INTRODUCTION

Placement of orthodontic appliances in a healthy oral cavity can induce a continuous accumulation of dental plaque,1 alter the normal oral microbiota,23 cause lesions in the buccal mucosa,4 exacerbate periodontal disease, and consequently cause infection.5 It is known that ulceration in the buccal mucosa is one of the most frequent complaints of patients because of the friction between bracket and mucosa, which causes discomfort for the patient.67 Thus, when these ulcerations persist during treatment, the orthodontist refers the patient to another specialist, who performs additional tests such as taking a biopsy and doing exfoliative cytology, which can detect alterations in the buccal mucosa caused by this irritation.

The use of exfoliative cytology in the diagnosis of buccal lesions was more common during the period from 1955 to 1975. Since then, a decline in its clinical application has occurred because of the subjective nature of its interpretation, and because few abnormal cells can be identified in smears. However, this technique has stirred renewed interest because of the possibility of its being complemented with other laboratory techniques such as molecular biology, cytomorphology, and immunohistochemistry.8 In addition, it offers the advantage of being minimally invasive and painless, without the need for local anesthetic, and it is easy to perform.

Another major advance in cytopathology was the development of liquid-based cytology, which provides a series of advantages in relation to the conventional type, featuring (1) better evidence of epithelial cells, (2) slides with fewer inflammatory cells and red blood cells, less cell debris, and fewer undesirable artifacts,9 (3) less cell overlapping, and (4) more representative samples for reading.10 Because liquid-based cytology mixes the complete sample in liquid, probably fewer false negatives occur, compared with conventional cytology, wherein only 20% of collected cells are transferred to the slide.9

The necessity of offering patients a fixed orthodontic treatment with no significant risk of damage to buccal mucosa cells and the lack of reports in the literature on the cytologic analysis of buccal mucosa adjacent to metal and ceramic brackets were the reasons for this study.

The aim of this investigation was to study and compare the epithelial cells of the buccal mucosa adjacent to metal and ceramic brackets at three time points: baseline, 60 days after placement, and 30 days after removal of the brackets. These cells were examined for morphometric alterations in the area of the nucleus and cytoplasm, alterations in the nuclear/cytoplasmic ratio, morphologic alterations in the nucleus and cytoplasm, and alterations in the cytologic criteria for malignancy.

MATERIALS AND METHODS

Individuals who were referred to the Dental Clinic for dental treatment were invited to participate in this study before beginning their treatment. When they agreed to participate, individuals or their legal guardians signed an informed consent form.

Selected individuals had no related history of smoking, alcoholism, diabetes, anemia, or debilitating diseases and were not being treated with antibiotics or steroids during the study period. They did not use alcohol-based mouthwashes, did not wear prostheses or have tooth restorations with sharp edges, and did not have any type of lesion on the buccal mucosa. The sample of this study comprised 21 Brazilian individuals (mean age, 14 years; range, 7.6 to 53.7 years)—7 males and 14 females.

The locations chosen for bracket placement were second deciduous molar, second premolar, and first and second permanent molars, all on the upper arch. The teeth chosen varied according to the stage of dentition for each at the time of bracket placement.

Four premolar brackets were bonded in each individual with Transbond XT adhesive (3M Unitek Orthodontic Product, Monrovia, Calif). Two were standard edgewise Dyna-lock metal brackets (3M Unitek Orthodontic Product) placed on the right side, and two were standard edgewise Transcend Series 6000 ceramic brackets (3M Unitek Orthodontic Product) placed on the left side (Figures 1 and 2).

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Cytomorphometric Analysis

The slides were examined under a binocular light microscope (Olympus BX50, Olympus, Tokyo, Japan) adapted with WH 10X-H/22 oculars and a PLAN 40X/ 0.65 objective (Olympus). Prior to reading, the identification number of the slides was covered to avoid bias. Fifty cells on each slide were selected randomly for examination.11–14 Areas with cells folded over and/ or clumped were avoided15 because of the difficulty involved in determining cell boundaries. The image of the cytologic fields was captured at a magnification of 400 times (Figures 3A through E) with the use of a Sony CCD Iris Color Video camera, model DXC-107A (Sony Electronics Inc, Tokyo, Japan). To measure nuclear (NA) and cytoplasmic (CA) areas, the image analysis system Image Pro Plus, version 4.5.029 for Windows 98/NR/2000 (Media Cybernetics Inc, Silver Spring, Md), was used to enhance precision and speed of measurement.16 After NA and CA were measured,1112 the nuclear/cytoplasmic ratio (N/C) was determined for each cell.111417

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RESULTS

Cytomorphometric Analysis

After placement of the brackets, a significant decrease in NA and N/C and an increase in CA of buccal mucosa cells adjacent to ceramic or metal brackets were observed (P < .01). Cells adjacent to metal brackets did show a lower NA and N/C compared with those next to ceramic brackets (P < .01). Although a greater increase was noted in the CA of cells adjacent to metal brackets than in those next to the ceramic bracket, it was not significant (P > .05).

When the brackets were removed, the buccal mucosa cells adjacent to metal brackets still showed a smaller nucleus, a larger cytoplasm, and a lower nuclear/cytoplasmic ratio than at T0 (P < .01), although with fewer alterations than at T1 (P < .01). On the other hand, the buccal mucosa cells adjacent to the ceramic brackets returned to their initial size (P > .05) (Tables 1 through 3).

Table 1.  Nuclear Area According to Bracket and Time

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Cytomorphologic Analysis

With respect to the predominance of cells present in the smears, on the basis of their staining, no slide was found with a predominance of cells of the spinosum and basale stratum.

