In vivo investigation of gingival health and oxidative stress changes in patients undergoing orthodontic treatment with and without fluoride use

Abstract

Background and Aims:

We aimed to investigate gingival index changes and oxidative stress in orthodontic patients with and without the use of fluoridated agents over a 6-month period.

Materials and Methods:

Ninety subjects divided into three groups (30 untreated controls, 30 with fixed appliances using nonfluoridated toothpaste, and 30 with fixed appliances using fluoridated toothpastes and mouthwashes ) comprised the sample. The Loe gingival index was used to rank gingival health at four specific time periods in the groups to determine differences (before, at 7 days, 30 days, and 6 months). Gingival crevicular fluid was evaluated for cytokines (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) to determine differences in oxidative status between the groups.

Results:

Controls showed no changes in gingival index throughout the 6-month observation period. There was a deterioration in gingival health in both the fluoridated and nonfluoridated groups till 6 months. IL-1β levels in the fluoridated group increased from the 7^th day, reached a peak at 30 days, and remained slightly elevated at 6 months. The nonfluoridated group also showed elevated levels at all tested time periods, but levels were lower as compared to fluoridated samples. The differences in IL-1β values between the fluoridated and nonfluoridated treated groups were significant. TNF-α levels in the three groups also showed a similar pattern with elevated levels seen in both the treated groups at the 7^th, 30-day, and 6-month periods. The fluoridated group showed the highest levels at three time periods which was statistically significant.

Conclusions:

Gingival health of subjects treated with fixed orthodontic appliances deteriorated from appliance placement till a 6-month time period. Cytokines such as IL-1β and TNF-α associated with oxidative stress during orthodontic treatment increased in both the treated groups, with higher levels in fluoridated subjects. Long-term consequences of oxidative stress changes need further investigation.

INTRODUCTION

Fixed orthodontic appliances are known to cause plaque retention with enlargement of gingival tissues due to inflammation. Periodontal diseases including gingivitis, periodontitis, and dental caries are the most commonly occurring dental diseases which have plaque as an etiological factor.[1] In order to prevent deterioration in gingival health during orthodontic treatment, plaque control using mechanical methods in conjunction with oral hygiene agents such as toothpastes and mouthwashes has been found most effective. Adequate plaque control in orthodontic patients is difficult due to the presence of metal bands, archwires, elastomeric modules, and stainless steel ligatures.[2] Findings of studies related to gingival tissue changes during orthodontic therapy have been variable. Gingival enlargement during orthodontic treatment was supposed to be due to bands, chemical irritation from cement, and food impaction.[3] Plaque microbiota and related byproducts were also listed as a factor for gingival enlargement during orthodontic treatment.[4] Positive associations between orthodontic treatment and development of gingivitis and gingival enlargement were confirmed by multiple researchers.[5,6] Orthodontic patients with fixed appliances have increased plaque retention and varying amounts of gingival enlargement due to difficulty in maintaining optimum oral hygiene.[7] Fluoride-containing toothpastes and mouthwashes are commonly prescribed during orthodontic treatment to prevent enamel demineralization and white spots. Adolescents with orthodontic appliances using mouthwashes with chlorhexidine showed reduced gingivitis and plaque.[8] Gingival health in subjects using different fluoride toothpastes was evaluated in order to determine if there was any demonstrable effect on gingivitis.[9] The findings in the study by Pedersen et al.[9] showed that toothpastes with fluorides, enzymes, and proteins were superior to regular fluoride toothpastes in maintaining gingival health and also for parameters such as gingival inflammation, plaque formation, and gingival bleeding.

