archives of oral biology 59 (2014) 1242–1248
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Effect of radiotherapy on the eruption rate and morphology of the odontogenic region of rat incisors Amanda Maria Medeiros de Araujo a,*, Carolina Cintra Gomes b, Solange Maria de Almeida b, Carla Beatriz Klamt b, Pedro Duarte Novaes c a
Department of Clinic and Social Dentistry, School of Dentistry, Federal University of Paraı´ba, Joa˜o Pessoa, Paraı´ba, Brazil b Department of Oral Diagnosis, Division of Oral Radiology, Piracicaba Dental School, State University of Campinas, Piracicaba, Sa˜o Paulo, Brazil c Department of Morphology, Piracicaba Dental School, State University of Campinas, Piracicaba, Sa˜o Paulo, Brazil
article info
abstract
Article history:
Objective: The goal in this study was to evaluate the results of doses of 5 and 15 Gy of
Received 6 November 2013
radiation in odontogenic region of the rats inferior mandibular-incisors by a histological
Received in revised form
analysis and the rate of eruptions.
26 June 2014
Design: Animals were divided into three groups: control, radiotherapy 5 Gy and radiothera-
Accepted 13 July 2014
py 15 Gy. In which tooth-eruption-rate was measured every two days.
Available online
Results: Animals in Group 5 Gy presented values similar to those of the control group. Animals in Group 15 Gy presented reduction in tooth-eruption-rate as of the sixth day of the
Keywords:
experiment, vast disorganization of odontoblasts and ameloblasts, apparent reduction in
Tooth eruption
cell population in the follicle region and alterations in cervical loop formation of the dental
Odontogenesis
organ.
Ionizing radiation
Conclusions: It was concluded that there was a difference between the researched doses, and histological alteration at 15 Gy lead to statistical reduction in tooth-eruption-rate. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Cancer is a set of diseases that have in common the disorderly growth of cells that invade tissues and organs and may spread by metastasis to other regions. Paediatric tumours account for approximately 2.5% of the total number of tumours expected
among Brazilians.1 In the United States, cancer in childhood occurs in fewer than 2% of cases.2 Various modalities of therapies have been applied in children with cancer, which are associated with toxic effects resulting in complications.3 Radiotherapy is widely used as an efficient form of treatment, either alone or associated with chemotherapy and/or surgery.4 There is concern due to the
* Corresponding author at: Rua Joakim Schuller, 40/Edf Oasis Plaza – Apt 304, 58.037-760 Bairro Jardim Oceania, Joa˜o Pessoa, Paraı´ba, Brazil. Fax: +55 8332167409. E-mail addresses: [email protected], [email protected] (A.M.M. de Araujo). http://dx.doi.org/10.1016/j.archoralbio.2014.07.004 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
archives of oral biology 59 (2014) 1242–1248
high percentage of acute or delayed oral complications that may occur during and/or after radiotherapy treatment.5 In cancer of the head and neck, the applicability and efficacy of radiotherapy is determined by parameters such as the histological type of the neoplasia (slow rapid response), location and volume of the area to be irradiated, dose, rhythm of application, type of radiation and the patient’s general conditions.6 The most frequent oral complications in patients submitted to radiotherapy in the region of the head and neck and/or chemotherapy are haemorrhage, infection, exacerbation of pre-existent infection, mucositis, xerostomia, radiation caries and interruption in tooth development. They lead to alterations in odontogenesis3 such as enamel hypoplasia, cessation of tooth development, non formation of teeth,7,8 microdontia and alterations in rhizogenesis, such as interruption, thinning and widening of the pulp chamber.9,10 The effect of radiation on permanent teeth and its degree of severity depend on the stage of tooth formation, as well as the dose and extent of the irradiated area. Innumerable dental alterations may occur in children submitted to antineoplastic treatment, capable of affecting shape, number or root formation, such as hypoplasia, microdontia, hypodontia, taurodontia, conical roots, among other.8 Odontogenesis is the stage of tooth development that involves morphogenesis, histogenesis and cell differentiation. The consecutive stages of tooth development are the bud, cap and bell stages.11,12 Tooth development is a complex process and it can undergo various alterations during its formation.12 Rat incisors have an odontogenic region that presents cells with elevated metabolism and constant differentiation. This region has a compartment of stem cells and one of proliferative cells with innumerable molecules involved in kinetic processes such as cell proliferation, migration, differentiation and cell death.13,14 In the epithelium of the cervical loop intense cell proliferation occurs, from which cells migrate and differentiate into ameloblastic and odontoblastic cells.9 The growth, calcification and eruption of rat incisors occur continuously throughout life, showing all the stages of formation in a single tooth, and is therefore a model for evaluating the effect of X radiation on progenitor cells.9 Few studies have been conducted to evaluate the effects of radiation on the in the stage of development. As there is a scarcity in the literature of studies on the irradiated odontogenic region, it is important to evaluate the effect of radiotherapy on the odontogenic region of rat incisors. The aim of this study was to evaluate the morphologic alterations caused by the action of radiation on the odontogenic region of rat incisors, with doses of 5 and 15 Gy, and find out whether these alterations may interfere in the eruption rate of these teeth.
2.