In T0, the number of slides with a predominance of surface and subsurface cells was similar, and for T1 and T2, a greater number of slides showed a predominance of surface cells. A statistically significant difference was found between T0 and T1 in the buccal mucosa cells adjacent to the ceramic and metal brackets, and between T0 and T2 in the buccal mucosa cells adjacent to the ceramic brackets (P < .05) (Table 4).

Table 4.  Number of Slides According to Cell Predominance

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Smears that were examined exhibited no instances of Papanicolaou Classes 0, III, IV, and V when the cytologic criteria for malignancy were determined. Classes I and II were observed at all time points, and no significant difference was noted among the groups (P > .05).

DISCUSSION

In this study, ceramic and metal brackets were bonded without the presence of arches, ligatures, or rubber bands because these materials could bias the findings.20 However, it is known that these accessories are used routinely in treatment, and they can protect the buccal mucosa from direct friction caused by the bracket.

Ulcerations in the buccal mucosa are frequent complaints among orthodontic patients. Studies indicate that approximately 76%6 to 95%7 of patients report ulcers in the buccal mucosa during treatment, and only between 16.5%7 and 21.1%6 of patients report ulcers only once. Therefore, because the epithelium of the buccal covering is exposed to aggressive agents, as in the case of brackets that are capable of causing alterations at various times during treatment, exfoliative cytology can be an effective tool in diagnosis to detect and evaluate these alterations, assuming that its limitations are well elucidated and applied.2122 The clinician should be knowledgeable about this technique because the cells are studied individually and cannot be evaluated with regard to tissue conformation, as in a biopsy.21 In addition, only the most surface cells of the epithelium are collected for exfoliative cytology.23 Therefore, the use of brushes to collect epithelial cells allows collection of samples that include cells of all the buccal mucosa stratified squamous epithelium,92425 in addition to providing a thinner and more dispersed, homogeneous distribution of cells on the slides.26

In the present study, placement of brackets in the buccal cavity caused diminution of the nucleus, an increase in cytoplasm, and a lower nuclear/cytoplasmic ratio of buccal mucosa cells that were in contact with the brackets. These results corroborate the findings of Shabana et al,27 who also reported a statistically significant increase in the size of cells of traumatic keratosis lesions when these were compared with normal cells of the buccal mucosa.

However, in the buccal cells of individuals with malignant lesions1113 or of smokers,1215 alterations distinct from those in the present study were found. In individuals with a tobacco-chewing habit and in those with smoking and tobacco-chewing habits combined, an increase in nuclear diameter and a decrease in cell diameter were observed,15 as were seen in samples of individuals with tumors in the mouth floor.13 Cowpe et al11 did not find size changes in the nucleus in samples of suspicious lesions of the buccal mucosa and the mouth floor but did observe a decrease in cytoplasmic area in lesions of the buccal mucosa. Ogden et al12 observed an increased nuclear area only in the buccal mucosa cells of smokers and did not note an alteration in the cytoplasmic area. Normal cells of the buccal mucosa have abundant cytoplasm and a single, small centralized nucleus; malignant cells have a broad, enlarged nucleus that occupies a large area of the cytoplasm, with well-stained chromatin and an irregular nuclear membrane.21 Therefore, the cellular changes that occurred in the buccal mucosa adjacent to the metal and ceramic brackets in the present study do not suggest malignancy. When this diagnosis was confirmed by the evaluation of cytologic criteria for malignancy, smears of only Classes I and II of Papanicolaou were noted.

Alterations in sizes of the nucleus and cytoplasm as demonstrated here suggest hyperkeratinosis of the stratified squamous epithelium of the buccal mucosa adjacent to the brackets. This would cause an increase in the number of cells in the corneum stratum of the epithelium that show abundant cytoplasm and smaller nuclei than cells from deeper layers. This hyperkeratinosis can be confirmed by an increase in the number of slides with a predominance of surface cells at T1 and T2.

Greater cell alterations on the side with the17-4 stainless steel bracket may have been caused by trauma to the buccal mucosa caused by the physical characteristics of brackets, in other words, because of the fact that the wings were less rounded than those of the ceramic brackets, or because of the cytotoxicity of stainless steel, which has been observed in other studies.28–32

In this study, buccal mucosa cells were evaluated only 30 days after removal of the brackets, because Jones et al22 recommend that if a lesion persists for longer than 14 days after removal of the causative factors, a biopsy should be performed immediately. Therefore, within 30 days, cells should have returned to their initial size. In future studies, the buccal mucosa cells should be analyzed after longer periods to determine whether these alterations persist in the buccal mucosa.

This was the first study undertaken to describe cellular changes in the buccal mucosa adjacent to metal and ceramic brackets. Prior to this finding, orthodontists had no way of defending against allegations that linked the use of brackets to oral cancer. Because brackets are essential components of fixed orthodontic appliances, biocompatibility is needed to prevent irreversible deleterious damage to tissues. Although results of this investigation suggest that brackets do not cause any malignant changes in the buccal mucosa, the origin of the observed changes remains uncertain. Future studies in this regard will explore ways to prevent these alterations.

CONCLUSIONS

• Placement of metal and ceramic brackets in the buccal cavity induces cellular alterations. These alterations do not suggest malignancy.

• Buccal mucosa cells adjacent to the metal brackets show greater changes than are seen in those adjacent to the ceramic brackets.

• Buccal mucosa cells adjacent to the metal and ceramic brackets tend to return to the initial morphology after removal of the brackets.