The potential cytotoxic effect of recent universal adhesives with modified monomeric compositions on human gingival epithelial cells
Omar Abd El-Maksoud, Nessma Sultan, Hoda Saleh Ismail, Ramy Ahmed Wafaie, Hamdi H. Hamama, Salah Hasab Mahmoud
TL;DR
This study compares the toxicity of new dental adhesives on human gum cells, finding some are less harmful than others.
Contribution
The study introduces a comparison of modified monomeric dental adhesives' cytotoxicity against traditional ones.
Findings
SBP, ZBU, and TNB significantly reduced cell viability compared to the control at both concentrations.
CSQ showed the highest cell viability and was less toxic than other adhesives.
ROS levels varied significantly among all tested adhesives, indicating composition-dependent cytotoxicity.
Abstract
This study aimed to assess the cytotoxicity of three recent universal adhesives (UAs) with modified monomeric compositions compared to a typical adhesive containing bisphenol A-glycidyl methacrylate (Bis-GMA) and hydroxyethyl methacrylate (HEMA) on human gingival epithelial cells. Disk-shaped specimens of Scotchbond Universal Plus (SBP), Zipbond Universal (ZBU), Clearfil TRI-S Bond Universal Quick (CSQ), and Tetric N-Bond Universal (TNB) were fabricated and then kept in Dulbecco’s Modified Eagle Medium (DMEM) for 24 h to prepare the extract medium. Cytotoxicity was evaluated with MTT, SRB, and ROS analyses. Data were analysed by ANOVA and Tukey’s post hoc multiple comparison tests (P ≤ 0.05). At both tested concentrations (50% & 100%), MTT results showed that SBP, ZBU, and TNB significantly reduced cell viability compared with the control, in contrast to CSQ, which exhibited the highest…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Fig. 2
Fig. 3
Fig. 4- —Mansoura University
Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsSurgical Sutures and Adhesives · Dental materials and restorations · Drug Solubulity and Delivery Systems
Introduction
Adhesive dentistry is one of the prominent dental branches that relies on establishing a durable bond with tooth structure using adhesive materials^1^. Dental adhesive systems have achieved widespread popularity and attracted considerable research interest, paving the way for minimally invasive and more conservative approaches^2,3^. Dental adhesives were initially classified based on their development timeline^4^ and were later reclassified into etch-and-rinse (E&R) and self-etch (SE) adhesives based on their adhesion strategy^5,6^. Following the trend of SE adhesives and aiming for versatility, universal adhesives (UAs) were introduced as multi-mode systems. UAs have become widely adopted due to their simplified application, which mitigates procedural complexity^7^. Moreover, these adhesives can be used in E&R, SE, or selective enamel etching (SEE) modes, allowing clinicians to adapt the bonding strategy to specific clinical situations^8,9^. What further distinguishes UAs is the inclusion of specific carboxylate and/or phosphate functional monomers such as 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP), which promote chemical bonding and reduce the likelihood of adhesive hydrolytic degradation^10^.
Beyond durability and versatility, the biological compatibility of UAs is a property of supreme importance^11^. The leaching of monomers from dental adhesives has raised concerns regarding their potential biological effects on different tissues^12^. Therefore, the biocompatibility of monomers has been extensively scrutinized, and released methacrylate monomers were found capable of disrupting cellular regulation and inducing acute toxicity, even at low concentrations^1,13^. Typical UAs contain hydrophobic cross-linking monomers such as Bis-GMA^14^, as well as hydrophilic monomers, most commonly HEMA^15^. These monomers have been associated with pronounced biological risks^16,17^, with HEMA reported to induce cell apoptosis and necrosis^18,19^, whereas Bis-GMA induces teratogenicity^20^. Moreover, the synergistic interactions between both monomers further amplify their adverse effect compared to each monomer solely^21,22^.