Materials and methods
Thirty adult male rats (Rattus norvegicus, Albinus Wistar), with a mean weight of 220 grams, obtained from the central vivarium of UNICAMP were used in the study. While the research was conducted, the rats were maintained in polycarbonate cages, in which they remained in an
1243
environment with controlled temperature and humidity, and an alternating 12-h light–dark cycle. The animals’ diet consisted of standard balanced rations and water ad libitum, daily. This study was approved by the Ethics Committee on Animal Experimentation of the State University of Campinas, Protocol Number 1843-1. The animals were divided into three groups: Group 1; Group 2 in which the animals were submitted to a 5 Gy dose of radiotherapy in the region of the head and neck; and Group 3 in which the animals were submitted to a 15 Gy dose of radiotherapy in the region of the head and neck. The experiment lasted 16 days, in which the tooth eruption rates were measured every two days. On the second day of the experiment, the animals were submitted to radiotherapy in the region of the head and neck. The eruption rate was measured in all the animals. To do this, a linear mark was made on the vestibular face of the mandibular incisors with cylindrical diamond bur adapted to a high speed handpiece under cooling. With the aid of a millimetre eyepiece (Ernest Leitz Wetzlar Germany 12.5) adapted to a stereoscopic loupe, and the use of a millimetre ruler for calibration (Carl Zeiss 5+ 100/100 mm), the distance from the gingival margin up to the mark was measured. To obtain the measurements, the animals were anesthetized with Halothane (Crista´lia, Brazil). When the mark approximated the incisal edge of the tooth that was being worn by the friction, a new mark was made. For better confirmation of the data, the eruption rate was verified both on the mandibular right incisor and on the left and the final daily eruption value was given by the mean of these values. The values were transformed into millimetres and statistically analyzed by ANOVA. Before all the irradiation procedures, all the animals were anesthetized with intramuscular injection of 80 mg/kg of Ketamine Hydrochloride (Dopalen1 Agribrands do Brasil Ltda.; Paulı´nia, Sa˜o Paulo, Brazil) and 8 mg/kg of Xylazine Hydrochloride (Rompum1, Bayer S.A., Sa˜o Paulo, SP, Brazil). The animals irradiated after beginning the tooth eruption rate measurement received a single dose of 5 Gy and 15 Gy of X radiation, depending on their group, from a linear accelerator, Varian brand, model Clinic 6/100 at a focal distance of 100 cm. The collimation field was 15 30 cm, so that only the head and neck region was irradiated. All the animals were sacrificed by cervical dislocation at the end of the experiment. The rat hemimandibles were removed and fixed by immersion in 10% buffered formalin. After collection, each hemimandible was divided into two parts, anterior and posterior, using a high concentration, dual faced cutting disc, measuring 400 0.1200 1/2 diamond (102 mm 0.3 mm 12.7 mm) (EXTEC Corp., Diamond Wafering Blade) and kept in 10% buffered formalin for 24 h under agitation to promote better fixation. The posterior half of each hemimandible was decalcified with 4.13% EDTA in phosphate buffer pH 7.4, for 30 days. After decalcification, the odontogenic region was separated with a scalpel from the mesial face of the third molar, dehydrated in increasing grades of ethanol solutions, diaphanized and embedded in Paraplast Plus1: tissue embedding medium (McCormickTM Scientific, Leica Biosystems St. Louis LLC, USA). Transverse cuts in relation to the long axis of the incisor were made from the beginning of the odontogenic region in
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archives of oral biology 59 (2014) 1242–1248
Fig. 1 – Disposition of the transverse histological cuts in the three selected regions (A–C).
the direction of the incisal edge of the incisor (Fig. 1), allowing the hemimandible to be divided into three parts: The region corresponding to the beginning of the odontogenic region; region B that comprises the median portion of the odontogenic region; and region C corresponding to the odontogenic region with reference to the distal face of the third molar. These were semi-serial cuts 5 mm thick, and were obtained using a Leica RM 2155 microtome (Leica – Fl?ssigkeit, German). For morphological evaluation the slides were stained with haematoxylin and eosin, mounted on Canada balsam and analyzed under a light microscope.
3.
Results
On the day of sacrifice, it was possible to observe that the rats with a single dose of 15 Gy presented burns, alopecia and weight loss (Fig. 2) when compared with the animals of the control group and those irradiated with 5 Gy. Table 1 presents the results of the mean and standard deviation of the tooth eruption rate in rats of the control, 5 Gy
Table 1 – Means and standard deviation of the values of tooth eruption rates of rat incisors in the control group and of those that received the radiotherapy dose of 5 and 15 Gy*. Control Day Day Day Day Day Day Day Day Day
0 2 4 6 8 10 12 14 16
0.52 0.50 0.55 0.54 0.54 0.55 0.56 0.55 0.56
(0.05) (0.06) (0.05) (0.04) (0.06) (0.05) (0.05) (0.03) (0.04)
Aa Aa Aa Aa Aa Aa Aa Aa Aa
5 Gy 0.54 0.50 0.56 0.53 0.52 0.55 0.50 0.51 0.50
(0.04) (0.06) (0.05) (0.07) (0.07) (0.05) (0.06) (0.08) (0.08)
15 Gy Aa Aa Aa Aa Aa Aa Aa Aa Aa
0.52 0.49 0.51 0.45 0.43 0.41 0.40 0.37 0.36
(0.05) (0.06) (0.04) (0.04) (0.05) (0.04) (0.05) (0.06) (0.05)
Fig. 2 – Mean value of the weight of rats (in grams). The arrow indicates the day on which the animals were submitted to radiotherapy.
