The present study aimed to investigate the occurrence of micronuclei and the degenerative nuclear alterations indicative of apoptosis and necrosis in the exfoliated buccal and palatal mucosa cells of children and adolescents using orthodontic appliances made of self-curing acrylic resins.
The micronuclei and nuclear alterations were evaluated in a minimum of 2000 cells collected from the cheeks and palates of 30 patients of both sexes, aged between 6 and 12 years. The cell evaluations were performed before appliance installation and 15 to 21 days after installation. A conditional test was used. In all analyses, the significance level was 5%.
No differences in the occurrence of micronuclei or the nuclear degenerative changes indicative of apoptosis and necrosis were observed in the cheek cells between the 2 time points ( P > 0.05). However, evaluation of the palate cells revealed a significant increase in the occurrence of micronuclei and the nuclear alterations indicative of apoptosis ( P < 0.01) but not of karyolysis ( P > 0.05).
Direct contact of orthodontic appliances made of acrylic resins with the oral mucosa increases the incidence of chromosomal damage and degenerative nuclear alterations.
We monitored children and adolescents who were using appliances made of acrylic resin.
Direct contact with acrylic resins induced chromosomal damage and apoptosis.
Local trauma might stimulate cell renewal and interfere with observed differences.
Acrylic resins have been widely used in dental practice since the mid-20th century, having been used in the making of removable orthodontic appliances since the orthodontics field was introduced. Orthodontic acrylic resins are organic compounds that are classified as polymers that consist predominantly of methyl methacrylate monomers.
Despite the various methods used in the polymerization of acrylic resins, the monomer to polymer conversion is never complete. , This results in varying amounts of methyl methacrylate monomers being released into the oral cavity during orthodontic appliance use. ,
In addition to altering the final physical properties of the resin, these residual monomers can lead to local and systemic tissue reactions when coming into contact with saliva and oral soft tissues. , These reactions can manifest as local chemical irritation, hypersensitivity, and/or inflammation of the mucosa. The potential genotoxic effects of methyl methacrylate are not yet entirely understood. Consequently, a study in this area is required because genetic damage at an early age can lead to the development of health problems throughout life , ; biomonitoring is one approach.
Among the methods used for biomonitoring children and adolescents, the micronucleus test, applied to exfoliated buccal mucosa cells, has been a frequent choice because of the comfort and speed associated with collecting the sample material. , Micronuclei are structures that result from whole chromosomes or chromosomal fragments that, because they do not bind to spindle fibers, are not included in the nuclei of daughter cells, remaining instead in the cytoplasm of interphase cells.
In this context, the present study aimed to investigate the occurrence of chromosomal (ie, micronuclei) damage and the degenerative nuclear changes that indicate apoptosis and necrosis in exfoliated cells of the buccal and palatal mucosa of children and adolescents using orthodontic appliances made of acrylic resin. In addition, the hypothesis that removable orthodontic appliances made of acrylic resin do not cause chromosomal damage or degenerative nuclear changes was investigated.
Material and methods
Before the start of the study, the project was submitted to the research ethics committee (Feira de Santana State University), from which it obtained a favorable opinion. Participation was voluntary, and the participants were given the freedom to remove themselves from the study at any time. All participants signed a consent form, which was also signed by their legal guardians. The study was carried out from January 2018 to April 2019.
Thirty patients, representing men and women and aged 6-12 years and with dental indications for the use of removable orthodontic appliances made of self-curing acrylic resin, participated in the study. The sample was randomly selected from a cohort of 353 patients from a school clinic. Definition of the sample size took previous studies that used the micronucleus test in exfoliated oral mucosa cells into consideration. In addition, a minimum of 20 patients has been recommended for studies in which chromosomal alterations are used as risk biomarkers in both exposed and control groups.
The exclusion criteria adopted were (1) use of any medication, (2) infection (viral or bacterial), (3) oral lesions, (4) use of oral antiseptics in the last month, (5) exposure to x-rays (radiography, tomography or any radiological procedure in the last 3 months) or to any other genotoxicity in the same period, and (6) chemotherapy or radiation therapy at any point.
After obtaining the superior models, removable orthodontic appliances were made. The apparatus consisted of an acrylic plate with posterior and anterior Hawley retainer clips ( Fig 1 ).
After making the staples and isolating the plaster model, the acrylic (Jet; Classico, Sao Paulo, Sao Paulo, Brazil), previously manipulated in a Paladon pot (Nova OGP, Bragança Paulista, Sao Paulo, Brazil), was adjusted from 2.5 parts polymer to 1.0-part monomer. After noting that the resin surface was dull, the appliances were immediately treated with an Ortho Class appliance (Classico), in which a hydraulic pressure of 25-30 pounds was applied. This was followed by finishing and polishing using drills and abrasive tips.
