Assessment of cytotoxicity, odontoblast-like differentiation, shear bond strength, and microhardness of four orthodontic adhesive composites

Abstract

Purpose: To determine and compare the cytotoxicity, odontoblast-like differentiation, shear bond strength (SBS) and Vickers microhardness of four commercial light-cured orthodontic adhesives.

Methods: The orthodontic resins selected were Transbond XT – GI, Transbond Plus Color Change – GII (both from 3M Unitek), Enlight – GIII and Blugloo – GIV (both from Ormco). Samples were prepared, and leached monomers were obtained. Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and deposited calcium was analyzed using Alizarin red staining (ARS). SBS and the adhesive remnant index (ARI) were determined using 120 human premolars. The Vickers microhardness test was performed on the resin discs.

Results: All adhesives showed moderate to severe cytotoxicity (21-37%) and promoted similar formation of calcium deposits. A SBS of 6-8 MPa was achieved only by Blugloo (7.1 ± 2.4 MPa), and Enlight showed the lowest Vickers hardness score (40 ± 2.5 HV). Transbond Plus Color Change (score 0 = 42.9%) and Blugloo (score 0 = 46.4%) showed better ARI scores than Transbond XT (score 0 = 7.1%) and Enlight (score 0 = 3.6%).

Conclusion: On the basis of the properties evaluated, Blugloo seems to be the best option.

Introduction

In current orthodontics practice, a number of materials, mechanics, and accessories may be combined for treatment of malocclusion. Materials including brackets, aligners, miniscrews, and other attachments, made mainly from metal alloys (nickel titanium, stainless steel), polymers and composites are often subjected to testing both in vitro and in vivo. Biocompatibility refers to the capacity of any given material to generate an adequate biologic response while performing a specific function, in the absence of any undesirable or harmful effects. Previous studies of metals and adhesives have revealed several effects [1,2,3,4] in the oral cavity, such as gingivitis, a metal taste, glossitis, gingival hypertrophy, and cheilitis.

As orthodontic bonding systems are a key element for successful long-term attachment of brackets and accessories to teeth, it is critical to investigate the properties of their composite materials. Mechanical properties such as the shear bond strength (SBS) and microhardness, as well as the biocompatibility of orthodontic adhesives in terms of cytotoxicity, are among the main features that clinicians should take in account when choosing an adhesive. In the past, different types of orthodontic adhesives were available – mainly self-cured resins, light-cured resins, and hybrid glass ionomer cements – but today light-cured adhesive systems are the standard in view of their advantages such as a longer working time, moisture tolerance, and color change for enhanced bracket positioning. The compounds contained in dental resins, including orthodontic adhesives, include the monomers bisphenol A-glycidyl methacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), and urethane dimethacrylate (UDMA) [5]. Although a number of authors have previously investigated the potential cytotoxic effects of leached monomers, most studies have focused on restorative resins or orthodontic adhesives that are no longer in use, such as self-curing resins. In this regard, cytotoxicity tests have relevance for biological studies of dental materials as they may initiate changes that lead to cell death. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide) assay [6,7] is based on the reduction of tetrazolium salts to formazan by active cells, being dependent on mitochondrial activity, and is a common method for assessing the in vitro cytotoxicity of drugs and materials.

Orthodontic therapy using clear aligners is becoming more popular, and attachments made from different composites are commonly placed on the tooth surface to exert the forces required for movement control. Although some manufacturers have developed specific composites for the production of these attachments, it is relevant to clarify the behavior of resins indicated for the bonding of orthodontic brackets in this context. The biocompatibility of the materials used during orthodontic treatment are a major concern for clinicians since these materials remain in close contact with intraoral tissues for long periods. Some previous studies have investigated the addition of materials to orthodontic adhesives to improve their properties [8], mainly mechanical. However, commercial manufacturers do not seem to be concerned about these components, probably because it would be more realistic to focus on the properties of available products to make better informed decisions about the choice of orthodontic adhesive. The ideal orthodontic composite for clinical use would combine suitable mechanical properties with a good biological response of oral tissues, as well as easy handling.