and 15 Gy Groups*. The animals of Group 5 Gy presented similar values to those of the control, however, there were no signs of alteration in the tooth eruption rate, whereas the animals submitted to the dose of 15 Gy presented statistical difference in the tooth eruption rate as from day 6 of the experiment. Histologically, in the dental organ of the control group, an aspect was observed similar to that of a horseshoe, fully surrounded by well vascularized conjunctive tissue, called a dental follicle. The odontoblasts were shown to be well organized, polarized and integral, disposed in a palisade, constituted of a single layer of cells contouring the dental papilla, with a columnar aspect. The pre-ameloblasts were organized as high, polarized cylindrical cells and uniformly disposed dental papilla cells. The dental papilla presents undifferentiated, fusiform or star-shaped cells, with numerous cytoplasmatic prolongations (Figs. 3–5). In Group 5 Gy, the odontoblasts were shown to be similar when compared with the control group. Whereas, in Group 15 Gy it was observed that apparently there was a reduction in cell population in the region of the follicle, adjacent to the lingual face of the tooth, and irregularities in the organization of the ameloblasts, alterations in the formation of the epithelial loop in the dental organ, in the odontoblasts with the inversion of polarity and matrix in different directions leaving the dentine irregular; in addition to a visible reduction in tooth size (Figs. 3–5).
4. Aa Aa Aa Bb Bb Bbc Bbc Bc Bc
* Means followed by different letters (uppercase letters in the horizontal and lowercase in vertical) differ by ANOVA and Tukey test (0.05).
Discussion
Deleterious effects may occur as a result of radiotherapy, such as malformations in tooth rhizogenesis and odontogenesis.7 The earlier the stage, the greater are the chances of alteration in tooth formation occurring.3,9,10 Studies have proved that alterations in the odontogenic region cause a reduction in the tooth eruption rate of the incisors, by the trauma caused in the periodontium related to enamel,15 resection of the odontogenic region16,17 or removal of the vestibular portion of the odontogenic region,18 indicating that the action of an external agent, whether it is trauma,
archives of oral biology 59 (2014) 1242–1248
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Fig. 3 – Histological cuts of portion A of the odontogenic region of rat incisors. The arrow points out the alteration in the formation of the epithelial loop and the star shows a reduction in the number of cells in the follicle region adjacent to the lingual face of the tooth. In (a) the value of the bar corresponds to 100 mm, in (b) to 50 mm and in (c) to 20 mm.
resection or radiotherapy may interfere in the tooth morphology and eruption rate. Therefore, some authors have discussed the effect of radiotherapy and the occurrence of alterations in tooth formation in rats,18–21 in the premolars of beagles22 and consequences in children submitted to antineoplastic treatment3,8–10,23 and in rat incisors.20,24–26 In the present study, in addition to the morphology, the daily tooth eruption rate values were evaluated in the incisors of rats submitted to radiotherapy in the head and neck region. In this study, we chose to use the rat incisor, because this tooth has continued growth and its cells are constantly renewing.16 Two radiotherapy doses were selected, as various studies have evaluated dental alterations in animals that received low doses19,22,24,25 and others in high doses.3,20,21,25,26 As some authors found morphological or structural alterations in the teeth at low doses of radiotherapy, we decided to use a dose of 5 Gy as well. With the purpose of evaluating the initial effect of radiotherapy in the odontogenic region, a single time of
sacrifice of 14 days after radiotherapy was chose, because it is an adequate time in which to evaluate the tooth eruption rate. On the day of sacrifice it was found that the animals that were treated with the 15 Gy dose presented lower weight, burns and alopecia when compared with the control and radiotherapy 5 Gy groups. These results are in agreement with authors3,20,21,25,26 who related that the higher the dose the more side effects would be observed in the animals, such as irregular dentine formation and disorganized odontoblastic cells. It was not possible to identify significant alterations in the tooth eruption rate when the animals were submitted to a single dose of 5 Gy, however, in the 15 Gy Group a significant reduction in the tooth eruption rate was verified as from the fourth day after radiotherapy. These findings differ from those of Coady et al.,26 who, when studying a cumulative dose of 12 Gy and 24 Gy observed no statistical difference, however, these authors used a fractionated dose of radiotherapy, which causes less tissue damage when compared with single doses. The study of Niehoff et al.27 justifies the application of a single
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archives of oral biology 59 (2014) 1242–1248
Fig. 4 – Histological cuts of portion B of the odontogenic region of rat incisors. The arrowhead shows the lack of enamel production in the 15 Gy Group and the cross shows the disorganization of the odontoblasts. In (a) the value of the bar corresponds to 100 mm, in (b) to 50 mm and in (c) to 20 mm.
