Material and methods

The study protocol was approved by the Human Research Ethics Committee of the Faculty of Dentistry, Chulalongkorn University, Thailand (HREC-DCU 2016-102, 2019-052).

The experimental flow and protocol are shown in Figure 1 . The sample size was calculated from previous studies. ^, The new appliance group had 36 sets of as-received brackets and tubes. Each set comprised 20 brackets and 4 tubes (Tomy International, Tokyo, Japan) with a slot size of 0.018-in. Used appliances (Tomy International) were obtained from 36 nonextraction orthodontic patients (10 males and 16 females, aged between 15-24 years) in the Department of Orthodontics, Faculty of Dentistry, Chulalongkorn University, Thailand. Each patient had 24 fully erupted teeth from the central incisors to the first molars that were bonded with 20 brackets and 4 molar tubes. During treatment, the patients repeatedly received oral hygiene instruction and motivation. They were directed to brush their teeth with a fluoride TP at least twice a day. No other anticarcinogenic product was recommended to the patients. The appliances were removed from the patients’ teeth after their treatment was completed. The average treatment duration was 23 ± 4 months. After debonding from the enamel surfaces, the brackets and tubes were washed with deionized water. To avoid the corrosive effect of typical sterilization techniques, the used appliances were sterilized in a hot air oven (180 ^o C, 60 minutes; Memmert, Schwabach, Germany) instead of using an autoclave or disinfectant.

Sample preparation and experimental flow; x and y indicate the areas of brackets and wires scanned by SEM.

The new and used brackets and archwires were immersed in deionized water and cleaned in an ultrasonic cleaner (Biosonic UC300; Coltene, Altstätten, Switzerland) at 40 kHz for 15 minutes. Each set of brackets and tubes were ligated with two 0.016-in × 0.022-in preformed archwires to simulate full fixed orthodontic appliances from both dental arches. The samples in the used and unused groups were divided into 3 groups (n = 12) by 3 metal types of archwires SS, NT, and TMA (Ormco; Accord, Orange, Calif). The compositions of the archwires are shown in Table I . The archwires were ligated to the brackets with 0.010-in ligature wires (Ormco; Accord). Each set of samples was weighed on a semimicro analytical balance (New Classic MS-S; Mettler Toledo, Greifensee, Switzerland).

The 12 sets of samples in each archwire group were divided into 3 media subgroups (n = 4); artificial saliva ( Table II ) as the CON group and solutions of TP or APF gel. The volume of the CON medium, 1 mL per 0.2 g of the weight of the set, was calculated from ISO10993-5. The TP medium was a mixture of TP containing 1000 ppm of NaF (Colgate Total; Colgate-Palmolive, Thailand) and artificial saliva at a 1:4 (w/v) ratio. The APF medium was prepared by mixing 1.23% APF gel (Pascal International, Bellevue, WA) and artificial saliva at a 1:1.4 (v/v) ratio.

The samples were immersed in their respective media in an incubator shaker at 37 ^o C and 80 rpm. The immersion period was estimated from the recommended time that each fluoride product would be clinically used for 3 months, as described in a previous study.

• 1.

  CON groups: 12 samples were immersed in artificial saliva without fluoride for 5 hours 36 minutes, calculated as in no. 2.

• 2.

  TP groups: 12 samples were immersed in TP solution for 5 hours 36 minutes, simulating the total recommended tooth brushing time for 3 months (ie, 2 minutes per time, twice per day).

• 3.

  APF groups: 12 samples were immersed in APF solution for 4 minutes and dried for 30 minutes, mimicking the recommended APF gel application time (ie, 4 minutes per time, once every 3-6 months).

The samples were washed with deionized water and cleaned in an ultrasonic cleaner at 40 kHz for 15 minutes. Each set of samples was then immersed in 10 mL cell culture medium (Dulbecco’s Modified Eagle’s Medium [DMEM]; Gibco; Thermo Fisher Scientific, Waltham, Mass) at 5 ^o C for 7 days. The DMEM was divided into 2 parts for metal ion measurement and cytotoxicity testing.