Orthodontic force application leads to tissue injury and an inflammatory response.[10] Mediators of inflammation called cytokines are released during fixed orthodontic treatment when mechanical forces are generated, which in turn are transferred to cells.[11] The presence of cytokines is supposed to accurately predict local microenvironmental factors in periodontal tissues and also play an important role in bony remodeling.[12,13] The cells that play a role in inflammation during orthodontic treatment migrate to periodontal tissues where oxidative stress due to orthodontic forces may cause the release of interleukin (IL)-1β and tumor necrosis factor (TNF)-α, both pro-inflammatory cytokines.[14] Cytokines which are anti-inflammatory are also produced at the same time which together regulate the inflammatory process.[15]

Oxidative stress-related changes during fixed orthodontic treatment have also been attributed to brackets, wires, and bands which contain nickel and chromium in varying proportions.[16] Nickel leaching from fixed appliances with subsequent damage to DNA was discussed in previous studies.[17,18] Therefore, there is some evidence linking nickel release during orthodontic treatment to development of oxidative stress. The rationale of the study was: (1) to determine changes in the gingival index of subjects undergoing fixed orthodontic treatment using fluoridated and nonfluoridated oral hygiene agents and (2) to assess changes in specific cytokines, if any, to determine oxidative stress damage when subjects used these oral hygiene agents.[12,13,14,15] The gingival index of Loe and Silness was used to rank gingival health in three groups of subjects. Oxidative stress levels were assessed in controls and orthodontic patients by analyzing gingival crevicular fluid (GCF) as a biomarker. The authors hypothesized that there would be no differences in gingival health among groups. Fluoride agents in toothpastes and mouthwashes were previously implicated in higher metal ion release[19] from orthodontic brackets and wires during treatment. The release of metal ions such as nickel and chromium could cause oxidative stress changes affecting cells in the oral cavity in the long term. In vivo studies of this nature have not been previously carried out. Information obtained could pave the way for better understanding of the inflammatory process that causes tooth movement and effects, if any, of oxidative stress changes on buccal mucosa cells.

MATERIALS AND METHODS

Ninety subjects (68 females and 22 males) were divided into three groups of 30 each. Power analysis utilizing GPower software determined sample size. Group 1 comprised untreated controls and Groups 2 (nonfluoridated) and 3 (fluoridated) were patients requiring fixed orthodontic treatment. Inclusion criteria were patients requiring nonextraction fixed orthodontic treatment between the ages of 12 and 35 years, all permanent teeth till 2^nd molars present, nonsmokers, teetotalers, and no medications like antibiotics used in the preceding 6 months. Ethical approval was granted prior to beginning and written informed consent obtained from all subjects participating. A single investigator treated all patients in the study. All patients had similar fixed appliances placed with a predetermined wire sequence till the 6-month study period was completed in order to make suitable comparisons. Fixed orthodontic appliances were bonded in patients of Groups 2 and 3 in both arches. Stainless steel brackets (Mini Twin 0.022 slot, Ormco Corporation, Glendora, CA, USA) were placed with Enlight light-cured adhesive (Ormco Corporation, Glendora, CA, USA). Only nickel–titanium (NiTi) archwires were used in the study. The archwires used were 0.014” NiTi, 0.016” NiTi, and 16 × 22” NiTi (Tru-Arch Align NiTi; Ormco) till 6 months of treatment. Each archwire was used for 2 months and then replaced with the subsequent preselected wire. Oral hygiene maintenance protocols were individually taught to all patients before commencement of treatment. The untreated control group and Group 2 patients used only nonfluoridated toothpaste (Dabur Red, Dabur India Ltd.) for oral hygiene maintenance twice daily. Patients in Group 3 used a fluoridated mouthwash (Colgate Plax, 225 ppm fluoride, Colgate Palmolive Co., India) and a fluoridated toothpaste twice daily (Colgate Strong Teeth, 1000 ppm fluoride, Colgate Palmolive Co., India).

Evaluation of the gingival index

The Silness and Loe gingival index[20] helped evaluate periodontal status at four specific time periods (before, 7 days, 30 days, and 6 months) in all subjects. The index was developed in 1963 to assess gingivitis severity and its location in four specific regions of gingival tissues with a qualitative examination. Instrumentation for measurement included a graduated periodontal probe and mouth mirror. Prior to measurements, the gingiva and teeth were air dried.