dose of 15 Gy comparable with a fractionated dose of 54 Gy, compatible with some antineoplastic treatments in the head and neck region in humans. The single dose of 15 Gy caused significant morphological alterations, probably inhibiting cell differentiation and consequently retarding the growth and eruption of the incisors. With the dose of 5 Gy it was observed that the odontoblasts were apparently shown to be organized and the ameloblasts were shown to be whole and appeared to continued to synthesize the enamel matrix actively, which is in disagreement with English and Tullis19 who studied the effects of radiotherapy on the morphology of swine molars in the stage of development and observed alterations in the ameloblasts in fractionated doses of 4 Gy. The results of the present study are in agreement with the finding of English et al.25 who found no significant morphological alteration in rat incisors with the dose of 5 Gy. In the teeth submitted to 15 Gy cell disorganization was observed, with highly altered, depolarized odontoblasts synthesizing dentine in different directions, or absence of
synthesis. Absence of enamel matrix and morphologically altered ameloblasts were also observed in certain regions. These results are in agreement with researches that have reported severe alterations in different types of cells with a radiotherapy dose of 15 Gy25,28,29 and that at higher doses of radiation the histological effects become more evident, therefore the more perceptible will be the deleterious effects caused on the dental organ.20,22,24,25 This may be compared with studies such as that of English et al.25 in which hair loss, skin lesions, ocular, weight and dental alterations were observed. Researches have proved that ameloblasts are cells that are less sensitive to radiation than odontoblasts,19,24 and that amelogenesis and dentinogenesis are progressively inhibited with the increase in therapeutic doses.19,21,25 In the present study it was observed that the odontoblasts suffered greater structural alterations than the ameloblasts at the dose of 15 Gy. At the beginning of the odontogenic region of the animals few alterations were observed in the experimental group compared with the control group. However, in the subsequent
archives of oral biology 59 (2014) 1242–1248
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Fig. 5 – Histological cuts of the odontogenic region of rat incisors, corresponding to the region of the distal face of the third molar in the control, radiotherapy 5 Gy and radiotherapy 15 Gy Groups. The circle indicates the visible reduction in size of the tooth, and the arrowhead, the formation of ‘‘islands’’ of dentine. In (a) the value of the bar corresponds to 100 mm, in (b) to 50 mm and in (c) to 20 mm.
regions, the alterations caused by radiotherapy were very evident in the 15 Gy Group, which suggests that the odontogenic region recovers over the course of time, and also due to eruptive tooth movement. At time intervals before 14 days, one could probably have observed more alterations in this region. Due to the fact of intense morphological alterations having been observed in the cells of the odontogenic region of the incisor, and consequently, a reduced eruption rate caused by the radiotherapy, this model may be indicated for future studies of radioprotective substances with the purpose of preventing or minimizing the effects results from radiotherapy.
5.
Conclusion
It was concluded that radiation caused histological alterations in the odontogenic region of the rat incisor and reduced the tooth eruption rate at the dose of 15 Gy.
Funding None.
Competing interests The author declares there is no conflict of interests.
Ethical approval Not required.
Acknowledgement The authors thank CAPES for the financial support.
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archives of oral biology 59 (2014) 1242–1248
references
1. Estimativas 2010. Incideˆncia de caˆncer no Brasil. Rio de Janeiro: Instituto Nacional de Caˆncer (INCA); 2010. 2. American Cancer Society. Cancer facts & figures. Available on: http://www.cancer.org [accessed 05.07.10]. 3. Kaste SC, Goodman P, Leisenring W, Stovall M, Hayashi R, Yeazel M, et al. Impact of radiation and chemotherapy on risk of dental abnormalities. Cancer 2009:5817–27. 4. Otmani N. Oral and maxillofacial side effects of radiation therapy on children. J Can Dent Assoc 2007;73(3):257–61. 5. Hong CHL, Napen˜as JJ, Hodgson BD, Stokman MA, MathersStauffer F, Elting LS, et al. A systematic review of dental disease in patients undergoing cancer therapy. Support Care Cancer 2010;18(8):1007–21. 6. Cardoso MFA, Novikoff S, Tresso A, Segreto RA, Cervantes O. Prevenc¸a˜o e controle das sequelas bucais em paciente irradiados por tumores de cabec¸a e pescoc¸o. Radiol Bras 2005;38(2):107–15. 7. Albuquerque RA, Morais VVL, Sobral APV. Protocolo de atendimento odontolo´gico a pacientes oncolo´gicos pedia´tricos – revisa˜o da literatura. Rev Odont UNESP 2007;36(3):275–80. 8. Lopes NNF, Petrilli AS, Caran EMM, Franc¸a CM, Chilvarquer I, Lederman H. Dental abnormalities in children submitted to antineoplasic therapy. J Dent Child 2006;73(3):140–5. 9. Kaste SC, Hopkins KP, Jones D, Crom D, Greenwald CA, Santana VM. Dental abnormalities in children treated for acute lymphoblastic leukemia. Leukemia 1997;11: 792–796. 10. Vasconcelos NPS, Caran EMM, Lee ML, Lopes NNF, Weiler RME. Dental maturity in children with acute lymphoblastic leukemia after cancer therapy. Forensic Sci Int 2009;184:10–4. 11. Lesot H, Brook AH. Epithelial histogenesis during tooth development. Arch Oral Biol 2009;54S:S25–33. 12. Ten Cate AR. Desenvolvimento do dente e seus tecidos de suporte. In: Ten Cate AR, editor. Histologia Bucal: desenvolvimento, estrutura e func¸a˜o. 5th ed. Rio de Janeiro: Guanabara Koogan; 2001. p. 76–100. 13. Ohshima H, Nakasone N, Hashimoto E, Sakai H, NakakuraOhshima K, Harada H. The eternal tooth germ is formed at the apical end of continuously growing teeth. Arch Oral Biol 2005;50:153–7. 14. Gomes JR, Omar NF, Neves JS, Narvaes EAO, Novaes PD. Increase of MT1-MMP, TIMP-2 and Ki-67 proteins in the odontogenic region of the rat incisor post-shortening procedure. J Mol Histol 2010;41:333–41.