Considerable efforts have been devoted to modifying the typical formulations in UAs. Accordingly, new modified UA systems have been launched in the dental market. Given the variations in their monomeric content across different types, it is critical to assess how these compositional differences directly affect their biocompatibility^22^. Among various in vitro methods to evaluate the biocompatibility of dental adhesives, cytotoxicity testing is considered one of the most effective approaches with standardized methodologies and limited variables^23^. Performing cytotoxicity tests is a basic screening step, essential for assessing newly introduced materials intended for direct application in the oral cavity^1^. Since the cytotoxicity of dental adhesives is reported to be linked to the release of residual monomers^24,25^, primary emphasis has been directed toward evaluating the cytotoxic potential of modified UAs, with selecting their monomeric composition as the main variable.
In most clinical scenarios, dental adhesives are directly applied to the walls/floors of prepared cavities. Consequently, they may come into close contact with soft and hard tissues for long intervals, potentially reaching the dental pulp through dentinal tubules. Moreover, a frequently overlooked aspect is their potential direct contact with oral mucosal epithelial cells^26^, where the gingival tissue around prepared cavities, especially in deep Class II cavities, could be exposed to dental adhesives in situations when rubber dam isolation is not applicable. In addition, residual monomers may leach into saliva after curing as a result of resin degradation, further increasing the probability of tissue contact^14^. Thus, the cytotoxic effect of dental adhesives toward gingival epithelial cells is a concern that should not be underestimated. To the best of our knowledge, this aspect has been negligently addressed in the literature. More specifically, there is a scarce of data on the cytotoxicity of modified UAs on gingival epithelial cells.
Therefore, the current study aimed to assess the cytotoxicity of three recent UAs extracts compared to a typical UA containing both Bis-GMA and HEMA monomers on human gingival epithelial cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, sulforhodamine B (SRB) assay, and reactive oxygen species (ROS) via enzyme-linked immunosorbent assay (ELISA). The null hypothesis was that there would be no substantial difference among the tested UAs in terms of the cytotoxicity on human gingival epithelial cells.
Materials and methods
Tested universal adhesives
This study tested four commercially available UAs with different monomeric compositions; Scotchbond Universal Plus Adhesive (SBP; 3M Oral Care), Zipbond Universal (ZBU; SDI Limited), Clearfil TRI-S Bond Universal Quick (CSQ; Kuraray Noritake Dental Inc) and Tetric N- Bond Universal (TNB; Ivoclar Vivadent AG). A full description of each adhesive is presented in Table 1.Table 1. Materials used in the study.MaterialsSpecificationManufacturerCompositionpHBatch NoScotchbond Universal Plus AdhesiveBis-GMA-free, HEMA-containing, one-step universal adhesive3M Oral Care, Neuss, GermanyHEMA (15%–25%), 10-MDP, 3M Vitrebond copolymer, BPA- free Dimethacrylate resins, silane, ethanol, water, CQ-based initiator2.710445962Zipbond UniversalBis-GMA- free, HEMA-free, acrylic-based one-step universal adhesiveSDI Limited, Victoria, Australia10-MDP, Acrylic monomer (40–50%) initiator, ethanol, water, fluoride2.51222448AClearfil TRI-S Bond Universal QuickBis-GMA- containing, HEMA-containing, amide-based one-stepUniversal adhesiveKuraray Noritake Dental Inc, Okayma, JapanBis-GMA (10–25%), HEMA (2.5–10%), 10-MDP, hydrophilic amide monomer, CQ, colloidal silica, silane couplingagent, sodium fluoride,ethanol, water2.3A20427Tetric N- Bond UniversalBis-GMA-containing, HEMA-containing, one-step universal adhesiveIvoclar Vivadent AG, Schaan, LiechtensteinBis-GMA (25- 50%), HEMA (10- < 25%), 10-MDP, D3MA, MCAP, CQ,Ethanol, Water2.5Z04LY6Bis-GMA, bisphenol A-glycidyl methacrylate; HEMA, hydroxyethylMethacrylate; MDP, Methacryloyloxydecyl dihydrogen phosphate; BPA, bisphenol A; D3MA, decandiolrylate dimethacrylate; MCAP, methac carboxylic acid polymer.
To minimize variability and risk of bias, all selected UAs shared the same functional monomer (10-MDP), solvent, photoiniator and fell within an approximate mild-acidity. This standardization aimed to focus more on their cytotoxic effects concerning the differences in monomeric composition. Additionally, to control parameters that may unwillingly influence the outcome, all specimens tested have underwent a standardized fabrication method, light-curing intensity and 40 s- photo curing time.