Oxidative stress evaluation

Collection of GCF was at four definite time periods, before treatment, 7 days after commencement, 30 days after commencement, and 6 months after commencement. The teeth were not cleaned or washed prior to sample collection. The samples were obtained from the maxillary canine area in all subjects [Figure 1]. The mandibular arch was not considered due to the difficulty in isolation. The opposite side maxillary canine sulcus was used to obtain GCF in cases where regular side samples showed evidence of contamination with blood. Standardized 5 μl graduated micropipettes (Sigma-Aldrich Co., St. Louis, USA) placed in the gingival sulcus were utilized for GCF collection. Collection time for 3–5 μl of GCF took between 10 and 35 min because healthy subjects, in general, had lesser flow rates and quantities. An Eppendorf tube with 2 ml of phosphate-buffered saline at a pH of 7.4 (Sigma-Aldrich, St. Louis, USA) was used to transfer GCF. The GCF samples were refrigerated at-15°C till they were assayed. Collected GCF was evaluated for IL-1β and TNF-α with enzyme-linked immunosorbent assay method in accordance with instructions of the producer (Elabscience, USA). Samples and standards were run in duplicate during the process of assaying, and cytokine concentrations for both were determined with the help of a calibration curve. The limit for detection for IL-1β and TNF-α was 4.69 pg/ml.

Figure 1.

Materials and Methods. (a) Eppendorf tube; (b) Micropipettes; (c) Enzyme-linked immunosorbent assay plate reader; (d) Gingival crevicular fluid collection

Statistical methods

The sample size was estimated using GPower v. 3.1.9.2 software(University of Dusseldorf, Dusseldorf, Germany). Considering the effect size to be measured (f) at 40%, power of the study at 80%, and margin of error at 5%, the total sample size needed was 66. The final sample size was rounded off to 75. Hence, each study group comprised 25 samples. An additional five samples per group were included to consider dropouts if any. The Statistical Package for the Social Sciences (SPSS) for Windows, version 22.0, 2013, Armonk, NY, USA, IBM Corp., was used for analysis of data. Descriptive statistics using frequency and proportions was used for categorical variables and means and standard deviation for continuous variables. Untreated controls showed no changes in the gingival index or levels of cytokines during tested periods.

Gingival index

Mean gingival index scores between all the three groups at specific intervals were assessed with Friedman's test. Kruskal–Wallis test followed by Mann–Whitney U-test compared mean gingival index scores at different times in each group. The level of significance (P value) was set at P < 0.05.

Oxidative stress

Repeated measures of analysis of variance and Bonferroni's post hoc analysis compared mean IL-1β and TNF-α in the groups at different time intervals. The level of significance (P value) was set at P < 0.05.

RESULTS

Demographic data are depicted in Table 1. There was no change in mean gingival index of the baseline group [Table 2]. However, a significant increase was noted in mean gingival index scores in Groups 2 and 3 from baseline to the 6-month period, as seen in Tables 3 and 4. The Kruskal–Wallis test comparing mean gingival index scores between the three groups at different time intervals revealed almost similar values in Group 2 nonfluoridated (1.07 ± 0.32 at 30 days) and Group 3 fluoridated (1.06 ± 0.38 at 30 days) subjects [Table 5]. Analysis of mean IL-1β showed significant differences between the tested groups (P < 0.0001) at 7 days with a mean difference of 0.053 (Group 1), 0.630 (Group 2), and 0.692 (Group 3) from baseline. At 30 days, mean differences in Group 1, Group 2, and Group 3 were 0.09, 1.765, and 6.746, respectively, with maximum difference in the fluoridated group. Group 1 showed significantly lesser levels than Groups 2 and 3 at all intervals. At the 30-day and 6-month period, mean IL-1β levels in Group 3(Fluoridated) reduced significantly as compared to Non- Fluoridated Group 2 (10.788, 11.480, 17.534, 12.582 at 6 months) with P < 0.001, as seen in Figure 2.

Table 1.