15. Merzel J, Novaes PD, Furlan S. The effects of local trauma to the enamel-related periodontal tissues in the eruption of the rat incisor. Arch Oral Biol 2000;45:323–33. 16. Merzel J, Novaes PD, Furlan S. A histological study of rootresects and root-transected rat incisor when eruption ceases, shortly before they are exfoliated from the socket. Arch Oral Biol 2000;45(2):315–22. 17. Merzel J, Nunes SF, Novaes PD. The effect of partial damage to the enamel-related periodontium combined with root resection on eruption of the rat incisor eruption. Arch Oral Biol 2004;49(3):209–16. 18. Merzel J, Novaes PD. Development of functional dentin incisors after a partial resection of the odontogeˆnica organ of rat incisors. Arch Oral Biol 2006;51(10):825–35. 19. English JA, Tullis JL. Oral manifestations of ionizing radiation. I. Oral lesions and effect on developing teeth of swine exposed to 2000 K.V. total body X-ray irradiation. J Dent Res 1951;30(1):33–52. 20. Sweeney WT, Elzay RP, Levitt SH. Histologic effect of fractionated doses of selectively applied 60Co Irradiation on the teeth of albino rats. J Dent Res 1977;56(11):1403–7. 21. Ubios AM, Piloni MJ, Cabrini RL. Mandibular growth and tooth eruption after localized x-radiation. J Oral Maxillofac Surg 1992;50(2):153–6. 22. Lee AC, Angleton GM, Benjamin SA. Hypodontia in the Beagle after perinatal whole-body 60Co g irradiation. Radiat Res 1989;118:467–75. 23. Maciel JCC, CastroJr CG, Brunetto AL, Leone LP, Silveira HED. Oral health and dental anomalies on patients treated for leukemia in childhood and adolescence. Pediatr Blood Cancer 2009;53:361–5. 24. Dale PP. The effect of x-ray irradiation on the rat incisor. J Dent Res 1953;32(1):117–25. 25. English JA, Schlack CA, Ellinger F. Oral manifestations of ionizing radiation. II. Effect of 200 KV. x-ray on rat incisor teeth when administered locally to the head in the 1,500 R. dose range. J Dent Res 1954;33(3):377–88. 26. Coady JM, Santangelo MV, Toto PD. Gamma-irradiated mouse incisor. J Dent Res 1967;46(4):681–5. 27. Niehoff P, Springer IN, Ac¸il Y, Lange A, Marget M, Rolda´n JC, et al. HDR brachytherapy irradiation of the jaw – as a new experimental model of radiogenic bone damage. J Craniofac Surg 2008;36(4):203–9. 28. Coppes RP, Zeilstra LJW, Kampinga HH, Konings AWT. Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists. Br J Cancer 2001;85(7):1055–63. 29. Boraks G, Tampelini FS, Pereira KF, Chopard RP. Effect of ionizing radiation on rat parotid gland. Br Dent J 2008;19(1):73–6.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Effect of radiotherapy on the eruption rate and morphology of the odontogenic region of rat incisors Amanda Maria Medeiros de Araujo a,*, Carolina Cintra Gomes b, Solange Maria de Almeida b, Carla Beatriz Klamt b, Pedro Duarte Novaes c a
Department of Clinic and Social Dentistry, School of Dentistry, Federal University of Paraı´ba, Joa˜o Pessoa, Paraı´ba, Brazil b Department of Oral Diagnosis, Division of Oral Radiology, Piracicaba Dental School, State University of Campinas, Piracicaba, Sa˜o Paulo, Brazil c Department of Morphology, Piracicaba Dental School, State University of Campinas, Piracicaba, Sa˜o Paulo, Brazil
article info
abstract
Article history:
Objective: The goal in this study was to evaluate the results of doses of 5 and 15 Gy of
Received 6 November 2013
radiation in odontogenic region of the rats inferior mandibular-incisors by a histological
Received in revised form
analysis and the rate of eruptions.
26 June 2014
Design: Animals were divided into three groups: control, radiotherapy 5 Gy and radiothera-
Accepted 13 July 2014
py 15 Gy. In which tooth-eruption-rate was measured every two days.
Available online
Results: Animals in Group 5 Gy presented values similar to those of the control group. Animals in Group 15 Gy presented reduction in tooth-eruption-rate as of the sixth day of the
Keywords:
experiment, vast disorganization of odontoblasts and ameloblasts, apparent reduction in
Tooth eruption
cell population in the follicle region and alterations in cervical loop formation of the dental
Odontogenesis
organ.