Cytotoxicity analysis
The Ethical Committee of the Faculty of Dentistry, Mansoura University, Mansoura, Egypt, approved this study (ethical approval No. A0801024CD). All procedures performed in this study were carried out in accordance with relevant guidelines and regulations of The International Organization for Standardization (ISO) no. 10993-5:2009 and 10993-12:2012.
Gingival epithelial cell culture
A human gingival epithelial cell line (ATCC CRL-3397) which was purchased from Nawah Scientific Inc. (Mokatam, Cairo, Egypt) was used in this study. Cells underwent cultivation in Dulbecco’s modified eagles medium DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with streptomycin (100 µg/mL), penicillin (100 µg/mL) (Biowest, USA), and 10% of heat-inactivated fetal bovine serum (FBS, Life science, UK) in a humidified atmosphere of 5% (v/v) CO_2_ at 37 °C^27^. The cell line was checked every day for growth, and the medium was changed twice weekly. Cell culture reached 70–80% confluence (logarithmic growth phase) within several days. An inverted microscope (Olympus, Tokyo, Japan) was used for the qualitative estimation of cell confluence at 10 × magnification (Fig. 1). Adherent cells were then detached for subculturing by adding a mixture of 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA)^28^. A cell analyzer was used to count the cells^27^.Fig. 1. Photomicrograph of gingival epithelial cells used in the study at 80% confluence under an inverted microscope showing a characteristic cobblestone appearance. scale bar 100 µm.
Preparation of adhesives extracts/eluants
Adhesive samples were prepared using custom-made molds with a thickness of 1.0 mm and a diameter of 4.0 mm^23,29,30^. For each adhesive, 10 µL was applied into the mold and gently air-thinned with oil-free air for 2–5 s. Light-curing was performed with a well-controlled light-emitting diode (LED) curing device (Elipar S10, 3M Oral care) at a wavelength of 430–480 nm and a light intensity equals to 1200 mW/cm^2^. The LED device tip was positioned perpendicularly close to the adhesive, which was photo-cured for 40 s^12,23^ until a solid disk of ≈ 0.8 mm thickness was obtained. Both surfaces of the disks were sterilized using 365 nm UV light for 60 min. Each disk was stored in a sterile 24-well plates in dry conditions for 24 h till complete polymerization, followed by adding DMEM culture medium and kept at 37°C for 24 h^29^.
When preparing the extract medium (eluate), ISO 10993-12 guidelines were followed, and a sample surface area/extraction volume ratio of 1.25 cm^2^/mL was used^31^. Accordingly, sample extracts were prepared at a 1:1 ratio, with a surface area of one disk (≈ 0.35 cm^2^) corresponding to an extraction medium volume of 280 µL per disk.
The selected surface area/extraction volume ratio was determined by considering the ranges recommended in ISO 10993-12 and the requirement that the volume of culture medium be sufficient to fully cover the adhesive disk. Taking into account that the extraction process should be appropriate to the nature and intended use of the tested materials, a ratio of 1.25 cm^2^/mL was adopted, balancing adherence to the standard with the purpose of the testing regarding the adhesives’ physicochemical characteristics, leachable components, and actual clinical use. The medium extracts/eluants were then filtered using 0.22 μm syringe filter till use.
Cell culture treatment with adhesives extracts/eluants
For each test, a volume of 100 μL of cell suspension was added to 96-well tissue culture plates at a density of 5 × 10^3^ cell/well and incubated for 24 h in complete culture medium till complete adherence. On the next day, culture medium was removed, and cells underwent washing twice with phosphate-buffered saline (PBS), then they were cultured with either 200 μL of undiluted filtered extract medium (100% or neat concentration) or 100 μL of the filtered extract medium + 100 μL of complete culture medium (50% concentration) and underwent incubation with 5% CO_2_ for 24 h at 37°C.