Demographic data

Age-wise distribution of study participants
Groups	n	Mean±SD	Minimum	Maximum	χ 2	P
Control	30	25.93±4.961	14	34	1.139a	0.566 (NS)
Nonfluoridated	30	26.13±4.652	15	34
Fluoridated	30	25.23±4.561	15	34
Gender-wise distribution of study participants
Groups	Male, n (%)		Female, n (%)	Total, n (%)	χ 2	P
Control	14 (46.7)		16 (53.3)	30 (100)	0.814b	0.665 (NS)
Nonfluoridated	11 (36.7)		19 (63.3)	30 (100)
Fluoridated	14 (46.7)		16 (53.3)	30 (100)

^aKruskal-Wallis test, ^bChi-square test. NS – Nonsignificant difference; SD – Standard deviation; n – Number of subjects; χ² – Chi- square; P – Probability value

Table 2.

Comparison of mean gingival index scores between different time intervals in control group using Friedman’s test

Group	Time	n	Mean±SD	Minimum	Maximum	P
Group 1	Before	30	0.40±0.17	0.10	0.70	0.015*
	7 days	30	0.42±0.11	0.15	0.75
	30 days	30	0.41±0.18	0.10	0.75
	6 months	30	0.40±0.17	0.10	0.70

*Statistically significant at P<0.05. Group 1 – Control; SD – Standard deviation; n – Number of subjects; P – Probability value

Table 3.

Comparison of mean gingival index scores between different time intervals in Group 2 using Friedman’s test

Group	Time	n	Mean±SD	Minimum	Maximum	P
Group 2	Before	30	0.58±0.16	0.3	0.8	<0.001*
	7 days	30	0.75±0.19	0.5	1.3
	30 days	30	1.07±0.32	0.8	1.8
	6 months	30	1.13±0.31	0.8	1.8

*Statistically significant at P<0.05. Group 2 – Nonfluoridated; SD – Standard deviation; n – Number of subjects; P – Probability value

Table 4.

Comparison of mean gingival index scores between different time intervals in Group 3 using Friedman’s test

Group	Time	n	Mean±SD	Minimum	Maximum	P
Group 3	Before	30	0.53±0.17	0.3	0.8	<0.001*
	7 days	30	0.79±0.17	0.5	1.0
	30 days	30	1.06±0.38	0.5	2.0
	6 months	30	1.15±0.36	0.8	2.0

*Statistically significant at P<0.05. Group 3 – Fluoridated group; SD – Standard deviation; n – Number of subjects; P – Probability value

Table 5.

Comparison of mean gingival index scores between three groups at different time intervals using Kruskal-Wallis test

Time	Group	n	Mean±SD	χ 2	P
Before	Group 1	30	0.40±0.17	17.504	0.001*
	Group 2	30	0.58±0.16
	Group 3	30	0.53±0.17
7 days	Group 1	30	0.42±0.17	45.335	0.001*
	Group 2	30	0.75±0.19
	Group 3	30	0.79±0.17
30 days	Group 1	30	0.41±0.18	58.629	0.001*
	Group 2	30	1.07±0.32
	Group 3	30	1.06±0.38
6 months	Group 1	30	0.40±0.17	63.873	0.001*
	Group 2	30	1.13±0.31
	Group 3	30	1.15±0.36

*Statistically significant at P<0.05. Group 1 – Control; Group 2 – Nonfluoridated group; Group 3 – Fluoridated group; SD – Standard deviation; n – Number of subjects; P – Probability value

Figure 2.

Mean interleukin-1β levels between three groups at different time intervals

The Bonferroni test is a multiple comparison test that ensures statistical significance of the data being presented. In the multiple comparison tests for IL-1β at four time periods, it can be observed that maximum mean differences in Group 2 (nonfluoridated) and Group 3 (fluoridated) are at 30 days. In Group 3, the time comparisons for before treatment and 30 days show a mean difference of −6.7. For 7 days and 30 days, the mean difference is −6.0. This points to maximal release of IL-1β at the 30-day posttreatment period which can be considered a significant finding [Table 6].

Table 6.