The aims of the present study were to evaluate and compare the cytotoxicity, odontoblast-like differentiation, SBS, adhesive remnant index (ARI) and microhardness of four commercial adhesive resins for orthodontic use as attachments under in vitro conditions.

Materials and Methods

Sample preparation

Four commercial light-curing orthodontic adhesives were evaluated (Table 1): Transbond XT – GI (3M Unitek, Monrovia, CA, USA), Transbond Plus Color Change – GII (3M Unitek), Enlight – GIII (Ormco, Brea, CA, USA), and Blugloo – GIV (Ormco). A total of 60 discs were prepared: 15 discs 10 mm in diameter and 1 mm thick from each of the orthodontic resins. The discs were formed in a made-to-measure polytetrafluoroethylene (PTFE) mold, pressed with a thin glass sheet to remove any excess, and light-cured on the upper surface for 20 s with a Bluephase LED curing lamp (Ivoclar Vivadent GmbH, Vienna, Austria). The surfaces of the discs were then gently polished with sandpaper sheets (1000, 2000, and 4000 grit). Five discs were tested for cytotoxicity, five for odontoblast differentiation, and five for microhardness. All specimens were prepared by the same operator.

Table 1 Compositions of the orthodontic adhesives examined, according to the individual safety data sheets (MSDS)

Material	Composition	Manufacturer/Batch no.
Transbond XT (GI)	silane-treated quartz (70-80%), bis-GMA (10-20%), bisphenol A dimethacrylate (5-10%), silane-treated silica (<2%), diphenyliodonium hexafluorophosphate (<1%), triphenylantimony (<1%)	3M Unitek, Monrovia, CA, USA/NE30598
Transbond plus color change (GII)	silane-treated glass (35-45%), silane- treated quartz (35-45%), 1,2,3-propanetricarboxylic acid, 2-hydroxy-, reaction products with 2-isocyanatoethyl methacrylate (5-15%), PEGDMA (5-15%), bis-GMA (<2%), silane-treated silica (<2%), diphenyliodonium hexafluorophosphate (<1%)	3M Unitek/NF25355
Enlight (GIII)	bisphenol A, ethoxylated, dimethacrylate (10-30%), TMSPMA (1-5%)	Ormco, Brea, CA, USA/9087691
Blugloo (GIV)	glass, oxide, chemicals (50-75%), bisphenol A, ethoxylated, dimethacrylate (≤10%), GMA (≤4.9%), propylidynetrimethanol, ethoxylated, esters with acrylic acid (≤3%), UDMA (≤3%), silica, amorphous, fumed, cryst.-free (≤3%), bisphenol A (<0.025%)	Ormco/8966982

Cell cultures

Human dental pulp stem cells (hDPSC) were used for tests of cytotoxicity and odontoblast differentiation. The hDPSC were obtained from the cell bank of the Interdisciplinary Research Laboratory, Area of Nanostructures and Biomaterials of the ENES Leon, UNAM, and were established and characterized as reported previously [9]. The cells were inoculated onto culture plates with Minimum Essential Medium (MEM) (Gibco, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% glutamine (Sigma-Aldrich, Saint Louis, MO, USA) and 1% penicillin/streptomycin (Sigma-Aldrich), and incubated at 37ºC under 95% humidity and 5% CO₂, changing the culture medium every 2 days until a cellular confluence of over 80% was reached. The cells were then subcultured on sterile 96-well plates at a concentration of 1.7 × 10⁶ cells/mL and incubated under the same culture conditions before the cytotoxicity and differentiation assays.

Acquisition of leached monomers

For testing of cytotoxicity and odontoblast-like differentiation, 10 discs of each orthodontic adhesive resin were exposed to ultraviolet light for a total of 60 min (30 min per side) to eliminate any bacterial contamination. To obtain the substances leached from the resins, each disc was immersed in 1 mL of sterile MEM and incubated at 37°C and 95% humidity for 24 h. Using the formula 2πrh + 2πr², the surface area to volume ratio was calculated to be 1.88 cm²/mL, following the International Organization for Standardization (ISO) 10993-12: Sample preparation and reference materials [10], which recommends a surface area to volume ratio of 0.5-6.0 cm²/mL for generation of extracts for tests on cultured cells. The extracts were filtered to remove solid particles and stored until further use.