Ionizing radiation
Conclusions: It was concluded that there was a difference between the researched doses, and histological alteration at 15 Gy lead to statistical reduction in tooth-eruption-rate. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Cancer is a set of diseases that have in common the disorderly growth of cells that invade tissues and organs and may spread by metastasis to other regions. Paediatric tumours account for approximately 2.5% of the total number of tumours expected
among Brazilians.1 In the United States, cancer in childhood occurs in fewer than 2% of cases.2 Various modalities of therapies have been applied in children with cancer, which are associated with toxic effects resulting in complications.3 Radiotherapy is widely used as an efficient form of treatment, either alone or associated with chemotherapy and/or surgery.4 There is concern due to the
* Corresponding author at: Rua Joakim Schuller, 40/Edf Oasis Plaza – Apt 304, 58.037-760 Bairro Jardim Oceania, Joa˜o Pessoa, Paraı´ba, Brazil. Fax: +55 8332167409. E-mail addresses: [email protected], [email protected] (A.M.M. de Araujo). http://dx.doi.org/10.1016/j.archoralbio.2014.07.004 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
archives of oral biology 59 (2014) 1242–1248
high percentage of acute or delayed oral complications that may occur during and/or after radiotherapy treatment.5 In cancer of the head and neck, the applicability and efficacy of radiotherapy is determined by parameters such as the histological type of the neoplasia (slow rapid response), location and volume of the area to be irradiated, dose, rhythm of application, type of radiation and the patient’s general conditions.6 The most frequent oral complications in patients submitted to radiotherapy in the region of the head and neck and/or chemotherapy are haemorrhage, infection, exacerbation of pre-existent infection, mucositis, xerostomia, radiation caries and interruption in tooth development. They lead to alterations in odontogenesis3 such as enamel hypoplasia, cessation of tooth development, non formation of teeth,7,8 microdontia and alterations in rhizogenesis, such as interruption, thinning and widening of the pulp chamber.9,10 The effect of radiation on permanent teeth and its degree of severity depend on the stage of tooth formation, as well as the dose and extent of the irradiated area. Innumerable dental alterations may occur in children submitted to antineoplastic treatment, capable of affecting shape, number or root formation, such as hypoplasia, microdontia, hypodontia, taurodontia, conical roots, among other.8 Odontogenesis is the stage of tooth development that involves morphogenesis, histogenesis and cell differentiation. The consecutive stages of tooth development are the bud, cap and bell stages.11,12 Tooth development is a complex process and it can undergo various alterations during its formation.12 Rat incisors have an odontogenic region that presents cells with elevated metabolism and constant differentiation. This region has a compartment of stem cells and one of proliferative cells with innumerable molecules involved in kinetic processes such as cell proliferation, migration, differentiation and cell death.13,14 In the epithelium of the cervical loop intense cell proliferation occurs, from which cells migrate and differentiate into ameloblastic and odontoblastic cells.9 The growth, calcification and eruption of rat incisors occur continuously throughout life, showing all the stages of formation in a single tooth, and is therefore a model for evaluating the effect of X radiation on progenitor cells.9 Few studies have been conducted to evaluate the effects of radiation on the in the stage of development. As there is a scarcity in the literature of studies on the irradiated odontogenic region, it is important to evaluate the effect of radiotherapy on the odontogenic region of rat incisors. The aim of this study was to evaluate the morphologic alterations caused by the action of radiation on the odontogenic region of rat incisors, with doses of 5 and 15 Gy, and find out whether these alterations may interfere in the eruption rate of these teeth.
2.
Materials and methods
Thirty adult male rats (Rattus norvegicus, Albinus Wistar), with a mean weight of 220 grams, obtained from the central vivarium of UNICAMP were used in the study. While the research was conducted, the rats were maintained in polycarbonate cages, in which they remained in an
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environment with controlled temperature and humidity, and an alternating 12-h light–dark cycle. The animals’ diet consisted of standard balanced rations and water ad libitum, daily. This study was approved by the Ethics Committee on Animal Experimentation of the State University of Campinas, Protocol Number 1843-1. The animals were divided into three groups: Group 1; Group 2 in which the animals were submitted to a 5 Gy dose of radiotherapy in the region of the head and neck; and Group 3 in which the animals were submitted to a 15 Gy dose of radiotherapy in the region of the head and neck. The experiment lasted 16 days, in which the tooth eruption rates were measured every two days. On the second day of the experiment, the animals were submitted to radiotherapy in the region of the head and neck. The eruption rate was measured in all the animals. To do this, a linear mark was made on the vestibular face of the mandibular incisors with cylindrical diamond bur adapted to a high speed handpiece under cooling. With the aid of a millimetre eyepiece (Ernest Leitz Wetzlar Germany 12.5) adapted to a stereoscopic loupe, and the use of a millimetre ruler for calibration (Carl Zeiss 5+ 100/100 mm), the distance from the gingival margin up to the mark was measured. To obtain the measurements, the animals were anesthetized with Halothane (Crista´lia, Brazil). When the mark approximated the incisal edge of the tooth that was being worn by the friction, a new mark was made. For better confirmation of the data, the eruption rate was verified both on the mandibular right incisor and on the left and the final daily eruption value was given by the mean of these values. The values were transformed into millimetres and statistically analyzed by ANOVA. Before all the irradiation procedures, all the animals were anesthetized with intramuscular injection of 80 mg/kg of Ketamine Hydrochloride (Dopalen1 Agribrands do Brasil Ltda.; Paulı´nia, Sa˜o Paulo, Brazil) and 8 mg/kg of Xylazine Hydrochloride (Rompum1, Bayer S.A., Sa˜o Paulo, SP, Brazil). The animals irradiated after beginning the tooth eruption rate measurement received a single dose of 5 Gy and 15 Gy of X radiation, depending on their group, from a linear accelerator, Varian brand, model Clinic 6/100 at a focal distance of 100 cm. The collimation field was 15 30 cm, so that only the head and neck region was irradiated. All the animals were sacrificed by cervical dislocation at the end of the experiment. The rat hemimandibles were removed and fixed by immersion in 10% buffered formalin. After collection, each hemimandible was divided into two parts, anterior and posterior, using a high concentration, dual faced cutting disc, measuring 400 0.1200 1/2 diamond (102 mm 0.3 mm 12.7 mm) (EXTEC Corp., Diamond Wafering Blade) and kept in 10% buffered formalin for 24 h under agitation to promote better fixation. The posterior half of each hemimandible was decalcified with 4.13% EDTA in phosphate buffer pH 7.4, for 30 days. After decalcification, the odontogenic region was separated with a scalpel from the mesial face of the third molar, dehydrated in increasing grades of ethanol solutions, diaphanized and embedded in Paraplast Plus1: tissue embedding medium (McCormickTM Scientific, Leica Biosystems St. Louis LLC, USA). Transverse cuts in relation to the long axis of the incisor were made from the beginning of the odontogenic region in
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Fig. 1 – Disposition of the transverse histological cuts in the three selected regions (A–C).
