Association among pain, masticatory performance, and proinflammatory cytokines in crevicular fluid during orthodontic treatment


Orthodontic patients usually complain about masticatory limitations associated with the activation of fixed appliances. The aim of this investigation was to evaluate whether orthodontic pain reflects differences in the objective evaluation of mastication and in the levels of proinflammatory cytokines in the crevicular fluid of patients undergoing orthodontic treatment.


Twenty patients with malocclusions requiring orthodontic treatment were included in this prospective study. Their pain experience, masticatory performance, and levels of interleukin 1-beta and prostaglandin E 2 in crevicular fluid were evaluated at 3 times: before bracket placement, 24 hours after archwire placement, and 30 days after the initial appointment. All variables were compared with those of a control group of 25 subjects with normal occlusion.


The masticatory performance of the patients was significantly reduced at 24 hours after bracket placement, the period in which they reported higher values of pain and had higher levels of interleukin 1-beta. The levels of prostaglandin E 2 did not change in the periods evaluated, and there were no correlations between the levels of cytokines and the functional limitations observed. The only significant correlation was between pain and decreased masticatory performance.


The masticatory performance of orthodontic patients is significantly reduced only during the period of greatest pain. However, these alterations did not correlate with any measurement of interleukin 1-beta or prostaglandin E 2 in the crevicular fluid, suggesting that these solitary measurements are inadequate to predict the temporary pain and masticatory limitations experienced by patients undergoing orthodontic treatment.


  • Orthodontic pain is the main reason that patients decline treatment with fixed appliances.

  • Orthodontic pain can evoke significant objective and subjective responses in patients.

  • Patients exhibited significant increases in interleukin-1β after archwire placement.

  • Subjective pain and objective masticatory indexes were significantly correlated.

  • Pain-related biomarkers were not correlated with pain reports or masticatory indexes.

Numerous orthodontic procedures evoke pain sensations in patients, such as separator placement, archwire placement and activations, the application of orthopedic forces, and debonding procedures. This complex negative experience, known as orthodontic pain, can significantly affect patients’ daily activities, causing difficulty in chewing, sleep disturbances, and use of self-medication. The literature shows that about 95% of patients experience some kind of pain associated with orthodontic therapy, and this is the main reason for nonacceptance or withdrawal of treatment with fixed appliances. Although the pain is usually temporary, its management often requires the use of nonsteroidal anti-inflammatory drugs for relief. Depending on the dosage and time of use, these drugs can sometimes retard orthodontic tooth movement. Therefore, the current trend is toward the use of preemptive analgesics, which are administered at least 1 hour before every orthodontic procedure, followed by postoperative doses for 2 or 3 days.

Clinicians usually respond to the most frequently asked questions—“Will it hurt?” and “Will I be able to eat normally?”—with the answer: “There may be some discomfort after the appliance activations, but the level of pain varies from patient to patient.” In fact, it would be useful to have some objective measurement to predict possible functional limitations, such as pain and masticatory difficulties, induced by orthodontic procedures. At the same time, a specific preemptive analgesic protocol could be chosen appropriately for each patient.

Recently, several studies have focused on gingival crevicular fluid (GCF) and its biochemical mediators to establish correlations between the levels of some biomarkers and significant clinical responses. For example, Giannopoulou et al reported that the intensity of pain at 1 hour after placement of elastic separators was associated with prostaglandin E 2 (PGE 2 ) levels, whereas pain intensity at 24 hours was associated with interleukin 1-beta (IL-1β) levels. Yao et al found a correlation between pain intensity and PGE 2 levels in the GCF of lateral incisors at 12, 24, and 72 hours after tooth movement. However, the role of the cytokines in orthodontic treatment is still largely unknown, in particular because of variations in the ages of the studied subjects, different measurement scales and styles used, and different teeth and mechanics evaluated; these cause confusion and make it difficult to compare the results from the studies.

We designed this study to investigate the effects of fixed orthodontic appliances on pain and masticatory performance during orthodontic treatment. Moreover, the levels of the proinflammatory cytokines IL-1β and PGE 2 in GCF were evaluated and correlated with the objective (masticatory performance) and subjective (pain intensity) variables studied.

