Material and methods

Twenty-three patients (11 male, 12 female), aged 10 to 24 years (mean, 13.43 ± 2,64 years), seen in the Department of Pediatric Dentistry and Orthodontics, Faculty of Dentistry, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil, from August 2011 to July 2013, were selected to participate in this study. All patients required extraction of their premolars for orthodontic reasons, such as crowding, dental biprotrusion, or Class II malocclusion, before orthodontic treatment. The inclusion criteria were as follows: (1) healthy patients with no evidence of type 1 or type 2 diabetes mellitus or osteoporosis; (2) patients who had not taken systemic antibiotics, or anti-inflammatory or hormonal drugs for 6 months before the study; (3) patients who required tooth extractions before treatment with fixed appliances; and (4) patients with good periodontal health and no radiographic evidence of periodontal bone loss.

This study was approved by our institutional ethics committee (protocol number 175.320/2012) and conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from each participant and his or her guardian if the patient was less than 18 years of age. At the first appointment (baseline), the Silness and Löe plaque index and the gingival bleeding index were recorded to ensure that the patients had no periodontal alteration at the start of the study. The mandibular or maxillary premolars without orthodontic appliances were used as the controls. The experimental group consisted of the contralateral teeth from the same arch that previously received orthodontic mechanical loading. The orthodontic appliance consisted of 0.022 × 0.028-in light Roth tubes (Eurodonto, Curitiba, Paraná, Brazil) and brackets (Aditek do Brasil, Cravinhos, São Paulo, Brazil) bonded with Transbond XT (3M Unitek, Monrovia, Calif). A 0.017 × 0.025-in beta-titanium alloy cantilever and a 0.010-in metallic ligature (Morelli Orthodontics, Sorocaba, São Paulo, Brazil) were placed between the premolar and the first molar on the same side by an orthodontist (D.F.M.) ( Fig 1 , A ). A force in the apical direction was applied to the premolar. The force magnitude was 0.980 N, measured with a digital tensiometer (model FGV-1X; Nidec-Shimpo, Itasca, Ill) that was perpendicular to cantilever ( Fig 1 , B ). No other forces were applied to the teeth before or during this phase. The experimental teeth were randomly selected. If a patient had 4 premolars to be extracted, pairs of teeth were allocated to 2 time points. The patients were instructed about proper oral hygiene. After mechanical stimuli, the teeth were extracted at these time points: 1, 3, 7, 14, 21, or 28 days. Just before the extractions, the force magnitude was measured again. The experimental and control teeth were extracted at the same time. Several time points were chosen to allow the evaluation of cytokine fluctuation after mechanical stimuli concomitantly in the PDL and GCF. After the experimental period, the patients were referred to start orthodontic treatment.

A, An activated orthodontic appliance consisting of a 0.022 × 0.028-in bracket and tube bonded with light-cured adhesive and a 0.017 × 0.025-in beta-titanium alloy cantilever; B, the force was set at 0.980 N in the apical direction, and the force magnitude was measured with a digital tensiometer; C, the PDL before scraping.

GCF samples were collected using periopaper strips (gingival fluid collection strips; Oraflow, Smithtown, NY) at the mesiobuccal and distobuccal sites of the control and experimental premolars. The teeth were previously isolated with cotton rolls, cleaned of plaque deposits, and dried gently with air before the paper strips were carefully inserted 1 mm into the gingival crevice for 30 seconds. Samples of GCF absorbed by the periopapers were measured using a calibrated Periotron 8000 (model 400 8000 precision gingival crevice fluid meter; Oraflow). Readings from the Periotron were converted to an actual volume (microliters) by reference to a standard curve calibrated with human serum. The PDL of each extracted tooth was taken from all root surfaces and immediately scraped using a 13/14 Gracey curette (Maximus, Contagem, Minas Gerais, Brazil) ( Fig 1 , C ). All tooth extractions and sample collections were performed by 1 professional (D.F.M.).

Samples of GCF and PDL were separately placed in sterile tubes that were immediately immersed in liquid nitrogen, and then kept frozen at −80°C for further analysis. Afterward, the GCF and PDL samples were separately homogenized in phosphate-buffered saline solution (0.4 mmol/L of sodium chloride and 10 mmol/L of sodium phosphate containing protease inhibitors [0.1 mmol/L of phenylmethylsulfonyl fluoride, 0.1 mmol/L of benzethonium chloride, 10 mmol/L of ethylenediaminetetraacetic acid, 0.01 mg/mL of aprotinin A, and 0.05% Tween-20 at 1 mg/mL]). The mixture was agitated on vortex for 30 seconds and then centrifuged (12.000 g) for 10 minutes at 4°C. Supernatants were collected and assayed with the cytometric bead array (CBA). Analyses of PDL and GCF were determined using a BD CBA Human Th1/Th2/Th17 Cytokine Kit (Becton, Dickinson and Company, BD Biosciences, San Diego, Calif) and analyzed on a BD FACSCalibur flow cytometer (Becton, Dickinson and Company). The concentrations of analytes (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon-gamma [IFN-γ], and tumor necrosis factor-alpha [TNF-α]) were measured using a standard curve according to the manufacturer’s instructions. The sensitivity of the assays was 20 to 5000 picograms per milliliter for all cytokines. The results were expressed in picograms per milliliter for GCF and picograms per 100 mg of tissue for the PDL.