Next, 15 mL plastic centrifuge tubes (Corning; Sigma-Aldrich, St. Louis, Mo) were cleaned by soaking in 10% nitric acid overnight and rinsed with deionized water. A total of 3 mL of culture media from each subgroup was dispensed into a tube. Ni, Cr, and iron (Fe) ions were measured using inductively coupled plasma mass spectroscopy (ICP-MS) (iCAP RQ ICP-MS; Thermo Fisher Scientific, Waltham, Mass). Standard solutions (50, 100, and 200 μg/L) of each metal were prepared in DMEM for calibration. DMEM alone was used as a negative CON. The mean of 3 values was calculated in μg/L.

Primary human gingival fibroblasts were isolated from the healthy gingiva over impacted third molars of 3 young adult donors, 2 men and 1 woman, after obtaining informed consent. The average donor age was 18 years 9 months (standard deviation, 1.7 years). The isolated tissue was washed several times with DMEM and cut into 1-2 mm ³ pieces. Five explant pieces were placed into a 35-mm × 10-mm cell culture dish (SPL Life Sciences, Gyeonggi-do, Korea) and incubated with a growth medium (DMEM supplemented with 10% fetal bovine serum, 10,000 IU/mL penicillin G sodium, 100,000 μg/mL streptomycin sulfate, 25 μg/mL amphotericin B, and 1% L-glutamine) at 37°C in a 5% CO ₂ atmosphere. When the outgrown cells reached 80% confluence, the cells were subcultured using 0.25% trypsin- ethylenediaminetetraacetic acid solution at a 12.5:1 ratio. All experiments were performed using cells from the third to the fifth passage. All media supplements were purchased from Gibco.

The gingival fibroblasts were seeded in 24-well plates at a concentration of 40,000 cells per well, incubated as above for 24 hours (approximately 80% confluent), washed twice with phosphate-buffered saline, and the wells were divided into 2 CON groups and 9 experimental groups and treated for 3 days as follows:

• 1.

  Positive CON: cells incubated with 1 mL 20% ethanol in DMEM per well. ^,

• 2.

  Negative CON: cells incubated with 1 mL DMEM per well.

• 3.

  Experimental groups: cells incubated with 1 mL culture media immersed with samples (SS-CON, NT-CON, TMA-CON; SS-TP, NT-TP, TMA-TP; SS-APF, NT-APF, and TMA-APF).

Subsequently, an MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay was performed for cytotoxicity testing as previously described. Briefly, the medium was removed, the cells were washed twice with phosphate-buffered saline and incubated with 250 μL 1 mg/mL MTT solution (Molecular Probes, Eugene, OR) in DMEM without phenol red for 10 minutes. The MTT solution was removed, and the precipitated formazan crystals were dissolved in 500 μL dimethyl sulfoxide. The optical density was determined by measuring the light absorbance at 570 nm using an Epoch Microplate Spectrophotometer (BioTek Instrument, Winooski, Vermont). The background absorbance of dimethyl sulfoxide was subtracted from the sample absorbance, and the experiments were performed 3 times. The percent cell viability was calculated using the following formula:

Cell viability (%) = (OD of the experimental group/OD of the negative CON) × 100

Four maxillary central incisor brackets and pieces of the archwire that were in contact with the brackets were randomly chosen from each experimental group. The as-received and untreated brackets and wires were used as a negative CON. The samples were cleaned in deionized water in an ultrasonic cleaner at 40 kHz for 15 minutes, wiped and left until dry and scanned by scanning electron microscope ( SEM) (FEI, Eindhoven, Netherlands) at 20 kV and 5000× magnification. The bracket area near the slot was scanned at position x, and the middle area of the wires was scanned at point y, which was adjacent to x ( Fig 1 , A ).