Multiple comparison of mean differences of interleukin-1β levels between different time intervals in each study group using Bonferroni’s post hoc analysis

Group	Time (I)	Time (J)	Mean difference (I-J)	95% CI for difference		P
				Lower	Upper
Group 1	Before	7 days	−0.053	−0.084	−0.023	<0.001*
		30 days	−0.09	−0.125	−0.055	<0.001*
	6 months	−0.098	−0.138	−0.058	<0.001*
	7 days	30 days	−0.037	−0.056	−0.017	<0.001*
	6 months	−0.045	−0.066	−0.024	<0.001*
	30 days	6 months	−0.008	−0.018	0.001	0.11
Group 2	Before	7 days	−0.630	−0.745	−0.515	<0.001*
		30 days	−1.765	−1.961	−1.570	<0.001*
		6 months	−1.214	−1.371	−1.057	<0.001*
	7 days	30 days	−1.135	−1.333	−0.937	<0.001*
		6 months	−0.584	−0.766	−0.403	<0.001*
	30 days	6 months	0.551	0.466	0.636	<0.001*
Group 3	Before	7 days	−0.692	−0.82	−0.564	<0.001*
		30 days	−6.746	−6.991	−6.501	<0.001*
		6 months	−1.794	−2.072	−1.515	<0.001*
	7 days	30 days	−6.054	−6.245	−5.862	<0.001*
		6 months	−1.101	−1.33	−0.873	<0.001*

*Statistically significant at P<0.05. Group 1 – Control; Group 2 – Nonfluoridated group; Group 3 – Fluoridated group; CI – Class interval; P – Probability value

The test results also demonstrated similar findings for TNF-α levels. The three tested groups showed no differences before treatment (6.711 ± 0.150, 6.768 ± 0.143, and 6.716 ± 0.141, respectively) at P < 0.24. The values increased significantly in Groups 2 and 3 at 7 days as compared to levels in Group 1 (6.850 ± 0.098, 7.115 ± 0.128, and 7.141 ± 0.141, respectively) at P < 0.001. At 30 days and 6 months, the levels remained low in Group 1(untreated controls) but were higher in the other two groups with levels in Group 3(Fluoridated) even higher than in Non- Fluoridated Group 2 (6.716, 7.141, 9.279 at 30 days, 8.332) at P < 0.001 [Figure 3].

Figure 3.

Mean tumor necrosis-α levels between three groups at different time intervals

The Bonferroni test findings were similar to the IL-1β results. Maximal mean differences were noted for Group 3(Fluoridated) subjects at the 30-day comparison period where the mean differences ranged between 0.347, 1.229 and 0.733 which were higher as compared to other time periods. Similar, though lesser values at the same 30 day time period were noted in Group 2 (Non-Fluoridated) [Table 7].

Table 7.

Multiple comparison of mean differences of tumor necrosis factor-α levels between different time intervals in each study group using Bonferroni’s post hoc analysis

Group	Time (I)	Time (J)	Mean difference (I-J)	95% CI for difference		P
				Lower	Upper
Group 1	Before	7 days	−0.140	−0.194	−0.085	<0.001*
		30 days	−0.240	−0.303	−0.177	<0.001*
		6 months	−0.290	−0.359	−0.222	<0.001*
	7 days	30 days	−0.100	−0.14	−0.061	<0.001*
		6 months	−0.151	−0.192	−0.109	<0.001*
	30 days	6 months	−0.050	−0.071	−0.029	<0.001*
Group 2	Before	7 days	−0.425	−0.496	−0.354	<0.001*
		30 days	−2.563	−2.672	−2.454	<0.001*
		6 months	−1.616	−1.758	−1.475	<0.001*
	7 days	30 days	−2.138	−2.262	−2.014	<0.001*
		6 months	−1.191	−1.322	−1.06	<0.001*
	30 days	6 months	0.947	0.821	1.073	<0.001*
Group 3	Before	7 days	−0.347	−0.422	−0.271	<0.001*
		30 days	−1.229	−1.337	−1.121	<0.001*
		6 months	−0.733	−0.829	−0.637	<0.001*
	7 days	30 days	−0.882	−0.95	−0.814	<0.001*
		6 months	−0.386	−0.452	−0.32	<0.001*
	30 days	6 months	0.496	0.417	0.575	<0.001*

*Statistically significant at P<0.05. Group 1 – Untreated controls; Group 2 – Nonfluoridated group; Group 3 – Fluoridated group; TNF-α – Tumor necrosis factor-α; CI – Class interval; P – Probability value

