To investigate the effect of aging on orthodontic bands, two commercial brands, American Orthodontic (AO) and 3 S-dental (T.S.H, Iran) were used. First, artificial saliva (AS) was prepared with the following composition per milliliter: 4.1 mM KH₂PO₄, 4.0 mM Na₂HPO₄, 24.8 mM KHCO₃, 16.5 mM NaCl, and 0.25 mM CaCl₂ (all from Merck, Germany) [27]. The acidic (pH = 4.5) and neutral (pH = 7.4) salvia were used in this experiment. To achieve an acidic environment, the pH was modified to 4.5 by incrementally adding ascorbic acid. pH measurements were conducted using a portable pH meter (Crison, Spain). Microporous sterilizing grade membrane filters (0.2 μm, GET Biofil, Korea) was employed to sterilize the artificial saliva. Orthodontic bands were incubated in saliva for 48 h before thermocycling. The incubation time was set according a study by Splaj S. et al. [20].

The bands were divided into four groups: Group 1: AO molar bands submerged in artificial saliva at pH = 7.4 for 48 h at 37 °C, followed by thermocycling. Group 2: AO molar bands submerged in artificial saliva at pH = 4.5 for 48 h at 37 °C, followed by thermocycling. Group 3: 3 S-dental molar bands submerged in artificial saliva at pH = 7.4 for 48 h at 37 °C, followed by thermocycling. Group 4: 3 S-dental molar bands were submerged in artificial saliva at pH = 4.5 for 48 h at 37 °C, then subjected to thermocycling. Three bands were used in each group.

The bands were weighed sterilized following the protocol specified by ISO 10993-5 (2009) [21], and immersed in a complete culture medium [Dulbecco’s Modified Eagle Medium (DMEM) containing 15% Fetal bovine serum (FBS) and 1% penicillin/streptomycin] (all from Gibco, USA) at a concentration of 0.2 g/ml. Each sample was stored for 48 h in a separate 50 ml falcon tube (Max well, China) while immersed in distilled water at 37 °C. The tubes were then subjected to thermocycling (VAFAEI industrial thermocycler, Iran) as previously described [22]. Briefly, the thermocycling process involved cycling in water with temperatures ranging from 5 °C to 55 °C for a total of 500 cycles. Each cycle involved of a 20-second immersion in each bath and a 5-second transfer time.

Human gingival fibroblast-like (HGF1-PI 1) cells was obtained from National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, Iran) and cultured in complete culture media at 37 °C in a humidified CO2 incubator (Binder, Germany). These cells are spindle-shaped, and have a bipolar morphology (Fig. 1). The medium was replaced 2 to 3 times per week until the cells reached to 70 − 0% confluency.

Morphology of fibroblast-like HGF cell line

For subculturing, the medium was completely removed and rinsed with 1× phosphate buffer saline (PBS). Then, 1 mL of trypsin/EDTA solution (Bisera, France) was added, and the flask was allowed to sit at 37 °C until the cells were detached. Fresh culture medium containing FBS was added and the cells were collected. The cell suspension was then transferred to a tube and centrifuged at 1500 rpm for 5 min. The supernatant was removed, and 1–2 mL of complete culture media was added to the cell pellet, which was thoroughly mixed to create a uniform cell suspension for counting.

To differentiate between viable and dead cells, trypan blue dye exclusion staining was employed. Ten microliters of cell suspension were mixed with 10 µL of trypan blue dye (Biosera, France) and placed on a Haemocytometer. Under a microscope (Leitz, Germany), live cells, which remain unstained by trypan blue, were counted in a set of 16 squares. Dead cells, taking up the dye, were also counted using the same method to estimate cell viability.

To assess the count of live cells per milliliter, the average cell count from 16 corner squares was multiplied by 10,000 and then by 2 to account for the 1:2 dilution caused by adding trypan blue. This result provided the number of viable cells per milliliter in the original suspension. The total cell count was obtained by summing the live and dead cell counts. To evaluate cell viability, the count of live cells was divided by the total number of cells, with cells exhibiting more than 95% viability being selected for further assessments.

To determine the appropriate number of HGF cells for cytotoxic assay, cell suspensions with varying concentrations (ranging from 781 to 100,000 cells in 100 µl) were prepared and seeded in a 96-well plate (SPL, Korea) in triplicate manner. The plates were then incubated at 37 °C in a CO₂ humidified incubator for 96 h. Following the incubation period, the MTT assay was conducted. An absorption curve was generated by plotting optical density (OD) against cell concentration, and the appropriate cell concentration was determined based on an OD range of 0.75 to 1 during the log phase of growth.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxic effect of each condition on HGF1-PI 1 cells. MTT is a color-based method employed to evaluate cell metabolic activity, cytotoxicity, and the effects of medicinal or toxic agents. In this assay, the yellow MTT dye is converted into an insoluble purple formazan by mitochondrial reductase enzymes, indicating cell viability and metabolic function [23]. The method was done based on previously described protocol [24]. Briefly, 15 × 103 cells suspended in 100 µl of complete culture medium were seeded in each well of a 96-well plate and incubated for 24 h to attach. The cells were then subjected to various treatments (AO & TSH; pH = 7.4 & 4.2) in triplicate manner. Copper-treated wells and untreated wells served as positive and negative controls, respectively. Following 72 h of incubation at 37 °C in a CO₂ incubator, the medium was replaced with 100 µL of DMEM containing 0.5 mg/mL of MTT solution (Sigma, Germany). The plate was incubated for 3–4 h at 37 °C to facilitate the formation of purple formazan crystals. Afterward, the MTT solution was removed, and 150 µl of DMSO (Tetra-chem, China) was added to dissolve the crystals. The absorbance of each well was then measured at 490 nm using an ELISA reader (Biotek, USA) after a 30 min incubation at 37 °C in dark.

The absorbance measurement of the blank was deducted from the readings of each sample. The absorbance values from the test samples were subsequently normalized by dividing them by the control values and multiplying them by 100 to determine the percentage of cell viability or proliferation. Absorbance values greater than the control indicated enhanced cell proliferation, whereas lower values indicated decreased cell viability or a reduction in proliferation.

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Genotoxic effects of aged bands in each condition on the HGF1-PI 1 cell line were evaluated using the comet assay. This approach refers to a single-cell gel electrophoresis assay which assesses DNA damage by measuring the migration of DNA fragments under an electric field. Intact, supercoiled DNA remains within the cell, while damaged DNA fragments migrate out, forming a “comet” tail.

For the assay, 200,000 HGF1-PI 1 cells, suspended in 2 mL of complete culture medium, were treated with media from different conditions (AO & TSH; pH = 7.4 & 4.2). Untreated cells served as negative controls, and cisplatin-treated cells (1 µM) were used as positive controls. As previously described [25], the cells were then incubated for 20 min at 37 °C with 5% CO₂, then mixed with low melting point agarose (LMPA), and applied on slides pre-coated with normal melting point agarose (NMPA). After setting the agarose, the slides were treated with a lysis solution, followed by electrophoresis at 24 V and 300 mA. The slides were then neutralized, dehydrated in ethanol, and stained with propidium iodide (Sigma, Germany). DNA damage was visualized under a high-resolution fluorescent microscope (Olympus, Japan) at 20X magnification, and images were examined with Olympus CellSens software.

Statistical analyses were conducted using IBM SPSS Statistics (Version 20). All results are expressed as means ± standard error of the mean (S.E.M.). The nonparametric Mann–Whitney U test was employed to assess the statistical differences between the groups, and P values less than 0.05 (two-tailed) considered significant.