MTT assay
Cytotoxicity was evaluated through measuring cell viability as represented by the ability of viable cells to reduce a yellow water-soluble MTT dye to insoluble purple formazan crystals by succinate dehydrogenase (SDH) in the mitochondria. The assay was performed using a Vybrant MTT Cell Proliferation Assay Kit (Molecular Probes Inc., Thermo Fisher Scientific). After the cells were incubated with the adhesive extracts (50% and 100% concentrations) for 24 h, the medium was aspirated and 10 μL of MTT stock solution (5 mg/mL MTT in 1 ml PBS) was added to each well and the plates were then incubated at 37 °C for 4 h. After incubation, the MTT solution was removed and 100 µL of sodium dodecyl sulfate with hydrochloric acid was added to solubilize the formazan crystals. Thereafter, the plate was shaken via an orbital shaker for 15 min. The dye absorbance was measured within 1h using a microplate reader (FLUOstar Omega, BMG Labtec, Ortenberg, Germany) at 570 nm. A negative control consisting of gingival cells treated with 200 μL of fresh complete culture medium was used as the baseline (100%). The results of cell viability were normalized against the viability of negative control cells and expressed as percentages relative to control^27,28,32^.
SRB assay
Cell viability was also assessed using SRB assay (Sigma-Aldrich). The assay utilizes a bright pink aminoxanthene dye that has two sulfonic groups, which binds to cellular basic amino acid residues under a mild acidic condition and then be extracted under a basic condition. The amount of dye extracted is directly proportional to the total cell mass. After the cells were incubated with the adhesive extracts (50% and 100% concentrations) for 24 h, they were then fixed at the bottom of wells through replacing the medium with 150 μL of 10% trichloroacetic acid solution (TCA) and underwent incubation at 4 °C for 60 min. Subsequently, removal of TCA solution was done, and cells underwent washing for 5 times using distilled water and were dried at room temperature. They were then stained with aliquots of 70 μL SRB premixed solution (0.4% SRB dissolved in 0.1% acetic acid) and underwent incubation in dark for 10 min at room temperature. To remove any unbound excess dye, plates underwent washing three times with 1% acetic acid and were allowed to air-dry overnight. 150 μL of TRIS solution (10 mM) was added to the wells, and plates were placed on an orbital shaker for 10 min to solubilize the dye. After an additional 1h of incubation, the microplate reader was used to measure the absorbance at 540 nm. The results were also analysed and expressed as percentages relative to negative control (gingival cells treated with fresh complete culture medium, used as the baseline (100%)^12,23,30,33,34^.
ELISA assay
The production of intracellular ROS was quantitatively analyzed using a commercially available ROS ELISA kit (Nova Lifetech Limited Company, Hong Kong, China) according to the manufacturer instructions. After 24 h of incubating the cells with the 50% concentrations of the adhesive extracts, the culture supernatants were centrifuged at 2000g for 20 min to remove the cells. The centrifuged supernatants were added to the 96-well plate coated with an ROS antibody. The supernatants were incubated with enzyme-conjugated Avidine and biotin-conjugated antibody at 37 °C for 1h, forming an immune complex. Upon the reaction, unbounded ROS was washed off for six times. 90 µL of tetramethylbenzidine (TMB) substrate was added, followed by 50 µL sulfuric acid stop solution for yellow color development. The absorbance was measured spectrophotometrically at a wavelength of 450 nm using the microplate reader^35,36^.
Statistical analysis
The evaluations were carried out in triplicate for each assay and concentration tested, and to ensure reproducibility, the tests were conducted twice. The Statistical Package for the Social Sciences (IBM-SPSS, V 24, Armonk, NY, US) was utilized to analyze the data. Extracted data was checked for normality with the Shapiro–Wilk test, the homogeneity of variances was also checked using Levene’s test. Since the normal distribution of data and homogeneity of variances were confirmed, parametric analysis of variance (ANOVA) tests were utilized to test the significance of difference between group variability, followed by Tukey’s post-hoc multiple comparison tests for in-between group comparisons. The level of significance was set at P ≤ 0.05.
Results
MTT assay results (Fig. 2)
*Mean ± standard deviation of cell viability (%) for each adhesive extract at 100% and 50% concentrations as evaluated by MTT assay. Different uppercase letters indicate statistically significant differences among the 100% concentration groups (P ≤ 0.05). Different lowercase letters indicate statistically significant differences among the 50% concentration groups (P ≤ 0.05). Asterisks denote statistically significant differences between the two concentrations for each adhesive (*P ≤ 0.05, **P ≤ 0.001).