DISCUSSION

The primary methods to evaluate the release of metal ions from orthodontic appliances are in vitro, examination of samples aged in vivo (ex vivo) and in vivo. The majority of published literature is of the in vitro type which makes it potentially difficult to match the findings to actual clinical situations. This study, by contrast, attempted to relate changes in the gingival condition, cytokine release, and changes in oxidative stress using an in vivo approach. The study aimed to categorize (a) differences in gingival health in three groups of subjects (untreated controls, patients using nonfluoridated toothpaste for oral hygiene maintenance, and patients using fluoridated toothpaste and mouthwashes). Gingival condition was assessed in all the three groups at four specified time periods using the gingival index of Silness and Loe. Deterioration in gingival index scores with time for both the treated groups was evident in the results obtained. The maximum increase in scores was noticed at the 1-month posttreatment commencement period in Groups 2 and 3. The fluoridated patient group presented with almost similar gingival index scores as compared to the nonfluoridated group. The higher amount of metal ions was shown to be released into GCF 30 days after commencing treatment in patients using fluoride agents in a previously published study. The findings of maximum increase in gingival index scores are in concordance with the previously published study by Chitra et al.[19]

Fluoridated mouthwashes are usually recommended by orthodontists to reduce white spot lesions and enhance enamel remineralization. However, previous studies by Chitra et al.,[19] and Petoumenou et al.,[21] demonstrated increased release of nickel, chromium, titanium, and manganese ions in patients exposed to fluoridated oral hygiene agents. Petoumenou et al. found increased metal ion release immediately after archwire placement. Both results were, however, not statistically significant.[21]

A higher metal ion load could affect gingival tissues and cause higher amounts of inflammation with deterioration in the scores for gingival health. Gingival health studies of orthodontically treated patients have shown deterioration in overall health during the course of treatment. A landmark study on the gingival condition during orthodontic treatment found that most children developed moderate levels of gingivitis a month or two after commencing treatment. The interproximal areas were more affected than the buccal.[22] The periodontal condition of orthodontically treated patients was compared with untreated subjects where findings indicated that orthodontically treated patients had significantly greater amounts of loss of attachment as compared to untreated subjects.[23] Periodontal pathogens were also deemed to play a role in development of orthodontically induced gingival enlargement.[24] IL-1β and TGF-β1 were supposed to be contributing factors. Orthodontically induced gingival enlargement was also linked to treatment duration,[25] where longer treatment periods led to greater amounts of enlargement. It can be concluded that gingival enlargement during orthodontic treatment is due to a combination of factors such as plaque accumulation around appliances,[26] increased release of metal ions,[19] and reduced ability to maintain adequate oral hygiene with subsequent changes in the gingival index scores. The effects of toothpastes and mouthwashes were also evaluated in multiple studies with variable findings. A systematic review on various mouthwashes with respect to gingivitis or biofilm formation pointed to the superior efficacy of mouthwashes with essential oils and chlorhexidine on reducing gingivitis and plaque.[27] Fluoride-containing mouthwashes had good anticariogenic properties, but their ability to reduce plaque and gingivitis remains questionable, as seen from the results of our study.

A landmark meta-analysis which assessed periodontal health status in orthodontic patients treated with fixed appliances and clear aligners concluded that periodontal health in patients using clear aligners was significantly better than in patients using fixed orthodontic appliances.[28] However, the variable clinical situations with multiple oral microbiota types and differences in oral hygiene maintenance protocols cause some confusion. Another study evaluated the periodontal health status in orthodontic patients treated with fixed appliances and clear aligners and concluded that no differences between the two modalities could be ascertained at the end of treatment, provided regular periodontal follow-ups were carried out by a dental hygienist.[29]

Additionally, (b) levels of two cytokines associated with the inflammatory response during orthodontic movement, namely IL-1β and TNF-α, were assessed using samples of GCF obtained at the same specified time periods in all the three groups. During force transfer to cells surrounding teeth, several mediators of inflammation called cytokines are released in variable quantities. They reflect the condition of the periodontal tissues which are affected by force application orthodontically and have a role in bony remodeling. IL-1β and TNF-α are associated with development of oxidative stress. Degree of oxidative damage during orthodontic tooth movement was determined in GCF using oxidative stress biomarkers (IL-1β and TNF-α). Comparisons of these biomarker levels in the three groups were made, and differences in oxidative stress damage in the groups using both nonfluoridated and fluoridated oral hygiene agents were evaluated. A review of literature on fluoride agents causing gingival inflammation during orthodontic treatment with increased oxidative stress did not show any results.

