancer treatment in children, this concentration is sometimes used in clinical therapy, such as for those in high-risk groups or extremely high-risk groups, depending on the risk or type of cancer [20]. Furthermore, it is known that the total dose of an alkylating agent is associated with its cytotoxicity. In clinical protocols, CY is sometimes administered daily. If the total dose of CY exceeds the amount compatible with normal root formation, there is a possibility of exacerbation of root malformation. Most previous studies on the effects of CY on developing teeth have involved continuously growing adult rat incisors. Those studies have shown that the effects of the drug were restricted to undifferentiated epithelial and mesenchymal cells, and that the extent of disruption of odontogenesis was related to the dose of the drug [11, 21, 22]. This model, using incisors, facilitates the observation of continuous changes in odontogenesis, but it does not incorporate monitoring of the formation of the root apex. The advantage of studying developing mouse molars is that it is possible to examine the effects of the cytostatic agent on crown and root formation, and the effects can be followed for a long period.
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Based on a small number of CY studies performed using rat molars, Na¨sman et al. [14, 15] reported that CY reduced the root length in first and second molars, and reduced the size of the third molar. These changes indicate that the effects of CY are related to the developmental stage of the teeth [14, 15]. The mice in our study were injected with CY at PN12, when the crowns of the first molars were fully formed; the roots of the first molars had already divided into two, and each mesial and distal root had formed. The R/C ratio results in this study suggest that CY inhibits molar root development in a dose-dependent manner, and the roots in the 100-CY group elongated little from PN16 to PN76. Shorter root length in the 100-CY group was also apparent in the HE-stained sections. We surmised that this reduction in root length may be caused by cessation of the downward growth of the epithelial root sheath, and performed immunohistochemical staining to investigate the morphology of HERS. HERS was observed as a two-layer structure of cytokeratinpositive cells at the apical region, at PN12 (data not shown). With regard to chronological observations, the HERS in the 100-CY group had already changed shape by PN14, and subsequently broke up and disappeared early, as compared to the control group. The severity of the disturbances induced by CY was positively associated with dose. Tooth development requires complex interactions between the epithelium of the enamel organ and the mesenchyme of the dental papilla, known as epithelial– mesenchymal interactions. Tooth crown formation precedes root development through epithelial–mesenchymal interactions; then, root development begins under the control of HERS. The role of HERS cells in root formation is widely accepted, including the size, shape, and number of roots [23]. Various growth factors and cytokines regulate the elongation and proliferation of HERS, including fibroblast growth factor-2 [24], sonic hedgehog [25], and insulin-like growth factor [26]. Inhibitory effects of CY on HERS development, which requires active proliferation of cells, would disturb the regulatory signaling involved in root formation. This result could explain why the loss of HERS changes the morphology of roots at an early stage, disrupting their development, such that the tooth root may not be elongated. Not only did the roots of these teeth fail to reach normal lengths, but closing of the apices also occurred. Anton [27] showed that non-dividing columnar odontoblasts and ameloblasts were not affected by 300 mg/kg CY administered as a single injection in the rat incisor. The early closing of the apices in the 100-CY group might have been caused by the formation of odontogenesis by the lessaffected odontoblasts in this area. It may be that V-shaped roots were built up by this continuous odontogenesis, in spite of the cessation of root elongation.
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Interestingly, in the 100-CY group cementum began to be deposited on the root surface immediately after HERS broke up, and the amount of cementum remained greater than that of the control group up to PN76. This was one of the reasons for the low increase in R/C ratio in the 100-CY group. Although the different functions attributed to HERS affect induction and regulation of root formation, the fate of HERS and the precise function of it are debatable [28]. Diekwisch [29] reported that HERS disintegrated prior to any cementum deposition, thus HERS might play a role in regulating cementum deposition. In this study, cementogenesis in the 100-CY group started earlier than it did in the control group. The early rupture and loss of HERS might be involved in the early formation of cementum. However, cementum accumulation after the completion of occlusion may be another possible reason; it may facilitate avoidance of harmful movement resulting from the short V-shaped root. Though the network of HERS and surrounding cells is crucial for tooth root development and the modeling of periodontal tissue, the mechanism of cementogenesis at an early stage observed in this study remains uncertain. Reduction in root size, and deviations from normal root morphology have also been reported in young humans treated with CY for different types of cancer [6, 7, 10, 17, 18]. Several studies have shown that such patients had significantly reduced root sizes in comparison to healthy children [8, 9, 19, 30, 31]. With regard to morphology, arrested root development resulting in short V-shaped roots and apical closure were relatively common in the CYtreated children [18, 32]. It is reasonable to assume that these abnormalities are due to disturbances in cell activity similar to those seen in the mice given CY in the present study; however, there are numerous potential causes of disturbances in the tooth development of long-term survivors of malignant diseases that may alter growth. Further studies should elucidate dose, age, and time-related effects of anticancer treatment on dental development, since many questions still remain. Dental development in humans and mice cannot be directly compared, nevertheless, the present results indicate that administration of CY may explain some of the effects seen in human teeth after CY therapy. Considering that the numbers of long-term survivors of childhood cancer will increase steadily in the future, an oral care program to prevent periodontal disease is important, given the irreversible short tooth roots of these survivors. In conclusion, this study indicates that CY can induce a defect in HERS, and that it causes early loss of HERS. Since HERS plays an important role in tooth root formation and periodontal tissue modeling, disruption of the epithelial sheath inhibits normal root formation, and this may cause irreversible short-root development.
Odontology Acknowledgments This work was supported by Grants-in-Aid for Scientific Research (C) 20592416 and (C) 23593048 from the Japan Society for the Promotion of Science, Tokyo, Japan. Conflict of interest related to this study.
The authors report no conflicts of interest
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