Two-way ANOVA test indicated that the UA type, the concentration, and the interaction between the two variables had a significant effect on the cell viability results (P ≤ 0.05). For the 100% concentrations, the Tukey’s post-hoc multiple comparisons test indicated that SBP, ZBU, and TNB induced a statistically signficant decrease in the cell viabilities as compared to the negative control (P ≤ 0.05). On the other hand, only CSQ showed no statistically significant difference in cell viability compared to the negative control group (P = 0.08), exhibiting the highest viability values. Regarding the 50% concentrations, CSQ also showed the highest cell viability, with no statistically significant difference compared to the negative control (P > 0.99). Conversely, SBP, ZBU, and TNB demonstrated a statistically significant decrease in cell viability compared to the negative control and to each other (P ≤ 0.05), with TNB exhibiting the lowest values. Comparing the cell viabilities of the 50% and 100% concentrations for each UA revealed statistically significant differences for SBP, ZBU, and TNB (P ≤ 0.001), as well as for CSQ (P = 0.049).
SRB assay results (Fig. 3)
*Mean ± standard deviation of cell viability (%) for each adhesive extract at 100% and 50% concentrations as evaluated by SRB assay. Different uppercase letters indicate statistically significant differences among the 100% concentration groups (P ≤ 0.05). Different lowercase letters indicate statistically significant differences among the 50% concentration groups (P ≤ 0.05). Asterisks denote statistically significant differences between the two concentrations for each adhesive (*P ≤ 0.05, **P ≤ 0.001).
Two-way ANOVA test also confirmed that cell viability was significantly influenced by the UA type, the concentration, as well as by the interaction between the two variables (P ≤ 0.05). The results of Tukey’s post-hoc test revealed that SBP, ZBU, and CSQ induced no significant differences in cell viability when compared to the negative control, as well as to each other (P > 0.05) at both tested concentrations. Meanwhile, a statistically significant decrease in cell viability was observed for TNB at both concentrations compared with the negative control and the other tested adhesives (P ≤ 0.001). Regarding the effect of UAs’ concentrations, a statistically significant difference was observed when comparing the cell viabilities of SBP, CSQ, and TNB at 100% and 50% concentrations (P ≤ 0.05). In contrast, there was no signifcant difference between both concentrarions of ZBU (P = 0.244).
ELISA results (Fig. 4)
Mean ± standard deviation of ROS concentration (pg/ml) for each adhesive extract at 50% concentration as evaluated by ELISA. Different lowercase letters indicate statistically significant differences among groups (P ≤ 0.05).
One-way ANOVA demonstrated a statistically significant difference among the 50% concentrations of all tested UAs extracts (P ≤ 0.05). The Tukey’s post-hoc test indicated that there was a statistically significant difference between all tested UAs (SBP: 242.33 ± 2.4, ZBU: 219.67 ± 2.8, CSQ: 149.96 ± 1.6 and TNB: 177.83 ± 1.5) when compared to the negative control (122.11 ± 2.1) as well as to each other (P ≤ 0.05). CSQ revealed the lowest ROS values in comparison to all tested groups, while SBP exhibited the highest ROS values.
Discussion
This in vitro study evaluated the cytotoxic effects of three recent UAs compared with a typical one on human gingival epithelial cells to assess how modifications in the conventional Bis-GMA/HEMA content influence the adhesives’ biocompatibility. The results of the current study demonstrated that the tested UAs induced variable cytotoxic effects on gingival epithelial cells across the three used assays. The MTT findings showed that all tested adhesives, except CSQ, decreased cell viability at both tested concentrations. Regarding SRB assay, the modified UAs maintained cell viability comparable to the control, while TNB impaired cell viability. Additionally, the ELISA assay revealed distinct variations in ROS levels among all tested UAs. Accordingly, the null hypothesis was rejected.