Studies evaluating levels of cytokines during orthodontic treatment have been few.[30,31] A landmark study by Basaran et al.[30] has provided evidence of increases in levels of two inflammatory cytokines, namely IL-1β and TNF-α in GCF, when orthodontic forces were applied. Results of these studies showed a maximum increase in cytokine levels within the first 24 h which reduced to baseline values thereafter. The findings were in contrast to our study which showed a maximal increase in levels of cytokines 30 days after commencement of orthodontic treatment. Though the levels were elevated when tested 7 days after treatment commencement, the values were higher at 30 days. A possible explanation for this could be attributed to higher metal ion release into the oral cavity at 30 days’ time period causing greater amounts of gingival inflammation and, in turn, increased oxidative stress which manifests with increased cytokine release. However, levels of cytokines in our study remained elevated even at 6 months and did not return to baseline levels. A possible explanation for this is that NiTi archwires were replaced with new heavier dimension wires every 2 months, causing increased leaching of nickel ions, which in turn could have an effect on gingival inflammation and release of pro-inflammatory cytokines. The possible explanation for lower levels of cytokines at 6 months as compared to the 30-day period could be formation of biofilms on metal orthodontic brackets reducing leaching of nickel or chromium ions. Thus, though cytokine levels were elevated, they remained lower than that seen at the 30-day test period. Oxidative stress changes in rat oral mucosal cells due to fluorides have been linked to DNA damage, apoptosis, and cell cycle changes in a study by He and Chen.[32] Since fluorides are commonly prescribed during the course of orthodontic treatment, a further investigation into the possible long-term effects of fluorides modifying the oxidant–antioxidant balance with effects on buccal mucosal cells is warranted.

CONCLUSIONS

There were increased levels of gingival inflammation with higher gingival index scores in both the treated groups from appliance placement till the 6-month observation period. The gingival index scores were highest at 30 days post appliance placement. Placement of fixed orthodontic appliances results in higher amounts of plaque formation and accumulation which, together with metal ion release from orthodontic brackets and archwires, causes increased gingival inflammation. This, in turn, leads to release of pro-inflammatory cytokines which are mediators of the inflammatory process with increased oxidative stress. This phenomenon is exacerbated in patients using fluoridated oral hygiene agents due to a link between fluoride use and increased release of metal ions. The use of fluoridated toothpastes and mouthwashes in orthodontic patients with metal appliances may be restricted, and chlorhexidine mouthwashes with nonfluoridated toothpastes could be used as equally efficient substitutes to prevent the cascade of events leading to gingival inflammation and oxidative stress damage. Oral mucosal cells could be affected by oxidative stress due to fluoride exposure during orthodontic treatment causing long-term changes, as yet unknown. Fluorides additionally cause the release of metal ions from orthodontic brackets and wires due to their acidic nature which can lead to higher levels of gingivitis, higher amounts of cytokine release, and significant changes in oxidative stress. The use of mouthwashes containing chlorhexidine instead of fluoride could be suggested as an alternative in orthodontic patients with low risk of caries.

Study limitations

Conclusive data on the relationship between orthodontic treatment and oxidative stress in the oral cavity are at present limited. The data are also not consistent over a long duration. The study demonstrated clear-cut differences in oxidant status on exposure to fluoride agents, which is probably the first of its kind, as no published literature on this aspect can be ascertained from a literature search. The increase in IL levels observed at the 30-day period normalized to an extent later. The exact mechanism for this could not be pointed out. Furthermore, the effects of vigorous toothbrushing and certain constituents in the diet affecting cytokine release cannot be ruled out. Further studies could shed light on these aspects.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.