The participants were instructed to use the orthodontic appliance continuously (ie, 24 hours per day) and remove it only during meals and clean the teeth. Each participant was given a form on which to note the periods when the appliance was not in their mouth.
The material for cytologic analysis was collected by gently scraping the buccal mucosa of the cheek and the mucosa of the palate with a cytobrush (CooperSurgical, Trumbull, Conn) rinsing the mouth with filtered water. These samples were taken immediately before the installation of the orthodontic appliance (Moment I) and then 15-21 days after (Moment II).
The collected material was smeared onto a clean glass slide on which were 2 drops of saline (0.9% NaCl). The slides holding the samples were dried at room temperature. After drying, they were fixed in a methanol/acetic acid solution (Reagen, Rio de Janeiro, Rio de Janeiro, Brazil) at a 3:1 ratio. After 24 hours, hydrolysis was performed in a hydrochloric acid (5 N HCl) solution for 20 minutes, followed by washing in distilled water 3 times.
After again drying the slides at room temperature, the samples were stained with Schiff reagent (US Merck, Kenilworth, NJ) for 90 minutes and then rinsed and dried. Finally, 1% Fast Green counterstaining was performed (Sigma-Aldrich), after which the slides were washed with absolute alcohol, and coverslips were affixed using Entelan (US Merck, Kenilworth, NJ).
The cytologic analysis was performed in a blind test by a single, trained evaluator using binocular optical microscopy at 20×, 40×, and 100× magnification. Micronuclei were identified as being rounded structures, distinctly separate from the nucleus, with well-defined borders and measuring about one-third to one-fifth of the size of the nucleus. They possessed a chromatin structure, were colored similarly to that and were viewable in the same plane.
At least 2000 cells from each region were analyzed per individual. Only cells with intact cytoplasm were counted. For the palate cells, because of keratinization, there were cells that presented only the stained cytoplasm, and so 2 slides had to be analyzed from each individual to allow the minimum number of cells to be counted.
In addition to the micronuclei, degenerative nuclear alterations, indicative of apoptosis (pycnosis—reduced nuclear structure; karyorrhexis—fragmentation of the nucleus into small round or oval bodies within the intact cytoplasm; condensed chromatin–more intensely stained in relation to the nucleus; and necrosis (or karyolysis)—the dissolution of the nucleus, visually marked by its absence) were also recorded ( Fig 2 , A – E ).
In addition to these changes, nuclear projections (broken eggs and nuclear buds) were also recorded. The broken eggs had rounded structures, color, and chromatin distribution similar to that of the nucleus and were connected to the nucleus by a thin chromatin filament ( Fig 3 , A ). Nuclear buds were observed as projections resulting from the strangulation of small areas of the nuclear surface, causing a rounded protuberance to emerge ( Fig 3 , B ).
The results of the cytologic analysis were evaluated using the conditional test for the comparison of proportions in rare-event situations—an alternative test to the chi-square significance test—in line with Fischer’s exact test. This test is considered to be suitable for cytogenetic evaluation when the detection of a given cytologic alteration requires the computation of a large number of cells. , In all analyses, the significance level was 5%.
The sample consisted of 30 patients (18 men, 12 women) aged 6-12 years (mean, 9.47 ± 1.77 years). Table I shows the distribution of the participants by sex and average age. The average daily extent of appliance use was 19.45 ± 2.089 hours. All of the participants who were present at the start of the study stayed until the study was complete.
|Sex||n||%||Age ± standard deviation|
|Male||18||60||10.04 ± 1.33|
|Female||12||40||8.61 ± 2.05|
|Total||30||100||9.47 ± 1.77|
The analysis of differences in the occurrence of micronuclei in cells collected from the mucosa of the cheeks between Moment I (before installation) and Moment II (15-21 days after installation), using the conditional test for the comparison of proportions in situations of rare events, did not show any significant differences (χ 2 = 0.9782; degree of freedom (df) = 1; P > 0.05). These data are presented in Table II .
|Moment||n||Total cells||Micronuclei (obs)||Micronuclei (exp)||χ 2||Apoptosis ∗ (obs)||Apoptosis ∗ (exp)||χ 2||Necrosis † (obs)||Necrosis † (exp)||χ 2|
|I||30||61709||28||31.96||0.9782||1415||1465.99||3.5426||1612||1665.22||χ 2 = 3.3971|
|II||30||61878||36||32.04||df = 1||1521||1470.01||df = 1||1723||1669.78||df = 1|
|Total||60||123587||64||64.00||P > 0.05||2936||2936.00||P > 0.05||3335||3335.00||P > 0.05|