MTT cytotoxicity assay

The cytotoxic effects of the adhesive extracts were determined using the MTT assay (Sigma-Aldrich kit). Cells in the 96-well plates were treated with 100 µL of the extract from each adhesive resin, and dilutions ranging from 100% to 0% were made. As a negative control, the cell lines were maintained only in culture medium. Extracts and controls were incubated for 24 h at 37°C and 5% CO₂. Subsequently, the supernatants were removed and the cells were incubated with 100 µL of MTT (0.2 mg/mL in fresh MEM) for 7 h. Formazan was dissolved with 100 µL of dimethylsulfoxide (DMSO) (JT Baker, Phillipsburg, NJ, USA) and the optical density (OD) was measured at 570 nm using a spectrophotometer (Bio-Rad, Hercules, CA, USA). Cell viability was calculated according to the formula: cell viability (%) = (OD of test group/OD of negative control group) × 100. Data were obtained from three different experiments carried out in triplicate (n = 9).

Cytotoxicity was classified on the basis of cell viability according to Sjögren et al. [11], i.e., non-cytotoxic (cell viability >90%), slightly cytotoxic (60-90%), moderately cytotoxic (30-59%), and severely cytotoxic (<30%). The 50% cytotoxic concentration (CC₅₀) was considered to be the concentration of test material required to reduce cell viability by 50%, relative to the negative control.

Odontoblast-like cell differentiation

Odontoblast-like differentiation was tested using 96-well plates containing hDPSC. The cells were incubated in odontoblast differentiation medium [12,13] (α-MEM supplemented with 10% FBS, 1% penicillin/streptomycin, 10 mM β-glycerophosphate, 50 mM ascorbate 2 phosphate, and 0.1 mM dexamethasone, all from Sigma-Aldrich) with the resin extracts in a 1:1 ratio, at 37°C under 5% CO₂ and 95% humidity. Cells incubated in odontoblast differentiation medium alone were used as a negative control, and as a positive control cells were maintained in MEM medium. For all groups, the medium was refreshed every other day for 15 days.

After this period, mineral deposition activity, an indicator of ongoing osteogenesis, was assessed by Alizarin red staining. Stained cells were observed with a light microscope (Leica Microsystems, Wetzlar, Germany) and images were taken (×20). For quantification of calcium concentration, ARS was extracted with 5% 2-isopropanol and 10% acetic acid (both Sigma-Aldrich) for 16 h and the absorbance at 550 nm was measured using a spectrophotometer (Bio-Rad).

Shear bond strength

One hundred twenty premolars extracted for orthodontic reasons were collected after obtaining informed consent from the patients and approval from the ethics committee of the School of Dentistry, Autonomous University State of Mexico (UAEMex), Toluca, Mexico (CEICIEAO-2023-019), and stored in 0.01% thymol until use. The buccal surfaces of the teeth were cleaned using a rubber cup and fluoride-free prophy paste, rinsed with water for 15 s, and dried with compressed air for 10 s. The teeth were randomly divided into four groups (n = 30) according to the orthodontic resin to be tested: Group I Transbond XT, Group II Transbond Plus Color Change, Group III Enlight, and Group IV Blugloo, and then mounted vertically on self-curing acrylic bars.

To test the resins, a bracket-shaped minimold (Fig. 1) with a base area of 17.5 mm² (5.0 mm × 3.5 mm) was used (Ortho Technology, Lutz, FL, USA). The tooth surfaces were conditioned with 35% phosphoric acid etching gel (Scotchbond Universal, 3M ESPE, St Paul, MN, USA) for 15 s, rinsed with water for 15 s, and dried with compressed air until a chalky white appearance was evident on the treated surface. Then, using a microbrush, the primer corresponding to each adhesive system was applied, sprayed with air for 5 s, and light-cured with a Bluephase LED curing light (Ivoclar Vivadent GmbH) for 10 s. Using a resin spatula, each resin was placed in the minimold, pressed onto the tooth surface, excess was removed, and light-curing was conducted for 20 s. Prior to the SBS test, the prepared samples were placed in distilled water in a 37°C incubator for 24 h. It should be noted that the last experimental condition (storage in water) did not approximate the clinical condition, and therefore may have influenced the results of the SBS test.