the direction of the incisal edge of the incisor (Fig. 1), allowing the hemimandible to be divided into three parts: The region corresponding to the beginning of the odontogenic region; region B that comprises the median portion of the odontogenic region; and region C corresponding to the odontogenic region with reference to the distal face of the third molar. These were semi-serial cuts 5 mm thick, and were obtained using a Leica RM 2155 microtome (Leica – Fl?ssigkeit, German). For morphological evaluation the slides were stained with haematoxylin and eosin, mounted on Canada balsam and analyzed under a light microscope.
3.
Results
On the day of sacrifice, it was possible to observe that the rats with a single dose of 15 Gy presented burns, alopecia and weight loss (Fig. 2) when compared with the animals of the control group and those irradiated with 5 Gy. Table 1 presents the results of the mean and standard deviation of the tooth eruption rate in rats of the control, 5 Gy
Table 1 – Means and standard deviation of the values of tooth eruption rates of rat incisors in the control group and of those that received the radiotherapy dose of 5 and 15 Gy*. Control Day Day Day Day Day Day Day Day Day
0 2 4 6 8 10 12 14 16
0.52 0.50 0.55 0.54 0.54 0.55 0.56 0.55 0.56
(0.05) (0.06) (0.05) (0.04) (0.06) (0.05) (0.05) (0.03) (0.04)
Aa Aa Aa Aa Aa Aa Aa Aa Aa
5 Gy 0.54 0.50 0.56 0.53 0.52 0.55 0.50 0.51 0.50
(0.04) (0.06) (0.05) (0.07) (0.07) (0.05) (0.06) (0.08) (0.08)
15 Gy Aa Aa Aa Aa Aa Aa Aa Aa Aa
0.52 0.49 0.51 0.45 0.43 0.41 0.40 0.37 0.36
(0.05) (0.06) (0.04) (0.04) (0.05) (0.04) (0.05) (0.06) (0.05)
Fig. 2 – Mean value of the weight of rats (in grams). The arrow indicates the day on which the animals were submitted to radiotherapy.
and 15 Gy Groups*. The animals of Group 5 Gy presented similar values to those of the control, however, there were no signs of alteration in the tooth eruption rate, whereas the animals submitted to the dose of 15 Gy presented statistical difference in the tooth eruption rate as from day 6 of the experiment. Histologically, in the dental organ of the control group, an aspect was observed similar to that of a horseshoe, fully surrounded by well vascularized conjunctive tissue, called a dental follicle. The odontoblasts were shown to be well organized, polarized and integral, disposed in a palisade, constituted of a single layer of cells contouring the dental papilla, with a columnar aspect. The pre-ameloblasts were organized as high, polarized cylindrical cells and uniformly disposed dental papilla cells. The dental papilla presents undifferentiated, fusiform or star-shaped cells, with numerous cytoplasmatic prolongations (Figs. 3–5). In Group 5 Gy, the odontoblasts were shown to be similar when compared with the control group. Whereas, in Group 15 Gy it was observed that apparently there was a reduction in cell population in the region of the follicle, adjacent to the lingual face of the tooth, and irregularities in the organization of the ameloblasts, alterations in the formation of the epithelial loop in the dental organ, in the odontoblasts with the inversion of polarity and matrix in different directions leaving the dentine irregular; in addition to a visible reduction in tooth size (Figs. 3–5).
4. Aa Aa Aa Bb Bb Bbc Bbc Bc Bc
* Means followed by different letters (uppercase letters in the horizontal and lowercase in vertical) differ by ANOVA and Tukey test (0.05).