Material and methods

Twenty healthy subjects (10 male and 10 female, ages 18 ± 5 years) participated in this prospective longitudinal study. The participants were chosen from the Department of Orthodontics of the Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, after an initial screening examination, and the following inclusion criteria were used: complete permanent dentition (except for third molars), an uneventful medical history and good oral health, and an approximately equal number of occlusal units (an occluding molar pair was counted as 2 occlusal units, and a premolar pair was counted as 1 occlusal unit) with malocclusions requiring orthodontic treatment. Bonding of at least 10 teeth in the maxillary arch and 0.014-in nickel-titanium archwires were used during the experimental period. One orthodontist (M.P.T.) performed the treatments. The brackets (Roth prescription, slot 0.022 in; Morelli, Sorocaba, Brazil) were placed by indirect bonding, and the second molars were not included in the appliance. All patients had mild or moderate crowding, and all treatments were performed without extractions. Nine patients had Class I malocclusions, 7 had Class II malocclusions, and 4 had Class III malocclusions. The type of malocclusion was not a confounding factor in this study because the pain experience was not related to the type or severity of the initial malocclusion. The exclusion criteria were previous orthodontic treatment and symptoms of temporomandibular joint dysfunction. Twenty-five volunteers aged 18 ± 4 years with normal occlusion and no fixed orthodontic appliances were selected from the students of the Faculty of Dentistry as the control group. The patients (evaluated by radiography) and the control group (evaluated by photography) were also homogeneous with regard to facial type. Informed written consent was obtained from all participants before enrollment in the study. The local ethics committee approved the study protocol (number 19067). The sample size was determined based on clinically relevant masticatory performance data from the literature, with a power of 80% and α = 0.05; 20 subjects were deemed adequate for this study.

Before GCF collection, each site was isolated with cotton rolls, any supragingival plaque was removed with cotton pellets, the tooth surface was dried gently with air, and the area was isolated with cotton rolls. GCF was collected with paper strips (Periopaper; Oraflow, Plainview, NY). Each strip was carefully inserted at the entrance of the gingival crevice for 30 seconds on the mesiolabial surface of the maxillary lateral incisors and on the mesiobuccal surface of the maxillary second premolars. The 4 strips were then placed into tubes that were sealed and immediately frozen at −70°C until the analysis. The samples in the strips were eluted with 750 μL of buffer containing 50 mmol/L of phosphate buffer, pH 7.2, and 0.1 mmol/L of phenylmethylsulfonyl fluoride, and shaken at room temperature for 15 minutes. Subsequently, the strips were carefully removed, and the supernatant was divided into 2 aliquots for the determination of each biochemical compound. The amounts of IL-1β and PGE 2 were determined by enzyme-linked immunosorbent assay specific for each compound (Quantikine for IL-1β and Parameter for PGE 2 ; R&D Systems, Minneapolis, Minn). The assays were carried out in accordance with the manufacturer’s instructions, and the levels of the biomarkers were reported as total amount of prostaglandin per 30-second sample. This is in accordance with the suggestion of others who used total amounts rather than concentrations of biochemical parameters because of inherent problems of accurate determination of GCF volumes. The results were calculated against a standard curve set for each biomarker. All samples and standards were assayed in duplicate. Before orthodontic treatment, all patients received oral hygiene instructions about the correct use of a toothbrush and dental floss. The instructions were also reinforced during the treatment.

Masticatory performance was evaluated by the individual capacity of fragmentation of an artificial test food (Optocal-based on Optosil Plus; Heraeus Kulzer GmBH, Hanau, Germany). The subjects were given 17 cubes (3.0 g) and instructed to chew for 15 cycles, which were monitored visually by a trained examiner and timed using a digital stopwatch. After the 15 strokes, the subjects spat the particles into a plastic cup, rinsed their mouth with water, and spat the remaining mouth contents until all particles were removed. The particles were washed and dried for 24 hours in a stove at 60°C. After that, they were sieved through a stack of up to 10 sieves, with square apertures decreasing from 5.6 to 0.5 mm, for 5 minutes. Median particle sizes (X50) were determined as previously described. X50 is defined as the aperture of a theoretical sieve through which 50% of the weight can pass. Disturbances in performance are detected by an increase in X50.