The debonding resistance of the attachments was measured using a universal testing machine (Autograph AGS-X, Shimadzu, Kyoto, Japan) with a cross-head speed of 0.5 mm/min until failure occurred. The unbonding force was recorded in Newtons and converted to MPa.

Fig. 1 Bracket-shaped minimold used for preparation of specimens for testing of shear bond strength (SBS) and adhesive remnant index (ARI).

Adhesive remnant index

Once the attachments had been unbonded, the surface of each tooth was examined with a stereomicroscope (Nikon, Tokyo, Japan) at a magnification of ×10 to assess the amount of adhesive remaining on the enamel. ARI scores were determined as follows:

1. 0 = no adhesive remaining on the enamel
2. 1 = less than 50% of the adhesive remaining on the enamel
3. 2 = more than 50% of the adhesive remaining on the enamel
4. 3 = all of the adhesive remaining on the enamel

Evaluation of microhardness

Surface microhardness was determined using a Vickers micro-hardness tester (Sinowon SXHV-1000TA, Dongguan, PR China) with a Vickers elongated diamond pyramid indenter. A constant load of 9.8 N was applied to the surface for 10 s, and the Vickers hardness (HV) values were recorded. Ten indentations across each disc (five disks per resin) were performed, for a total of 50 indentations per resin (n = 5).

Statistical analysis

Data were analyzed using SPSS statistical software version 25 (SPSS Inc., Chicago, IL, USA). For all tests, means and standard deviations were calculated, and normality and homoscedasticity tests (except for chi-squared) were performed. Normality of the data was assumed, and parametric tests were followed. One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to compare the results for cytotoxicity, odontoblast-like differentiation, and SBS. Homogeneity of variances was assumed except for the data related to 12.5%, 25% and 50% dilutions. The chi-squared test was used to determine the significance of differences in the ARI scores. Microhardness values were analyzed by ANOVA followed by Scheffé post-hoc test. All statistical tests were run at a significance level of P ≤ 0.05.

Results

Cytotoxicity

Cytotoxicities for hDPSC at 100% concentration for the four groups were: GI 37 ± 18.2, GII 36 ± 5.7, GIII 32 ± 22, and GIV 21 ± 28.1. There were no significant differences in cell viability between the groups at each dilution, but intragroup comparisons revealed statistically significant differences between the 100% dose and all the other doses for all resins (P ≤ 0.05). Dose-response results for the adhesives are shown in Fig. 2.

According to Sjögren et al., the 100% dose in the GI, GII and GIII groups demonstrated moderate cytotoxicity (30-59%), and GIV showed severe cytotoxicity (<30%). Doses of 1.5%, 3.1%, 6.2%, 12.5%, 25% and 50% in all the study groups showed non-cytotoxic (>90% cell viability) to slightly cytotoxic (60-90%) activity. CC₅₀, from the most to the least cytotoxic, was as follows: GIV 43.8%, GIII 65.9%, GI 76.3%, and GII 80.8%.

Fig. 2 Cell viability of hDPSC exposed to leached monomers of the orthodontic resins. Geometrical forms correspond to different concentrations of the extracts. MTT assay was performed after 24 h of exposure. Data are expressed as means ± standard deviations for three different experiments carried out in triplicate. Error bars represent the standard deviation.

Detection and quantification of mineralization induced by odontoblastic differentiation

Mineralization in differentiated hDPSC was detected by Alizarin red staining. Calcium deposits were evident in all of the groups treated (Fig. 3). The results of ANOVA comparing the amount of calcium deposition after extraction of the Alizarin red staining are shown in Table 2. Starting with the highest amount, the results were as follows: GI (118 ± 13.1), GIII (103 ± 5.9), GII (102 ± 7.7), Control (100 ± 10.5) and GIV (96 ± 4.5). Statistically significant differences were found between the control and GI, and between GIV and GI (P ≤ 0.05).

