Effects of mouthwash exposure to stainless steel temporary anchorage device on cytotoxicity and inflammation levels of BHK-21 cells

Abstract

Stainless steel temporary anchorage device (SS TAD) has toxic risk due to the content that may be released when exposed to the oral environment, and the mouthwash being used. This research aims to analyze the cytotoxicity of SS TAD and measure the inflammation level in cells after exposure to three types of mouthwash. SS TADs (n = 28) were divided into four groups (n = 7 per group) and immersed in the following mouthwash solutions for 90 days. The resulting eluates were then applied to BHK-21 fibroblast cell cultures and incubated for 24 h. Matrix metalloproteinase-8 (MMP-8) levels in the supernatants on days 1 and 7 were measured using enzyme-linked immunosorbent assay MMP-8 kit. BHK-21 fibroblast cells showed significant differences in reactivity (P < 0.05) after exposure to SS TAD eluate in povidone–iodine (PVP-I) and chitosan mouthwash, compared to control groups without SS TAD. Viability test revealed significant differences (P < 0.05) after exposure to SS TAD eluate in PVP-I mouthwash compared to PVP-I alone. The reactivity of BHK-21 fibroblast cells exposed to fluoride and distilled water was not significantly different (P > 0.05) from control groups without SS TAD. The viability of BHK-21 fibroblast cells exposed to fluoride and distilled water did not differ significantly from control groups without SS TAD. MMP-8 levels differed significantly between SS TAD eluate groups (P < 0.05) on day 1 and day 7, with day 7 levels significantly higher than day 1. The most recommended mouthwash is chitosan for TAD SS users rather than fluoride and PVP-I.

INTRODUCTION

Stainless steel (SS) material poses a risk of allergic reactions due to its nickel content.[1] Biocompatibility is essential for any metallic material used in the oral cavity; effectiveness and safety must be ensured by preventing the release of any toxic ions and minimizing allergic reactions or tissue irritation.[2] SS temporary anchorage devices (SS TADs) are exposed to a variety of conditions in the oral environment, which can alter the metal’s chemical properties and induce corrosion.[3] Corrosion is evidenced by the release of metal ions into the oral cavity’s tissues and fluids.[4] Furthermore, the release of metal ions, particularly nickel and chromium, poses a health risk.[5,6]

Metal material toxicity can induce reversible reactions.[7] Nickel toxicity inhibits the proliferation of oral epithelial and fibroblast cells by apoptosis, stimulates gingival overgrowth,[8,9] and may manifest as allergic reactions.[10]

Orthodontic treatment, due to its extended duration, can increase the risk of dental caries. Therefore, fluoride regimens (toothpaste, mouthwash) are commonly prescribed to orthodontic patients.[11] While fluoride is an effective anti-caries agent.[12] Povidone–iodine (PVP-I) solution is an antimicrobial agent.[13] Chitosan exhibits antibacterial and antiviral properties.[14]

Therefore, this study aims to evaluate the effects of fluoride, PVP-I, and chitosan mouthwashes on the biocompatibility of SS TADs, assessed through cytotoxicity parameters including reactivity, viability, and inflammation levels in BHK-21 cells.

MATERIALS AND METHODS

Samples of 28 pieces of SS TAD were immersed in 4 treatment groups (7 TAD each): The 0.2% sodium fluoride mouthwash solution, 1% PVP-I, 1.5% chitosan, and 0.2% distilled water (NaCl). It takes 1 ml of solution for every 200 mg of TAD weight. A total of 28 Eppendorf tubes containing SS TAD in the test solution and 4 tubes containing each test solution without SS TAD as control group were stored in an incubator at 37°C for 90 days. Time of 90 days is selected based on gargling instructions once daily. Each gargle obtained a solution retention of 6 h.[15] Based on the assumption that TAD use ranges from 6 months to 1 year, then in 1 year it is obtained as much as 2160 h, which is then converted into 90 days.

BHK-21 cells were cultured in Alpha-modified Eagle medium, pH 7.2, in an incubator at 37°C for 48 h to establish a monolayer. Each experimental and control group was tested in quadruplicate and incubated for 24 h. The reactivity of BHK-21 fibroblast cells exposed to the eluates or test solutions was assessed using a grading system adapted from ISO 10993-5 (2009), based on the presence of cell lysis and growth inhibition.[16]

Cell viability was determined using the MTT assay. Ten microliters of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for 3 h. Furthermore, 96 wells were then placed on an orbital shaker for 5 min at 50 rpm, and the absorbance was measured at 600 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. Cell viability percentage was calculated using the cell viability formula described by Vande Vannet et al.[17] Toxicity levels were categorized according to the cytotoxicity criteria defined by Sjögren et al.[16]

The supernatant was collected from each sample tube, and matrix metalloproteinase-8 (MMP-8) levels were quantified using a guinea pig MMP-8 ELISA kit (Indolisa, Indogen Intertama, Indonesia).[18] Absorbance was measured at 600 nm.