An established cell line rather than primary cells was selected for this study because of the ease of maintenance in culture, elimination of variability from different donors, and greater reproducibility^27,37^. The extract method was selected as it is the most commonly utilized and reliable in vitro technique for testing the cytotoxic effects of adhesive materials^28,38^. Extracts do not contain volatile elements, ensuring that only the actual chemical components of the investigated adhesive affect the cell viability. Additionally, maintaining a constant adhesive concentration is possible, as the extracts are homogeneous solutions^28^.
In order to provide a broad evaluation of the cytotoxic potential of the tested UAs, three different assays were used in the present study. MTT assay reflects the metabolic status of viable, mitochondrially active cells by evaluating the capacity of viable cells to reduce the MTT reagent to insoluble purple formazan via SDH activity^27^. SRB assay is one of the most frequently used colorimetric assays to quantitatively assess the cell viability based on cellular protein content, independent of metabolic activity and cell functionality. This assay provides a sensitive estimation of total cellular protein levels that is directly correlated to cell mass^12,33,39^. The biological assessment of UAs appears to involve measuring the ROS produced by leachable monomers as well. It has been demonstrated that ROS disrupt several cellular sites, leading to damage to nucleic acids, protein oxidation, and lipid peroxidation^40^.
Comparing the outcomes of SRB and MTT assays revealed inconsistent results. This discrepancy could be ascribed to the fundamental differences in the evaluation mechanisms between the two assays. The MTT assay reflects mitochondrial metabolic dysfunction. However, cells may still retain structural integrity and cellular protein mass, exhibiting high viability in the SRB assay. The lower MTT viabilities for SBP, ZBU, and TNB at 100% concentration may suggest early metabolic stress, where mitochondrial dysfunction precedes but does not necessarily decrease total protein mass or even lead to structural damage^29^, potentially reflecting a transient or reversible metabolic shift. Another plausible explanation is that the metabolic dysfunction may have already resulted in cellular lysis, with protein residues remaining attached to the plate or suspended in the medium. The SRB assay may have therefore overestimated viable cell count by staining the protein fractions corresponding to immediately lysed cells^41^, given its reported inability to clearly distinguish between live and dead cells^33,42^.
According to ISO 10993-5 standards^30,43^, a material is considered cytotoxic when cell viability decreases below 70% relative to negative controls. Therefore, the MTT results for SBP indicated satisfactory cell viabilities, even at the 100% concentration. Furthermore, the SRB assay revealed optimal cell viabilities for both concentrations of SBP. These results could be ascribed to the absence of Bis-GMA in SBP’s modified formulation. Several studies have indicated that Bis-GMA exhibits the highest cytotoxicity among common dental monomers^25,44,45^. Moreover, Bis-GMA is a high molecular**-**weight monomer, and its exclusion may have enhanced the adhesive’s polymerization rate and degree of conversion^46^, likely decreasing the amount of unreacted monomers and improving the cytocompatibility^44^. These outcomes contrast with those of Demirel et al.^47^ who reported that a Bis-GMA–free adhesive induced a severe reduction in cell viability after 24 h of exposure. This conflict may be attributed to variations in adhesive type, testing method, and the cell line evaluated.
In parallel, the SRB assay revealed excellent cell viability for ZBU at both concentrations. In the MTT assay, ZBU exhibited acceptable cell viability at 50%, but a dramatic decline at 100% concentration. This discordance suggests early mitochondrial-specific impairment without significant structural damage or reduction in total protein mass. Despite not containing Bis-GMA or HEMA, it is plausible that the manufacturers’ proprietary combination of monomers in ZBU^48^ might have interfered with the mitochondrial metabolic activity or the MTT reduction chemistry. In addition, the high solvent concentration (30–35% ethanol) in ZBU might have increased water sorption and solubility^49^, reducing degree of conversion and promoting the release of unreacted monomers, which further impaired mitochondrial metabolism and induced cytotoxicity^50^. This potential cytotoxic effect aligns with the findings of Lima et al.^48^ who reported that ZBU exhibited high cytotoxicity, with a remarkable reduction in mitochondrial metabolism within the first 24 h of contact with human dental pulp stem cells.