Fig. 3 Calcium deposition detected by Alizarin red staining after 15 days of odontoblast differentiation. Micrographs were taken at a magnification of ×20. A, positive control, cells in DMEM medium exhibiting a typical spindle shape; B, negative control, cells in odontoblast differentiation medium; C, D, E, F, Transbond XT, Transbond Plus Color Change, Enlight, and Blugloo, respectively, all in odontoblast differentiation medium.

Table 2 Results of Tukey test comparing the quantification of calcium deposits in hDPSC after odontoblast differentiation

Adhesive	Mean (%)	Standard deviation	Tukey test†
Control	100	10.5	A
Transbond XT (GI)	118	13.1	B
Transbond plus color change (GII)	102	7.7	AB
Enlight (GIII)	103	5.9	AB
Blugloo (GIV)	96	4.5	A

^†The same letters indicate that the P value was not significantly different (P ≤ 0.05).

 

Shear bond strength and adhesive remnant index

The results of ANOVA comparing the SBS of the resins to human teeth are shown in Table 3. From the highest to the lowest value, the SBS (MPa) in the groups was: GIV 7.1 ± 2.4, GII 5.8 ± 2.4, GI 5.7 ± 2.1, and GIII 4.6 ± 1.8. ANOVA demonstrated significant differences between the groups (P ≤ 0.01), and the Tukey post-hoc intergroup comparison demonstrated significant differences between groups III and IV.

The results of the chi-squared test for ARI are shown in Table 4. The outcomes indicated a relationship between the type of resin and the level of remnant adhesive, specifically for score 0 = no adhesive remaining on the enamel, where the following values were obtained: GI 6.7%, GII 40%, GIII 3.3% and GIV 43.3%, indicating statistically significant differences between groups I and III versus groups II and IV (P ≤ 0.002).

Table 3 Results of Tukey test comparing shear bond strength (SBS) between the groups

Orthodontic adhesive	Mean (MPa)	Standard deviation	Tukey test†
Transbond XT (GI)	5.7	2.1	AB
Transbond plus color change (GII)	5.8	2.4	AB
Enlight (GIII)	4.6	1.8	A
Blugloo (GIV)	7.1	2.4	B

^†Adhesives with different letters showed significant differences from each other (P ≤ 0.01).

 

Table 4 Distribution and results of chi-squared test for adhesive remnant index (ARI) scores

Orthodontic adhesive	ARI (%)				χ2†
	0	1	2	3	0	1	2	3
Transbond XT (GI)	6.7	53.3	26.7	13.3	A	A	A	A
Transbond plus color change (GII)	40	36.7	23.3	----	B	A	A	A
Enlight (GIII)	3.3	46.7	33.3	16.7	A	A	A	A
Blugloo (GIV)	43.3	30	16.7	10	B	A	A	A

0 = no adhesive remaining on the enamel; 1 = less than 50% of the adhesive remaining on the enamel; 2 = more than 50% of the adhesive remaining on the enamel; 3 = all of the adhesive remaining on the enamel. ^†Adhesives with different letters showed significant differences from each other (P ≤ 0.002).

 

Microhardness test

The results of ANOVA comparing the Vickers microhardness of the adhesives are shown in Table 5. Significant differences were found among all groups evaluated, except between GII and GIV (P ≤ 0.05). GI showed the highest microhardness score (74 ± 5.7 HV), whilst the lowest value was shown by GIII (40 ± 2.5 HV).

Table 5 Results of Scheffé test comparing Vickers microhardness of the orthodontic adhesives

Orthodontic adhesive	Mean (HV)	Standard deviation	Scheffé test†
Transbond XT (GI)	74	5.7	A
Transbond plus color change (GII)	66	6.5	B
Enlight (GIII)	40	2.5	C
Blugloo (GIV)	67	3.4	B

^†Adhesives with different letters showed significant differences from each other (P ≤ 0.05).