Intra- and interobserver reliability tests are performed in reactivity assessments. The normality of the viability data was assessed using the Shapiro–Wilk test. An independent t-test was used to assess reactivity and viability of BHK-21 fibroblast cells and MMP-8 levels in supernatants from day 1 and day 7.

RESULTS

The reliability analysis demonstrated satisfactory interobserver reliability, with an intraclass correlation coefficient of 0.969 and satisfactory Cronbach’s alpha reliability of 0.984. BHK-21 fibroblast cells exposed to the fluoride solution exhibited a distinct, ruptured star-shaped morphology, contrasting with the spindle-shaped control cells. No significant differences in cell shape or growth were observed between the two fluoride groups. Significant differences in cell morphology were observed between the PVP-I group and the SS TAD eluate in PVP-I group, with reduced cell growth in the eluate group. Compared to the control, the chitosan solution group showed minimal differences in cell morphology, but a slight reduction in cell proliferation was observed in the SS TAD eluate in chitosan solution group. The reactivity of BHK-21 fibroblast cells to the distilled water group did not show significant differences in cell shape between distilled water with and without SS TAD, and no visible inhibition of cell growth was observed [Figure 1].

Figure 1.

Cell reactivity of BHK-21 fibroblast cells after exposure to mouthwash only (blue) and elution of stainless steel temporary anchorage device in mouthwash (orange) for 90 days. (*): Cell reactivity was significantly increased in povidone–iodine and chitosan groups (P < 0.05). SS TAD: Stainless steel temporary anchorage device

Cell viability assays revealed no significant differences in the mean viability of BHK-21 cells between samples with and without SS TAD in the fluoride, chitosan, and distilled water groups. In contrast, the PVP-I groups with and without SS TAD showed highly significant differences in mean cell viability [Figure 2].

Figure 2.

Cell viability of BHK-21 fibroblast cells after exposure to mouthwash only (blue) and stainless steel temporary anchorage device elution in mouthwash (orange) for 90 days. (*): Cell viability was significantly decreased in povidone–iodine groups (P < 0.05). SS TAD: Stainless steel temporary anchorage device

The ELISA revealed elevated MMP-8 levels in the mouthwash groups compared to the control, with a further increase observed on day 7 compared to day 1 [Table 1]. Compared to the control, the highest MMP-8 levels were observed in the fluoride eluate group, while the lowest levels were found in the distilled water group.

Table 1.

Comparison of matrix metalloproteinases-8 levels between groups

Groups	Day 1 (average)	Day 7 (average)	P (independent t-test)
SS TAD + fluoride	8.30	10.46	0.001*
SS TAD + povidone–iodine	7.79	10.09	0.001*
SS TAD + chitosan	7.48	9.63	0.002*
SS TAD+ aquadest	6.82	9.23	0.000*

*P<0.05=significant. SS TAD: Stainless steel temporary anchorage device

Micrographs (×20 magnification) of BHK-21 fibroblast line cell reactivity after exposure to SS TAD eluates in the four test solutions are presented in Figure 3, and micrographs of cells exposed to the four solutions without SS TAD are presented in Figure 4.

Figure 3.

Cell reactivity image of BHK-21 fibroblast cells after exposure of stainless steel temporary anchorage device elution by: (a) Fluoride; (b) Povidone–iodine; (c) Chitosan; (d) Distilled water on an inverted light microscope magnification ×20 (line = 50 µm)

Figure 4.

Cell reactivity image of BHK-21 fibroblast cells to mouthwashes without stainless steel temporary anchorage device: (a) Fluoride; (b) Povidone–iodine; (c) Chitosan; (d) Distilled water on an inverted light microscope magnification ×20 (line = 50 µm)

DISCUSSION

Fluoride solutions, with and without SS TAD eluate, exhibited high reactivity and extremely low cell viability, indicating severe toxicity with near-complete cell death and damage. These results suggest that SS TADs may exacerbate the inherent toxicity of fluoride solutions. This aligns with findings by Abboodi and Al-Dabagh, who observed corrosion on SS TAD surfaces after fluoride exposure.[19] Fluoride is a halide that is known to be corrosive to SS. The proposed mechanism involves the destabilization of the SS oxide layer (Cr2O3) by fluoride ions, leading to its decomposition and reduced corrosion resistance.[20] Huang et al. found a decrease in the corrosion resistance of titanium alloys as well as in the stability of its passive film when the fluoride concentration increased.[12] The fluoride concentration used in this study was the standard level in the mouthwash solution of 0.2% (2000 ppm), but showed high reactivity and very low cell viability where cell death reached almost 100%. Previous studies have shown that the safe limit for fluoride to fibroblast cells is below 19 ppm.[21] However, we cannot conclude the relationship between the concentration of fluoride solution, metal ions released, and the toxicity of gingival fibroblasts in this study.

