INTRODUCTION

Clear aligners are produced from thermoplastic materials. One such thermoplastic material is multilayer aromatic thermoplastic polyurethane/copolyester (TPU).¹ Various orthodontic aligner materials, including TPU, have been tested and reported to exhibit some toxicity on gingival fibroblasts.² This raises concerns that TPU material could affect the oral tissues, as well as the content of saliva or gingival crevicular fluid (GCF). This is particularly relevant because clear aligners are constantly maintained in the mouth, except during eating, and are in contact with soft tissues.

The GCF can secrete various mediators known as cytokines, which assist in cell signaling related to periodontal disease and orthodontic treatment.³ Applying mechanical force to teeth leads to significant changes in periodontal tissue homeostasis, and alterations are supported by the release of chemical mediators in the GCF.³ In particular, interleukin-8 (IL-8), a prototype of the chemokine family, is released by neutrophils and macrophages in response to various stimuli.⁴ Interleukin-6 (IL-6) stimulates interferon production by T lymphocytes and supports immunoglobulin secretion by active B lymphocytes.⁵ Rhodus et al.⁶ have highlighted the roles of various cytokines, including IL-8 and IL-6, due to their involvement in angiogenesis and inflammation.

Using TPU materials for orthodontic treatment for extended periods could lead to biological and molecular changes, particularly in the oral cavity and specifically in GCF, that trigger immune, inflammatory, or cytotoxic alterations and the release of related mediators. This was supported by an in vitro study indicating that all clear aligner materials tested exhibited some degree of slight cytotoxicity to human fibroblasts, suggesting that these materials could cause cellular changes.⁷ Alhendi et al.² reported some toxicity on gingival fibroblasts with various aligner materials (Invisalign, Eon, SureSmile, and Clarity) they tested.

The current study was intended to compare changes over time in the levels of IL-8 and IL-6 in the GCF during the first 3 weeks of orthodontic treatment with TPU material-based clear aligners in individuals with mild to moderate lower and upper arch crowding. The null hypothesis (H0) for this study was that there would be no significant difference in the levels of IL-8 and IL-6 in the GCF during the first 3 weeks of orthodontic treatment with TPU-based clear aligners compared to preprocedure levels.

MATERIALS AND METHODS

The sample size was calculated using the G-Power statistical software package (Version 3.1, Franz Faul, Universität Kiel, Germany). The effect size (d) was set at 0.40, with a Type I error (α = 0.05) and a power value of 80%, resulting in a total of 15 participants. Approval for this study was obtained from the Van Yuzuncu Yil University Clinical Research Ethics Committee (05.04.2023/07). Conducted in accordance with the ethical principles of the Declaration of Helsinki, detailed information about the purpose and methodology of the research was provided to all individuals who agreed to participate in the study and written consent was obtained. A flow chart describing the study is reported in Figure 1.

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The inclusion criteria were: no history of any systemic disease, no pharmacological treatment received within the last 6 months, absence of missing or impacted teeth except for third molars, no parafunctional habits that could lead to traumatic oral lesions, indicated for orthodontic treatment without extractions, having a maximum arch length discrepancy of 4–6 mm in both jaws, and skeletal Class I (0° ≤ ANB ≤4°; ANB angle: angle between point A, nasion, and point B). Individuals who did not need planned movement for the sampled teeth during the first 21 days were preferred. The exclusion criteria were as follows: PPD >3 mm, BoP ≥ %10,⁸ and radiographic evidence of bone loss, poor oral hygiene, need for orthognathic treatment, temporomandibular joint problems, known allergies to any medication or substance, use of tobacco and alcohol, personal or family history of cancer, and having received chemotherapy or radiotherapy treatment.

The GCF samples were collected before the placement of the clear aligner and postplacement at the first hour, third day, seventh day, 14th day, and 21st day.⁹ During the collection of these samples, teeth numbers 16 (right upper first molar) and 31 (left lower central incisor) for each individual were gently cleaned of plaque and isolated with cotton rolls. Subsequently, a paper strip (PerioPaper, Oraflow Inc, NY, USA) was gently inserted into the gingival crevice and left in place for 60 seconds. Paper strips visually contaminated with blood or saliva during the procedure were discarded and not included in the study. Each pair of samples collected from teeth 16 and 31 of individuals was placed in the same microcentrifuge tube containing phosphate buffer solution (Eppendorf tubes; Scientific Inc., Orlando, USA) and stored at −40°C until the day of analysis.