Discussion

It is important to investigate the properties of adhesives used in orthodontics. One of these is their toxicity, in view of their proximity to soft oral tissues and tooth enamel, and the fact that they are places in the oral environment for long periods. It has been reported previously that various proportions of monomers do not polymerize, and that elution of these residual monomers causes some degree of toxicity [14]. Bis-GMA is one of the main monomers contained in orthodontic resins, and one that most frequently exhibits potential toxicity. The present evaluation of extracts from four light-curing orthodontic resins revealed that their cytotoxicity for human dental pulp stem cells ranged from moderate to severe. Ahrari et al. [1] reported that the cytotoxicity of three orthodontic resins (Unite, 3M Unitek; Denfil Flow, Vericom Laboratories Ltd., Anyang, Republic of Korea; Transbond XT, 3M Unitek) on human oral fibroblasts ranged from zero to moderate. Similarly, Jonke et al. [15] investigated the cytotoxicity of four orthodontic adhesive systems (Light Bond, Reliance Orthodontic Products, Itasca, IL, USA; Enlight, Ormco; Concise and Transbond XT, both 3M Unitek) on the L-929 murine fibroblast cell line and found that cell viability ranged between 66.8 ± 20.19% and 78.26 ± 13.42%, whereas in the present study cell viability ranged between 21 ± 28.1% and 37 ± 18.2% at a 100% extract concentration. Malkoc et al. [16] evaluated five orthodontic composites and reported a cell viability between 87.03 ± 10.66% and 91.94 ± 11.58% for L-929 murine fibroblasts, i.e. ranging between slightly cytotoxic and non-cytotoxic. The authors considered that Transbond XT was the most cytotoxic, unlike the present study in which Transbond XT was found to have the least cytotoxicity. Heravi et al. [8] evaluated Transbond XT resin with and without titanium dioxide (TiO₂) nanoparticles and reported a cell viability of 36 ± 6.53% and 63.1 ± 5.9% for human gingival fibroblasts (HGF) and mouse L-929 fibroblasts, respectively. These results are very similar to those reported here for the specific case of Transbond XT, with a viability of 37 ± 18.2%, considering that the extraction of monomer eluates also corresponded to 24 h.

GIV showed the highest cytotoxicity among the adhesives evaluated. According to the manufacturer, among its components is UDMA, a monomer that has previously been reported to have marked toxicity [17]. However, Transbond XT containing Bis-GMA, which has also been mentioned as a monomer with high toxic potential, showed lower cytotoxicity, although there was no significant difference between the two. This difference in the outcomes may be due to several reasons, including the difference in the adhesives studied, the period for extraction of adhesive eluates, the concentrations tested, and the cell lines used. The ISO recommends a surface area to volume ratio of 0.5-6.0 cm²/mL for generating extracts for tests on cultured cells, and the ratio reported in various studies has varied, thus potentially influencing the concentration and resulting cytotoxicity. The resins included in each study, and thus the components, would influence the results; the weight percent (Wt%) and type of monomers reported by the manufacturers of the resins tested in this study included Bis-GMA, PEGDMA (polyethylene glycol dimethacrylate), TMSPMA (3-[trimethoxysilyl] methacrylate), GMA (glycidyl methacrylate), and UDMA, and the Wt% of the monomers combined varied from 18 to 30. One of the main differences of this study relative to others was the use of human dental pulp stem cells, as monomer eluates have been reported to be capable of diffusing through the dentin layer to reach pulp cells [18,19,20] with the probability of causing some degree of pulp damage. It seems that few previous studies have considered the use of hDPSC for evaluating the cytotoxicity of orthodontic resins.

hDPSC are known to have the potential to differentiate into various cell lines; when differentiating to odontoblasts-like cells, they secrete extracellular matrix and initiate mineralization. Alizarin red staining can detect the formation of calcium deposits and is considered a standard method for assessment of dental pulp stem cell differentiation in vitro. The present results indicated that GI, GII, and GIII produced more calcium deposits compared than the control; only GIV induced fewer deposits. Although no other study has used this assay to evaluate orthodontic resins, the results may be comparable to those obtained in other studies [21,22] that evaluated the effect of silicate/mesoporous bioactive glass nanoparticles/graphene oxide composites on hDPSC mineralization, the evaluated resins producing a greater amount of calcium deposits than the controls. These results suggest that the orthodontic composites studied can promote mineralization during odontoblast differentiation to a greater extent than the control, which would be considered a desirable biological property.