Pain experience was evaluated by a 100-mm visual analog scale, in which the subjects were asked to mark their degree of discomfort (0, no pain; 10, worst pain imaginable) immediately after the masticatory performance test with the artificial food. The patients were asked whether they took any medications during the experimental period (acetaminophen was indicated for orthodontic pain), but no one reported taking analgesics during the study.

Statistical analysis

All variables were evaluated at 3 times: before bracket placement (T0), 24 hours after archwire placement (T1), and 30 days after the initial appointment (T2). Shapiro-Wilk tests were used to verify the normality of the data. Variables were analyzed by Friedman and Dunn tests because they were not normally distributed (intragroup comparisons). The Mann-Whitney test was used for the intergroup comparisons. SPSS statistical software (version 19.0; IBM, Armonk, NY) was used for all analyses, and the significance level was set at P <0.05.


Table I shows the levels of the proinflammatory cytokines IL-1β and PGE 2 in the GCF of the orthodontic patients and the control group in the 3 experimental periods, T0, T1, and T2. In the patient group, a significant increase in the level of IL 1-β was observed at T1 compared with the other periods (T0 and T2) ( P <0.05). The concentration of IL-1β at T1 was also higher in the patients than in the control group ( P <0.05). However, no differences were observed between the patients and the controls at T0 and T2. The PGE 2 levels had no statistical differences, in either the intragroup or the intergroup comparison ( P >0.05).

Table I
Means (and standard deviations) of the levels of proinflammatory cytokines IL-1β and PGE 2 (total amount expressed as prostaglandin per 30-second sample) at the 3 experimental times: before bracket placement (T0), 24 hours after archwire placement (T1), and 30 days after archwire placement (T2)
Cytokines in CGF Patients Control group (normal occlusion)
T0 T1 T2
IL-1β 13.7 (11.6) a 33.7 (19.7) b 23.9 (17.9) a 20.4 (17.6)
PGE 2 304.8 (142.8) a 355.7 (156.9) a 247.2 (159.9) a 332.1 (94.1)
Different letters indicate statistical differences between periods in the patient group (Friedman/Dunn, P <0.05).

Statistical differences in relation to the control group (Mann-Whitney test, P <0.05).

The data on masticatory performance, including the median particle size chewed for 15 cycles (X50), total chewing times and durations of each cycle, and pain experience during mastication, are shown in Table II . In the patient group, the masticatory performance was lower at T1—24 hours after archwire placement ( P <0.05)—and returned to normal at T2. The masticatory performance of the patients was lower than that of the control in all periods evaluated ( P <0.05). The orthodontic patients reported a significant increase in pain at T1 in relation to the other periods ( P <0.05), and all patient values were higher than those of the control group.

Table II
Means (and standard deviations) of pain experience, median particle size chewed for 15 cycles (X50), total chewing times, and durations of each cycle during mastication at the 3 experimental times: before bracket placement (T0), 24 hours after archwire placement (T1), and 30 days after archwire placement (T2)
Variables Experimental times Control group (normal occlusion)
T0 T1 T2
Pain intensity (visual analog scale) (mm) 9.6 a (16.7) 61.3 b (32.8) 13.0 a (22.3) 0.3 (0.8)
Masticatory performance (X50) (mm) 6.6 a (2.2) 8.7 b (2.0) 6.5 a (1.8) 4.7 (1.1)
Total chewing time (s) 11.4 a (1.9) 14.8 b (3.0) 10.9 a (1.4) 11.9 (2.7)
Duration of each chewing cycle (s) 0.8 a (0.1) 1.0 b (0.2) 0.7 a (0.1) 0.8 (0.2)
Different letters indicate statistical differences between periods in the patient group (Friedman/Dunn, P <0.05).

Statistical differences in relation to the control group (Mann-Whitney test, P <0.05).

Table III shows the correlations between masticatory performance (X50), pain, and the levels of IL-1β and PGE 2 in the GCF. A significant positive correlation between masticatory performance and pain was observed at T1 in the patient group. The correlations between masticatory performance or the pain experience and the levels of cytokines were not statistically significant in either the patients or the control group.

Apr 6, 2017 | Posted by in Orthodontics | Comments Off on Association among pain, masticatory performance, and proinflammatory cytokines in crevicular fluid during orthodontic treatment
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