The clinical periodontal parameters (Gingival index [GI],¹⁰ plaque index [PI],¹¹ probing pocket depth [PPD]; measured from the gingival margin to the bottom of the pocket, and bleeding on probing [BoP]) for the full mouth and the teeth from which GCF was collected were measured at the baseline and on the 21st day. Measurements were taken from four surfaces of each tooth using a Williams periodontal probe (Hu Friedy, Chicago, Illinois, USA), and the values obtained were recorded. Subsequently, the average of these values was calculated. All clinical periodontal parameters and GCF samples were collected by a single researcher (DA).

Orthodontic records, including cephalometric radiographs and intraoral photographs, were collected from individuals who met the criteria for orthodontic treatment. Digital orthodontic models were obtained from these individuals using a three-dimensional scanning device (iTero Element 2, Align Technology, San Jose, CA, USA). The records were uploaded to the ClinCheck software for treatment planning. When planning the appropriate treatment, the first three aligners were planned in such a way that there was no tooth movement in the GCF sampled teeth and only in the adjacent teeth. Subsequently, each patient started using three clear aligners, changed every 10 days. During this phase of treatment, no interproximal reduction was performed on the teeth.

The levels of IL-8 and IL-6 in the GCF were evaluated using the enzyme-linked immunosorbent assay (ELISA) method, according to the manufacturer’s recommendations (Human Interleukin 8 ELISA Kit and Human Interleukin 6 ELISA Kit, Bioassay Technology Laboratory, Shanghai, China). Absorbance was measured at 450 nm. The readings were taken using an ELISA reader (μQuantTM ELISA Microplate Reader, BioTek Instruments, Inc., VT, USA).

SPSS software (IBM Corp., Armonk, NY, version 21) was utilized for calculations, providing descriptive statistics including the number of participants, mean, standard deviation, minimum, and maximum values. Analysis of variance for repeated measures detected time differences, followed by Bonferroni correction for post hoc analysis. Mann-Whitney U-test identified differences in clinical periodontal parameters over time. Spearman’s correlation analysis evaluated time correlations. Statistical significance for measurement differences was set at P < .05.

RESULTS

This prospective clinical study included 15 patients (five women, ten men, mean age: 27.4 ± 6.52 years). IL-8 levels over time are presented in Table 1, and the comparative statistics are shown in Figure 2. A statistically significant difference was observed between the IL-8 levels pre-procedure (317.15 ± 67.39), on the third day (311.66 ± 47.94), and on the 7th day (314.63 ± 60.32), compared to the 21st day (406.69 ± 74.38) (P < .001).

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Table 1. IL-8 Levels (ng/mL) According to Time and Statistical Comparison

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The IL-6 levels over time are presented in Table 2, and comparative statistics are shown in Figure 3. The increases in IL-6 levels on the 14th day (214.19 ± 41.07) and 21st day (222.25 ± 38.98), compared to preprocedure (170.68 ± 19.74), were found to be statistically significant (P < .001). Similarly, a statistically significant difference was observed between the third day (174.74 ± 8.82) and the levels on the 14th and 21st days (P < .001).

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Table 2. IL-6 Levels (ng/mL) According to Time and Statistical Comparison

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The correlation values between the IL-8 and IL-6 levels are presented in Table 3, and the scatter plot is shown in Figure 4. Accordingly, a positive correlation (r = 0.668) was observed between IL-6 and IL-8 levels at the first hour and a negative correlation (r = −0.305) on the third day. Lower levels of positive correlation were observed between IL-8 and IL-6 levels on the seventh, 14th, and 21st days (r = 0.360, r = 0.371, and r = 0.254, respectively).

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Table 3. Correlation Analysis of IL-8 and IL-6 Levels Over Time

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Data regarding clinical periodontal parameters are provided in Table 4. No significant difference was found in the GI, PI, BoP, or PPD values for the full mouth and tooth number 16 between preprocedure and the 21st day. For tooth number 31, no significant difference was found in the GI, BoP, and PPD values between preprocedure and the 21st day, while a significant difference was found in the PI values (P < .01).