Discussion
Deleterious effects may occur as a result of radiotherapy, such as malformations in tooth rhizogenesis and odontogenesis.7 The earlier the stage, the greater are the chances of alteration in tooth formation occurring.3,9,10 Studies have proved that alterations in the odontogenic region cause a reduction in the tooth eruption rate of the incisors, by the trauma caused in the periodontium related to enamel,15 resection of the odontogenic region16,17 or removal of the vestibular portion of the odontogenic region,18 indicating that the action of an external agent, whether it is trauma,
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Fig. 3 – Histological cuts of portion A of the odontogenic region of rat incisors. The arrow points out the alteration in the formation of the epithelial loop and the star shows a reduction in the number of cells in the follicle region adjacent to the lingual face of the tooth. In (a) the value of the bar corresponds to 100 mm, in (b) to 50 mm and in (c) to 20 mm.
resection or radiotherapy may interfere in the tooth morphology and eruption rate. Therefore, some authors have discussed the effect of radiotherapy and the occurrence of alterations in tooth formation in rats,18–21 in the premolars of beagles22 and consequences in children submitted to antineoplastic treatment3,8–10,23 and in rat incisors.20,24–26 In the present study, in addition to the morphology, the daily tooth eruption rate values were evaluated in the incisors of rats submitted to radiotherapy in the head and neck region. In this study, we chose to use the rat incisor, because this tooth has continued growth and its cells are constantly renewing.16 Two radiotherapy doses were selected, as various studies have evaluated dental alterations in animals that received low doses19,22,24,25 and others in high doses.3,20,21,25,26 As some authors found morphological or structural alterations in the teeth at low doses of radiotherapy, we decided to use a dose of 5 Gy as well. With the purpose of evaluating the initial effect of radiotherapy in the odontogenic region, a single time of
sacrifice of 14 days after radiotherapy was chose, because it is an adequate time in which to evaluate the tooth eruption rate. On the day of sacrifice it was found that the animals that were treated with the 15 Gy dose presented lower weight, burns and alopecia when compared with the control and radiotherapy 5 Gy groups. These results are in agreement with authors3,20,21,25,26 who related that the higher the dose the more side effects would be observed in the animals, such as irregular dentine formation and disorganized odontoblastic cells. It was not possible to identify significant alterations in the tooth eruption rate when the animals were submitted to a single dose of 5 Gy, however, in the 15 Gy Group a significant reduction in the tooth eruption rate was verified as from the fourth day after radiotherapy. These findings differ from those of Coady et al.,26 who, when studying a cumulative dose of 12 Gy and 24 Gy observed no statistical difference, however, these authors used a fractionated dose of radiotherapy, which causes less tissue damage when compared with single doses. The study of Niehoff et al.27 justifies the application of a single
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Fig. 4 – Histological cuts of portion B of the odontogenic region of rat incisors. The arrowhead shows the lack of enamel production in the 15 Gy Group and the cross shows the disorganization of the odontoblasts. In (a) the value of the bar corresponds to 100 mm, in (b) to 50 mm and in (c) to 20 mm.
dose of 15 Gy comparable with a fractionated dose of 54 Gy, compatible with some antineoplastic treatments in the head and neck region in humans. The single dose of 15 Gy caused significant morphological alterations, probably inhibiting cell differentiation and consequently retarding the growth and eruption of the incisors. With the dose of 5 Gy it was observed that the odontoblasts were apparently shown to be organized and the ameloblasts were shown to be whole and appeared to continued to synthesize the enamel matrix actively, which is in disagreement with English and Tullis19 who studied the effects of radiotherapy on the morphology of swine molars in the stage of development and observed alterations in the ameloblasts in fractionated doses of 4 Gy. The results of the present study are in agreement with the finding of English et al.25 who found no significant morphological alteration in rat incisors with the dose of 5 Gy. In the teeth submitted to 15 Gy cell disorganization was observed, with highly altered, depolarized odontoblasts synthesizing dentine in different directions, or absence of
synthesis. Absence of enamel matrix and morphologically altered ameloblasts were also observed in certain regions. These results are in agreement with researches that have reported severe alterations in different types of cells with a radiotherapy dose of 15 Gy25,28,29 and that at higher doses of radiation the histological effects become more evident, therefore the more perceptible will be the deleterious effects caused on the dental organ.20,22,24,25 This may be compared with studies such as that of English et al.25 in which hair loss, skin lesions, ocular, weight and dental alterations were observed. Researches have proved that ameloblasts are cells that are less sensitive to radiation than odontoblasts,19,24 and that amelogenesis and dentinogenesis are progressively inhibited with the increase in therapeutic doses.19,21,25 In the present study it was observed that the odontoblasts suffered greater structural alterations than the ameloblasts at the dose of 15 Gy. At the beginning of the odontogenic region of the animals few alterations were observed in the experimental group compared with the control group. However, in the subsequent
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Fig. 5 – Histological cuts of the odontogenic region of rat incisors, corresponding to the region of the distal face of the third molar in the control, radiotherapy 5 Gy and radiotherapy 15 Gy Groups. The circle indicates the visible reduction in size of the tooth, and the arrowhead, the formation of ‘‘islands’’ of dentine. In (a) the value of the bar corresponds to 100 mm, in (b) to 50 mm and in (c) to 20 mm.
regions, the alterations caused by radiotherapy were very evident in the 15 Gy Group, which suggests that the odontogenic region recovers over the course of time, and also due to eruptive tooth movement. At time intervals before 14 days, one could probably have observed more alterations in this region. Due to the fact of intense morphological alterations having been observed in the cells of the odontogenic region of the incisor, and consequently, a reduced eruption rate caused by the radiotherapy, this model may be indicated for future studies of radioprotective substances with the purpose of preventing or minimizing the effects results from radiotherapy.
5.
Conclusion
It was concluded that radiation caused histological alterations in the odontogenic region of the rat incisor and reduced the tooth eruption rate at the dose of 15 Gy.
Funding None.
Competing interests The author declares there is no conflict of interests.
Ethical approval Not required.
Acknowledgement The authors thank CAPES for the financial support.
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