Material and methods

This longitudinal study was carried out with 72 samples of GCF, taken at 3 times from 24 patients (12 male, 12 female) with fixed orthodontic appliances. The times were (1) pretreatment (baseline), (2) 1 month after the initiation of treatment, and (3) 6 months after baseline.

The research committee of The Azad University approved the protocol and ethics (registered as an orthodontics master’s thesis #2013-t33 for M.S.). Written consents were taken from the participants or their parents. The subjects were sequentially acquired from a list of about 100 patients attending the Department of Orthodontics (in 2011 and 2012), until 2 groups of 12 female and 12 male patients were enrolled.

The inclusion criteria were patients who were willing to be part of the study and needed fixed orthodontic treatment in the maxillary arch. The exclusion criteria were any diseases, syndromes, allergies, metal restorations, consumption of medication or alcohol, smoking, or previous orthodontic treatment. All inclusion criteria had to be fulfilled during the study period.

Only the maxillary arch was treated. Stainless steel brackets (American Orthodontics, Sheboygan, Wis) were bonded using NoMix adhesive (3M ESPE, St Paul, Minn). To reduce the confounders, only nickel-titanium archwires (G&H Wire, Franklin, Ind) were used during the 6-month study (stainless steel archwires were not used). The archwires were replaced with greater sizes about every 4 to 6 weeks, depending on the treatment plan. Within the 6-month period of the study, in 15 patients, all wires were 0.014-, 0.016-, and 0.018-in round wires, used in that order. In 6 patients, the wires were 0.016- and 0.018-in round wires, used sequentially. In 3 patients, the archwires were 0.012-, 0.014-, and 0.016-in round wires followed by 0.019 × 0.025-in square nickel-titanium wires.

Oral hygiene maintenance was taught to the patients before the study, both orally and in written form. Patients were asked to use 1 type and brand of toothpaste (Crest Regular, Procter & Gamble, Cincinatti, Ohio) during the study period. The sampling session was scheduled in the morning. The subjects were told not to brush their teeth on the morning of the sampling session. The patients were instructed, orally and in written form, to avoid consuming nickel- and chromium-rich foods for 48 hours before the sampling visit; they were also asked not to eat or drink on the morning of the scheduled visit.

The sampling was carried out 3 times (before treatment, and 1 month and 6 months later) by a senior orthodontics graduate student (M.S.) trained and calibrated by a periodontist. The teeth and gingivae were not sprayed with water or rinsed to prevent removal of the GCF. To eliminate saliva contamination, the teeth and surrounding gingivae were gently air dried. The region was isolated with cellulose strips. A standardized cellulose acetate absorbent strip with 45-μm micropores (PerioPaper; Oraflow, New York, NY) was used for the GCF collection. It was gently placed in the sulcus for 60 seconds. In each patient, 4 sites for GCF collection were randomly selected in the first sampling session (from the midbuccal sulci and proximal embrasures of the maxillary central and lateral incisors, canines, and premolars) based on the lack of bleeding and the possibility of inserting the PerioPaper strip for at least 1 mm. If a site or tooth showed bleeding on sampling, after the bleeding stopped, other sites or teeth were randomly selected and sampled. In addition, if calculus did not allow inserting the strip, another site was randomly selected. Moreover, if a specific sulcus did not completely wet the strip within 60 seconds (determined macroscopically), the strip was discarded, and a new one was used to sample the gingival fluid from another randomly selected site. After each successful sampling, the PerioPaper strip was removed and placed in a glass container with a lid. Each bottle was filled with 4 strips, each soaked only in 1 site from each patient (no PerioPaper strip was used to sample from more than 1 site). The specimens were kept in a refrigerator at 1°C for a maximum of 1 week and then shipped to the laboratory. The sites of sampling in the first session were recorded for each patient to be sampled in the second and third sessions as well, if they maintained proper conditions for sampling. If the sites were not available, a new sampling site would be randomly selected instead of each site that had bleeding or calculus or insufficient GCF. The strips were weighed before and after GCF sampling (AB204-S; Mettler-Toledo International, Greifensee, Switzerland).

As an additional finding, the gingival index of Loe was used to evaluate and rank gingival health. This index categorizes the health level with the following scores: 0, no inflammation; 1, mild inflammation, slight discoloration, and slight edema, without ulceration or spontaneous bleeding; 2, moderate inflammation, redness, and bleeding on probing; and 3, severe inflammation and considerable hypertrophy, ulceration, and spontaneous bleeding.

The specimens were transferred to the Atomic Energy Organization for atomic absorption spectrophotometry with a calibrated device (AA280Z GTA120; Varian, Mulgrave, Australia). They were then burned at 550°C for 1 hour. The resulting powder was digested by adding hydrochloric acid and placing it in an ultrasonic bath, at the same time. Each specimen was examined 3 times, and the average ion concentration was recorded in parts per million (μg/g of GCF).

Results

The patients’ mean age was 16.3 ± 3.5 years (range, 13-22 years). There was no significant correlation between the ion levels except for the pair of nickel levels at the baseline and the sixth month ( Fig ; Table I ).

Box plots showing the medians, interquartile ranges, and minimums/maximums (excluding the outliers [ asterisk ]) for nickel ( Ni ) and chromium ( Cr ) levels (μg/g GCF) at baseline ( 0 ), the first month ( 1 ), and the sixth month ( 6 ).

Compared with the baseline levels, chromium increased about 200% and 700%, respectively, in the first and sixth months of treatment ( Fig ; Table II ). Nickel showed increases of about 150% and 510% compared with the baseline, respectively, in the first and sixth months ( Fig ; Table II ). The repeated-measures analysis of variance and Friedman tests identified the changes as significant (both P values <0.0001). All pairwise comparisons were significant ( Table III ).

Table III

Results of Bonferroni test, comparing the estimated marginal means (μg/g of GCF) of the chromium and nickel groups

Ion	Time (I)	Time (J)	Mean diff (I-J)	SE	P	95% CI diff
Cr	Baseline	Month 1	−2.158	0.347	0.000007	−3.053	−1.263
	Baseline	Month 6	−11.782	0.720	0.000000	−13.641	−9.924
	Month 1	Month 6	−9.625	0.746	0.000000	−11.551	−7.698
Ni	Baseline	Month 1	−2.019	0.609	0.001591	−3.590	−0.448
	Baseline	Month 6	−15.916	1.913	0.000018	−20.856	−10.976
	Month 1	Month 6	−13.897	1.884	0.000030	−18.761	−9.032

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