Table 4. Clinical Periodontal Parameters at Baseline and at 21 Days^a

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DISCUSSION

Significant increases in the levels of IL-8 and IL-6 at different times compared to preprocedure levels were noted, leading to rejection of the null hypothesis. Whereas cytokines are synthesized in certain amounts in healthy tissues, many factors influence their levels in the GCF; their levels vary, particularly in the presence of periodontal diseases. The literature indicates that the levels of IL-8 and IL-6 in the GCF were altered in periodontal diseases.^12,13 However, Goutoudi et al.¹⁴ reported no difference in the mean total amounts of these mediators in the GCF between healthy and diseased periodontal tissues. Tou et al.¹⁵ suggested that there was no correlation between the IL-8 and IL-6 levels in the GCF and clinical parameters during orthodontic treatment with spurs in children. On the other hand, Lagdive et al.¹⁶ found significantly higher levels of IL-8 in the periodontitis group compared to a clinically healthy control group and reported a positive correlation between clinical parameters and IL-8. Similarly, Giannopoulou et al.¹⁷ reported a positive correlation between the amounts of IL-8 and IL-6 and PPD. During the current study, to minimize the influence of periodontal parameters, periodontal parameters of the entire mouth and the teeth from which GCF samples were collected were recorded. Consequently, no significant difference was found, particularly in clinical periodontal parameters between the baseline and the 21st day.

Orthodontic force also causes an inflammatory response as a result of tissue injury. There are numerous studies indicating that forces applied to teeth for orthodontic treatment purposes lead to changes in the IL-8 and IL-6 levels. Van Gastel et al.¹⁸ suggested that IL-8 and IL-6 levels could cause potential inflammation during the orthodontic treatment process. Nunes et al.¹⁹ found that IL-6 levels were significantly higher on the seventh day in individuals who underwent conventional fixed treatment. Gujar et al.²⁰ reported increases in IL-8 and IL-6 levels after 21 days in groups treated with clear aligners and conventional fixed treatment, although minor differences were observed between the groups. Kamran et al.²¹ reported that aligners and traditional labial orthodontic treatment increased inflammatory cytokine levels, including IL-8 and IL-6, after one month. Ren et al.²² indicated that IL-6 levels peaked within 24 hours and IL-8 levels within a month, after which cytokines regulated to a new homeostatic state. Chami et al.²³ reported that IL-8 levels in the GCF remained constant during 21 days of orthodontic activation with a single clear aligner. Additionally, indirect resorption caused by orthodontic treatment started on the 21st day.³ It has even been reported that significant increases in various cytokine concentrations occur in both tension and compression areas 3 weeks after applying orthodontic force.⁹ Based on these findings, no force application was planned for only the GCF sampled teeth for the first 21 days. Therefore, any increase in the IL-8 and IL-6 levels in the GCF could be attributed to the effect of the TPU material in the clear aligner content.

The literature indicates that prosthetic, restorative, and orthodontic dental materials may have cytotoxic effects.^24–27 Specifically, Premaraj et al.²⁴ evaluated the response of oral epithelial cells exposed to clear aligner plastic. They reported that this material caused changes in membrane permeability and cell adhesion in epithelial cells. Alhendi et al.² also reported some toxicity of thermoplastic materials used in multiple clear aligner systems to gingival fibroblasts. Another study mentioned that various clear aligner materials exhibited slight cytotoxicity to human fibroblasts.⁷ Conversely, Eliades et al.²⁸ found no evidence of cytotoxicity on human gingival fibroblasts, focusing particularly on potential bisphenol-A release. In the current study, the levels of IL-8 and IL-6 in the GCF were examined. IL-8 acts as a chemokine and can be released in response to stimulation caused by the chemical environment, inflammatory reaction, or environmental stress.⁴ IL-6 is a lymphocyte chemotactic factor that can be secreted by myeloid immune cells, keratinocytes, endothelial cells, and fibroblasts.²⁹ The increase in the levels of these two cytokines over time could be due to a mild cellular response resulting from the content of the clear aligner material used.

Results of the current study should be interpreted with caution due to its limitations. These include the limited number of patients, the lack of observation of the material’s effects at the cellular level, and the assessment of cytokine levels only in GCF without evaluating their levels in saliva and serum. In addition, even if tooth movement was not planned in the sampled teeth, it should be kept in mind that the levels of cytokines in the GCF may have changed due to the pressure associated with the height of the clear aligner material or movement that may have occurred in the adjacent teeth. In addition, although the aligners used in the current study were designed not to cause tooth movement in the GCF sample teeth, it should be considered that the fluctuations in cytokine levels might stem from other subclinical tissue reactions independent of the material.

CONCLUSIONS

• Increase in the levels of IL-8 and IL-6 in the GCF was observed during the first 21 days of orthodontic treatment with TPU material-based clear aligners.

• Additional clinical and in vitro studies could help elucidate the potential allergic or toxic effects of TPU-based clear aligners at the cellular level.