Attachments are small composite devices that are affixed to some teeth during treatment with clear aligners to achieve better control and predictability over tooth movement. Different types of composites have been used to make these attachments and it is important to evaluate their strength of adhesion to the enamel surface, since they are subject to occlusion forces. In this study, four orthodontic resins normally used for bonding of brackets to the tooth surface were evaluated as composite attachments. Their use in this context was justified because they are resins with a higher viscosity and filler load than the flowable resins frequently used for these purposes, and therefore can better withstand the forces to which they are subjected. Most previous studies have investigated the SBS of brackets with different composites, but there is little information about the SBS of resin attachments used for aligner therapy, and practically no studies have evaluated the present composites as attachment materials. The present study revealed significant differences in the SBS between groups III and IV (P ≤ 0.01). Kircelli et al. [23] evaluated the bond strength of five composites used in the production of clear aligner attachments – two of them high viscosity and three flowable – and reported mean SBS values ranging from 16.6 ± 3.6 to 21.0 ± 4.0 MPa, with significant differences between the groups, although none of the resins evaluated were included in the present study. Arieli et al. [24] evaluated the SBS of metal brackets with four adhesive materials, including Transbond XT, at different time intervals. For Transbond XT at a debonding interval of 24 h, the SBS value was 8.1 ± 2.3 MPa, differing from the value of 5.7 ± 2.1 MPa obtained here. Another study [25] assessed the SBS and ARI of seven different metal brackets, all bonded with Transbond XT, and the values ranged from 3.8 ± 3.9 MPa (Morelli, Sorocaba, São Paulo, Brazil) to 9.8 ± 5.1 MPa (Tecnident, São Carlos, São Paulo, Brazil), the highest ARI scores lying between 0 and 1. Data from multiple studies suggest that there several factors can influence the SBS, such as the surface on which the materials are tested (enamel, porcelain, ceramic), the selected tooth (premolar, molar, incisor), the type of etching material (self-etching, acid etch, air abrasion, laser), the light curing system and time (high/low-intensity light, halogen light), the type of bracket (metal, ceramic), the design and size of the bracket base, and the adhesive used (composites, glass ionomers). Regardless of the conditions adopted for each study, it has been widely accepted that a clinically appropriate orthodontic adhesion strength should range between 6 and 8 MPa, as proposed by Reynolds [26], and in the present study only Blugloo met this standard. The ARI results indicated that a score of 1 (less than 50% of the adhesive remaining on the enamel) was the most frequent in the groups as a whole (41.7%), and a score of 0 accounted for a total of 65%. This could be beneficial, as it would mean that after debonding there would be less remnant adhesive to remove from the tooth enamel, thus minimizing any enamel loss or damage and saving time in the dental chair.

Assessment of Vickers hardness in orthodontic resins is valuable because higher hardness values are associated with a higher filler particle content, and in turn, greater wear resistance [27]. Regarding the present microhardness results, significant differences were evident between the evaluated adhesives, except between GII and GIV. Yilmaz et al. [28] studied some physical and mechanical properties of three orthodontic adhesives (Transbond XT, 3M Unitek; Grēngloo, Ormco; Light Bond Paste, Reliance Orthodontic Products), cured with different light units. For Transbond XT, they reported hardness levels of between 47.3 ± 3.6 HV and 50.6 ± 3.2 HV, while in this study the mean for the same adhesive was 74 ± 5.7 HV. In one more study [29], where they evaluated the effects of conventional and high-intensity halogen light on water sorption and microhardness, a value between 55.10 ± 2.46 HV and 55.42 ± 2.70 HV was obtained for the same resin. These variations in outcome could be related to the use of different light units or the thickness of the evaluated discs, since the first study employed specimens 5 mm in diameter and 2 mm thick, and the second one specimens 8.5 mm in diameter and 2 mm thick. In the present study, as resin discs 10 mm in diameter and 1 mm thick were tested, the light beam may have penetrated deeper and produced a better level of polymerization. The hardness values for the other adhesives assessed (in this study and the others) may not have been comparable as they differed in both composition and features.