Results

A total of 68 premolars were obtained (36 maxillary first premolars, 2 maxillary second premolars, 28 mandibular first premolars, and 2 mandibular second premolars). A mean of 5.67 (n = 5-6) pairs of teeth was allocated at each time point. The appliances were well tolerated. Demographic descriptions of the participants are in Table I . The clinical gingival conditions were satisfactory at baseline, and no significant difference was seen for the plaque index or the gingival bleeding index between the groups. Although no difference between GCF volumes was seen on days 1, 3, 14, or 28 ( P >0.05), increased volumes of GCF were seen on days 7 and 21 for the experimental group ( P <0.05). The initially applied force magnitude of 0.980 N was gradually reduced to a mean of 0.866 ± 0.166 N just before extraction of the experimental teeth. Sex, type of tooth, age of participants, and experimental force had no influence on the concentrations of the examined cytokines at any time ( P >0.05).

Figure 2 , A shows cytokine concentrations of Th2 response: interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-10 (IL-10). Th17, interleukin 17-A (IL-17A), and Th1, TNF-α, and IFN-γ levels are shown in Figure 2 , B . On day 1, a significant increase of IL-6 was seen in the PDL subjected to mechanical stress in relation to the control ( P = 0.043). Then, the IL-6 levels in the PDL were gradually reduced. In GCF, the peak of IL-6 was later than that observed for the PDL on day 3, with a subsequent decrease. On day 21, IL-6 concentration in the GCF of the experimental group was lower than that of the control ( P = 0.028). Descriptive analyses showed a similar phenomenon in the GCF for IL-10, IL-17A, and IFN-γ. Those cytokines seemed to increase at the beginning of orthodontic tooth movement in the experimental group, followed by a subsequent decrease at later stages, although there were no significant differences between the groups for any of these evaluated molecules ( P >0.05).

A, Time course of Th2 (IL-2, IL-4, IL-6, IL-10) cytokine production in the PDL and GCF. In the experimental group, increased levels of IL-6 in the PDL on day 1 and reduced levels of IL-6 in the GCF on day 21 were observed. The data are expressed as mean ± SD.* P <0.05 comparing the groups at the same time point. Paired Student t tests were used for IL-2 PDL; IL-2 GCF; IL-4 PDL; and IL-10 PDL; and Wilcoxon tests for IL-4 GCF; IL-6 PDL; IL-6 GCF; and IL-10 GCF. B, Time course of Th1 (TNF-α and IFN-γ) and Th17 (IL-17A) cytokine production in the PDL and GCF, in which no statistical difference was observed.* P <0.05 comparing the groups at the same time point. The paired Student t test was used for TNF-α PDL; Wilcoxon tests were used for TNF-α GCF; IFN-γ PDL; IFN-γ GCF; IL-17A PDL; and IL-17A GCF.

The correlations of cytokines in the PDL and GCF are described in Table II . Interestingly, a significantly strong positive correlation in the experimental group was observed on days 3 (IFN-γ), 7 (IL-10), 14 (IL-17A), and 28 (IL-17A, TNF- α), with significantly strong negative correlations on days 14 (IFN-γ) and 21 (IL-2, IL-10). For the control group, significantly strong negative correlations were seen on days 1 (IL-4), 21 (IFN-γ), and 28 (IL-2, IFN-γ)

Table II

Correlations of cytokine levels between the PDL and GCF

	Day 1	Day 3	Day 7	Day 14	Day 21	Day 28
Experimental, GCF vs PDL
IL-2	−0.355	0.669	0.209	−0.115	−0.916 ∗	−0.216
IL-4	−0.229	−0.527	0.714	0.447	−0.551	0.103
IL-6	−0.600	0.600	0.714	−0.400	0.029	0.500
IL-10	−0.224	0.200	0.899 ∗	−0.359	−0.899 ∗	−0.600
IL-17A	−0.395	−0.289	−0.185	0.894 ∗	−0.417	0.895 ∗
IFN-γ	−0.148	0.900 ∗	0.086	−0.949 ∗	−0.516	−0.300
TNF-α	−0.053	0.400	0.235	−0.400	0.058	0.900 ∗
Control, GCF vs PDL
IL-2	0.862	0.251	0.147	0.737	0.054	−0.891 ∗
IL-4	−903 ∗	0.708	0.800	0.505	0.549	0.052
IL-6	0.300	0.400	0.429	0.400	0.116	0.800
IL-10	−0.483	0.141	0.611	0.392	0.245	−0.041
IL-17A	−0.860	−0.287	−0.290	0.112	−0.417	−0.447
IFN-γ	−0.103	0.700	0.429	0.205	−0.899 ∗	−0.800
TNF-α	−0.414	−0.095	−0.168	−0.140	0.104	−0.301

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