PVP-I alone exhibited moderate reactivity and high BHK-21 cell viability, whereas the SS TAD eluate in PVP-I showed significantly lower viability. This indicates that while PVP-I is not inherently toxic, SS TAD may trigger its toxicity, leading to severe toxicity upon exposure. Ochar and Vermilyea reported that SS corrosion accelerates with increasing iodine ion concentrations.[22] Iodine will react with SS constituent ions forming iodide metal bonds, a hallmark of corrosion.[23] The iodine-metal bond and released metal ions are thought to cause toxic effects on cells. However, further investigation is needed by quantitatively evaluating the number and types of metal ions released in the test solution by using the inductively coupled plasma–optical emission spectrometry method.

In this study, PVP-I exhibited moderate toxicity, consistent with findings reported by Müller and Kramer and Xu et al.[24,25] The cytotoxicity of PVP-I depends on the dose of exposure and cell type.[26,27] The duration of exposure may also play a role in enhancing the toxic effects of PVP-I, as this solution will be temporarily toxic at the beginning of contact, but the phenomenon of cell revitalization occurs within 24 h of postculture.[24] Immersion was carried out continuously for 90 days; it is thought that the toxic effects may last without revitalization, but further research is needed to prove whether various durations of immersion are in line with the toxic effects of PVP-I.

Chitosan solution with a concentration of 1.5% showed low reactivity and moderate viability. Despite its anticorrosive properties, these results suggest that chitosan may not completely prevent the release of metal ions, which can exhibit toxicity towards fibroblast cells.[28,29] Despite claims of biocompatibility, the chitosan solution exhibited considerable toxicity in BHK-21 cells.[14] The results of this study are contrary to Utami et al., who stated that 1.5% chitosan was not toxic to gingival fibroblasts. That study also stated that the use of miniscrews did not increase the toxicity of 1.5% chitosan solution.[30] However, Narvaez-Flores et al. found that chitosan at concentrations greater than 0.19% can decrease the viability of gingival fibroblast cells.[14] There may be a positive relationship between concentration, time of exposure, and toxicity levels of chitosan, but the statement requires further research.

Distilled water showed low reactivity and very high viability of BHK-21 cells in both groups. This result is in line with Mihardjanti et al., who found no release of metal ions in the immersion of SS bracket in distilled water.[31] This may be due to the neutral pH of the distilled water which does not affect the release of metal ions.[32]

Studies conducted by Gursoy et al. and Gupta et al. proved that MMP-8 could be a promising biomarker for detecting alveolar bone destruction and periodontitis in saliva.[33,34] The release of metal ions is thought to be involved as one of several triggering factors associated with dental implant peri-implantitis.[35,36] Further enhancement of this effect occurs because titanium corrosion may be enhanced under inflammatory conditions.[37]

CONCLUSION

The most recommended mouthwash is chitosan for TAD SS users rather than fluoride and PVP-I. The results showed that the most toxic mouthwash solution in this study was fluoride. In addition, PVP-I mouthwash should not be used continuously in patients with SS TAD. This aims to give time for cell revitalization so that the effects of toxicity are reduced.

The inflammatory effect test showed high MMP-8 levels on both days 1 and 7, with a significantly higher level on day 7, indicating progressive and substantial tissue inflammation caused by the use of SS TAD and mouthwash.

Further studies are needed to examine the correlation between mouthwash concentration, metal ion release, and gingival fibroblast toxicity, as well as the impact of varying immersion duration on the cytotoxicity of SS TAD in mouthwash.

Key Highlight

The study investigates the toxicity of fluoride, povidone–iodine (PVP-I), and chitosan on BHK-21 fibroblast cells, focusing on their effects on stainless steel temporary anchorage devices (SS TADs). Fluoride at 0.2% concentration showed high toxicity, resulting in nearly complete cell death, while PVP-I exhibited moderate toxicity, with potential cell revitalization after 24 h. In contrast, chitosan demonstrated the lowest toxicity, causing minimal changes in cell morphology and slight reductions in cell proliferation compared to controls.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was funded by Universitas Indonesia through the PUTI Grant with contract number NKB-183/UN2.RST/HKP. 05.00/2023