In some studies it has also been mentioned that the color of the resin can influence the level of polymerization and therefore the microhardness [30]; Jafari et al. [Jafari Z et al., Daneshvar Medicine 22, 17-24, 2015] evaluated the microhardness of resins with different colors (blue, green, lemon, golden, silver and pink) and reported that a blue color represented the lowest level of hardness, whereas a silver color represented the highest degree of polymerization; the other colors represented average microhardness values. In the present study, two color-changing resins were evaluated; Transbond Plus Color Change is a pink adhesive allowing easy removal of excess during bracket placement and changes to a color similar to that of the tooth when light-cured; Blugloo, on the other hand, is a blue resin that also allows easy removal of excess during bracket bonding, as well as removal of remnants during debonding, since when it is light-cured and reaches body temperature it becomes transparent and remains so, and during debonding it changes back to blue for easy cleanup when cool water or air is applied. The microhardness for GII was 66 ± 6.5 HV and for GIV it was 67 ± 3.4 HV; only between these two adhesives was there no significant difference. This similarity in microhardness could be related to the color change exhibited by the two adhesives.

Within the limitations of this study, the present results indicate that some components of the tested adhesives affect the properties evaluated, and that their use may be appropriate in certain cases. It is concluded that all of the tested adhesives caused moderate to severe cytotoxicity on hDPSC and showed a comparable degree of calcium deposition when odontoblast-like differentiation was induced. An accepted SBS value of between 6 and 8 was met only by Blugloo, and Enlight showed the lowest score for microhardness. Transbond Plus Color Change and Blugloo exhibited similar degrees of microhardness.

These in vitro data suggest that the properties assessed here should be considered when selecting an orthodontic adhesive, and that Blugloo could be a better alternative for bracket bonding and may be used to produce attachments, as it combines an adequate degree of microhardness, greater SBS, less remnant adhesive on the enamel and better clinical control during bonding and debonding of brackets, due to its color change features. However, further investigation will be needed to evaluate the actual clinical performance of these materials.

Abbreviations

ARI: adhesive remnant index; ARS: alizarin red staining; Bis-GMA: bisphenol A-glycidyl methacrylate; CC₅₀: 50% cytotoxic concentration; DMSO: dimethylsulfoxide; FBS: fetal bovine serum; GMA: glycidyl methacrylate; hDPSC: human dental pulp stem cell; HGF: human gingival fibroblast; HV: Vickers hardness; ISO: International Organization for Standardization; MEM: minimum essential medium; MSDS: materials safety data sheet; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD: optical density; PEGDMA: polyethylene glycol dimethacrylate; PTFE: polytetrafluoroethylene; SBS: shear bond strength; SD: standard deviation; TEGDMA: triethylene glycol dimethacrylate; TiO₂: titanium dioxide; TMSPMA: 3-trimethoxysilylpropyl methacrylate; UDMA: urethane dimethacrylate

Ethical Statements

Ethical approval for this study was authorized by the ethics committee of the School of Dentistry, Autonomous University State of Mexico (UAEMex), Toluca, Mexico (CEICIEAO-2023-019). Donated teeth for shear bond strength tests were collected after obtaining written informed consent from the patients concerned.

Conflicts of Interest

The authors have no conflicts of interest to declare.

Funding

This research received no external funding.

Author Contributions

DBM: conceptualization, investigation, writing, and original draft; RJSV: conceptualization, writing, review, and editing; RCB: data curation and formal analysis; RGC: methodology, validation, and supervision. All authors reviewed and approved the final version of the manuscript.

ORCID iD

¹⁾DBM: [e-mail], ORCID iD: https://orcid.org/0000-0002-3216-2368

¹⁾RJSV*: [e-mail], ORCID iD: https://orcid.org/0000-0003-4671-0748

²⁾RCB: [e-mail], ORCID iD: https://orcid.org/0000-0003-1760-2000

³⁾RGC: [e-mail], ORCID iD: https://orcid.org/0000-0003-3504-5519

Data Availability Statements

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

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