CSQ showed the most favourable cell viability results in both SRB and MTT assays. This may be attributed to the relatively lower concentrations of Bis-GMA (10–25%) and HEMA (2.5–10%) in its formulation compared to those typically used in conventional UAs. Furthermore, incorporating new acrylamide technology, represented by the inclusion of a hydrophilic amide monomer, might contribute to its high cytocompatibility. This was supported by Ahmed et al.^51^ who observed favorable cell viability when evaluating the cytotoxicity of two experimental acrylamide monomers designed to replace HEMA in UA formulations. The incorporation of acrylamide monomers has also been reported to provide an efficient degree of conversion, thereby reducing the leachability of residual monomers and improving overall cell viability^9^. These results were consistent with Wawrzynkiewicz et al.^22^, who reported that CSQ exhibited no significant cytotoxicity, genotoxicity, or increase in apoptosis in the tested cell line.
TNB displayed the lowest SRB viabilities at both concentrations and similarly showed a substantial decline in the MTT assay. These findings could be linked to its typical monomeric composition, which includes relatively high concentrations of both Bis-GMA (25–50%) and HEMA (10– < 25%). Along with the cytotoxic potency of Bis-GMA, HEMA has been reported to reduce cellular metabolism and induce notable cytotoxic effects^47,52^. Moreover, it has been reported that combining Bis-GMA and HEMA may exert a synergistic effect, further exaggerating cytotoxicity^21^. This evident cytotoxic effect of TNB was supported by Panda et al.^53^, who revealed that TNB exhibited the lowest cellular viability after 24 h of exposure to human dental pulp stem cells. In contrast, Wawrzynkiewicz et al.^1^ reported minimal cytotoxicity and genotoxicity for a similar formula-based UA, Adhese Universal, when tested toward a monocyte/macrophage peripheral blood cell line. This variation in results may be due to differences in the assay used and cell line tested.
The ELISA analysis was included as a comparative assessment alongside MTT and SRB assays to explore an additional cellular parameter beyond metabolic activity and total cell mass. The 50% extract concentration was selected for this analysis, as it consistently showed higher cell viability than the 100% extracts, allowing a rational comparison. ROS production was therefore measured to investigate a potential link between the cytotoxicity of the tested UAs and ROS generation.
Under normal physiological conditions, cells produce ROS at levels that must be tightly regulated to maintain balanced redox homeostasis^54–57^. Resin monomers in dental adhesives may disrupt this balance by elevating ROS levels, potentially leading to oxidative stress^54,58–60^. Accordingly, the adhesives’ cytotoxicity could be attributable to elevated ROS levels induced by residual monomers. Based on ELISA results, CSQ was the only UA to exhibit corresponding ROS levels and cytotoxicity results. In contrast, no such consistency was observed for the other tested UAs. Therefore, a definite relationship between ROS generation and the cytotoxicity of UAs cannot be confirmed. These results suggest that the increase in ROS levels was either counteracted by cellular antioxidants or triggered adaptive signaling, such as activation of the NF-κB pathway, limiting oxidative stress and protecting cells from further cell death^19,61^.
It should be recognized that this current study has certain limitations. Only a single cell line was examined, and differences in cell line responses could lead to variability in the cytotoxicity results. The study utilized a 24h exposure time, which may not account for the long-term contact with tissues. Moreover, two concentrations were only highlighted in the current study. Further studies should consider diverse cell lines, multiple exposure times, and different concentrations to provide an extensive evaluation. Worth mentioning, the ideal conditions adopted in this in vitro study, including the absence of oral environmental factors and the use of optimal light-curing parameters, make direct correlation of these results with the clinical situation unrealistic. While standardizing these parameters is achievable in vitro, it becomes even more challenging in certain clinical scenarios where cavity depth and the presence of a matrix system may further hinder clinical handling and curing efficacy.
Conclusions
The cytotoxic potential of UAs is influenced by differences in monomeric compositions and proportions, in a concentration-dependent manner. The modified formulations exhibited relatively safer profiles compared to the typical formula-based adhesive reflecting the effect of modifying the monomeric composition in enhancing cytocompatibility. CSQ exhibited the most favourable profile with optimal cell viabilities and minimal ROS production, supporting its potential as the UA of choice in deep preparations, particularly where direct contact with gingival tissues is unavoidable.