The approximately 400 × 400 μm ² area at position x and y ( Fig 1 , A ) on the bracket and wire surfaces were scanned by a noncontact optical 3-dimensional surface characterization and roughness measuring device (Infinite focus SL, Alicona, Graz, Austria) to evaluate the surface roughness of the samples at 50x magnification. Each sample was scanned 3 times. The data was analyzed by IF-MeasureSuite software (Alicona, Graz, Austria).

Results

The mean and standard deviation values for the metal ion release, cell viability, and surface roughness of the brackets and wires are presented in Table III .

Table III

Mean and standard deviation of metal ion release and cell viability

Groups	Cr (μg/L)		Fe (μg/L)		Ni (μg/L)		Cytotoxicity(%)
	New	Used	New	Used	New	Used	New	Used
SS
CON	0.84 ± 0.10	35.32 ± 21.70	255.13 ± 11.0	756.02 ± 254	5.36 ± 0.07	84.02 ± 11.38	90.28 ± 7.19	70.29 ± 5.50
TP	0.60 ± 0.04	134.45 ± 22.62	281.99 ± 2.95	1327.1 ± 251.1	5.86 ± 0.41	148.37 ± 62.13	83.53 ± 6.09	53.71 ± 9.02
APF	112.01 ± 10.88	166.3 ± 19.58	641.36 ± 36.14	2236.1 ± 487.5	62.81 ± 5.85	845.32 ± 138.3	67.23 ± 7.08	53.71 ± 5.99
(−)	0.07 ± 0.02	0.07 ± 0.02	233.84 ± 6.00	233.84 ± 6.00	0.83 ± 0.10	0.83 ± 0.10	97.19 ± 1.8	96.4 ± 2.32
(+)	–	–	–	–	–	–	1.91 ± 0.33	1.83 ± 0.38
NT
CON	0.48 ± 0.02	26.60 ± 13.24	271.82 ± 12.61	270.61 ± 16.16	7.17 ± 0.42	121.02 ± 138.3	81.60 ± 4.32	65.97 ± 7.75
TP	0.54 ± 0.05	64.04 ± 8.66	264.37 ± 12.51	406.53 ± 293.5	8.38 ± 0.36	145.80 ± 42.82	70.51 ± 4.66	49.77 ± 8.04
APF	44.38 ± 4.40	105.46 ± 13.53	562.02 ± 26.7	1795.7 ± 69.86	265.64 ± 27.89	1844.8 ± 249.6	53.71 ± 5.99	30.54 ± 6.63
(−)	0.12 ± 0.07	0.12 ± 0.07	243.78 ± 4.53	243.78 ± 4.53	0.67 ± 0.09	0.67 ± 0.09	100.26 ± 5.94	101.19 ± 4.48
(+)	–	–	–	–	–	–	1.50 ± 0.37	1.36 ± 0.49
TMA
CON	1.01 ± 0.12	3.64 ± 2.57	256.49 ± 20.42	290.51 ± 4.29	6.27 ± 0.10	115.81 ± 34.86	73.51 ± 4.87	63.51 ± 6.27
TP	0.21 ± 0.02	13.45 ± 10.41	297.83 ± 18.83	377.6 ± 164.2	3.82 ± 0.35	300.02 ± 78.98	53.88 ± 13.56	41.38 ± 8.47
APF	34.22 ± 3.12	98.25 ± 8.12	383.03 ± 30.21	1240.0 ± 111.5	121.65 ± 9.69	402.79 ± 80.50	9.62 ± 3.76	2.77 ± 0.88
(−)	0.12 ± 0.02	0.12 ± 0.02	237.06 ± 8.90	237.06 ± 8.90	0.74 ± 0.04	0.74 ± 0.04	96.6 ± 3.95	99.4 ± 2.05
(+)	–	–	–	–	–	–	1.63 ± 0.51	1.62 ± 0.53

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