Evaluation of physicochemical characteristics of experimental resin infiltrant containing graphene oxide

Evaluation of physicochemical characteristics of experimental resin infiltrant containing graphene oxide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of physicochemical characteristics of experimental resin infiltrant containing graphene oxide Jade Laísa Gordilio Zago, Ana Laura Almeida Rodrigues, Sabrina Cândido Costa, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7011700/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Oct, 2025 Read the published version in Clinical Oral Investigations → Version 1 posted 9 You are reading this latest preprint version Abstract Objective This study aimed to evaluate the influence of 0.5% graphene oxide (GO) incorporated into an experimental resin infiltrant on its physicochemical and antibacterial properties compared to Commercial Icon (IC) and Experimental (E) groups. Materials and Methods Groups E and GO were manipulated, and specimens were prepared according to test requirements. Fourier-transform infrared spectroscopy (FTIR) was used to assess the degree of conversion (DC) (n = 5) before and after photoactivation. Sorption (So) and solubility (Sol) (n = 10) were evaluated after 7 days of water storage. Three-point bending tests determined the elastic modulus (EM) and flexural strength (FS) (n = 10). Initial carious lesions were induced in bovine enamel and analyzed through Confocal Laser Scanning Microscopy (CLSM) for penetration depth (n = 3). Antibacterial activity was assessed using antibiofilm assay (CFU), MTT metabolic test, and biofilm analysis via Scanning Electron Microscopy (SEM). Generalized linear models were applied for So, Sol, EM, and FS, while Kruskal-Wallis and Dunn’s tests analyzed DC, CFU, and MTT. Results GO differed from IC and E in DC after 80s photoactivation, exhibiting superior results. GO showed higher So and Sol values. IC demonstrated the best EM and FS. CLSM confirmed enamel infiltration for GO and IC. E showed the highest CFU, while GO had the lowest. E exhibited the highest MTT absorbance. Conclusion Incorporating GO into the experimental infiltrant is feasible, demonstrating good infiltration and antibacterial activity. Further studies are needed to optimize its properties. Clinical relevance: Infiltrants with graphene oxide and commercial formulations both demonstrated satisfactory antibacterial and infiltration performance. Dentistry Dental Caries Dental Enamel Graphene Oxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Dental caries is a significant public health issue and remains one of the most prevalent oral health disorders worldwide [ 1 ]. This sugar-dependent pathology involves various factors, including diet, dental anatomy, and salivary composition. When cariogenic acids diffuse through the enamel, they dissolve hydroxyapatite crystals, creating micropores within the dental tissue. These micropores, when filled with air or water, exhibit a different refractive index than healthy enamel, resulting in a white, opaque appearance of the lesion, which is clinically visible and serves as the first sign of dental caries [ 2 , 3 ]. Resin infiltration was introduced as a microinvasive technique to initial carious lesion treatment. The resin infiltrants are curing materials of low viscosity and high penetration coefficient, being capable of fulfilling the enamel’s micropores and consequently obliterating them [ 4 , 5 ]. This obliteration stops the interaction between biofilms’ acids and enamel structure, paralyzing the lesion progression [ 6 ]. Resin infiltrant Icon® is the first material in this category, and was released in 2009 by DMG (Hamburg, Germany) and it is composed mainly of triethylene glycol dimethacrylate monomer (TEGDMA) [ 7 , 8 ]. Since it came to the market, several studies have been carried out considering differents formulations that can improve mechanical and antibacterial properties, to improve and enhance the development of new infiltrants [ 3 , 7 , 9 , 10 ]. Thus, one proposal is the addition of graphene into the resin infiltrant, that is currently considered the most promising nanomaterial in biomedicine due to its chemical and physical properties, and because it has a wide application in medicine [ 11 ]. Graphene oxide (GO) is formed through the oxidation of graphite and has high biocompatibility [ 12 ]. Due to its versatility, it has been considered for new biomaterials with better mechanical properties, and it is possible to observe in dentistry several researches into applications of the material, such as in titanium implants, membranes, resin composites, cements and scaffolds [ 12 – 16 ]. Furthermore, a major highlight of GO is its antimicrobial capacity, through the production of oxidative stress and by interfering with the absorption of nutrients, it can combat bacterial growth in the oral cavity [ 14 , 15 , 17 ]. This characteristic is desirable in restorative materials; therefore, GO has become a viable alternative to provide these properties in materials such as resins, adhesives, and cements [ 15 – 17 ]. The development of experimental infiltrants with properties beyond the capacity of penetration in demineralized areas are important to improve their use. Properties such as better mechanical performance and possible antibacterial effect are very desirable and, therefore, it is worth considering the use of graphene oxide for this purpose. For this reason, the aim of the study was to evaluate the physochemical properties and antimicrobial capacity of an experimental infiltrant containing 0.5% graphene oxide. MATERIALS AND METHODS 1. Experimental infiltrants formulation The manipulation of the experimental infiltrant was formulated with a monomeric base of 75% TEGDMA and 25% BisEMA. Additionally, 0.5% camphorquinone and 1% EDAB were used as photoinitiator systems. In the group containing graphene oxide, 0.5% commercially acquired particles (Sigma-Aldrich, Steinheim, Germany) were incorporated into the experimental base [ 8 , 15 , 18 ]. The materials were weighed using an analytical balance (Chyo JEX-200, YMC Co Ltd, Tokyo, Japan), and the manipulation was performed in a controlled environment. The manipulated material was homogenized in a Speed Mixer (FlackTeck, INC, USA) at 3000 rpm for 5 minutes and stored under refrigeration at 4°C [ 10 ]. Specifically for manipulation of the resin infiltrant containing graphene oxide, after being homogenized it was taken to an ultrasonic bath for 10 minutes. Prior to the use of experimental infiltrants they were taken to magnetic homogenizer for 30 minutes [ 10 ]. The resin infiltrants used in the study are described in Table 1 . Table 1 Groups division and their composition. Group Composition Icon (I) Icon® Experimental (E) 75% TEGDMA, 25% BisEMA 0,5% CQ, 1% EDAB Experimental containing graphene oxide (GO) 75% TEGDMA, 25% BisEMA, 0,5% CQ, 1% EDAB + 0,5% Graphene oxide Experimental resin infiltrants: ethoxy bisphenol A glycidyl dimethacrylate (Bis-Ema – Sigma Aldrich, St. Louis, EUA); triethylene glycol dimethacrylate (TEGDMA - Sigma Aldrich, St. Louis, EUA); camphorquinone (CQ – Sigma Aldrich, St. Louis, EUA); tertiary amine dimethylaminoethyl benzoate (EDAB – Sigma Aldrich, St. Louis, EUA); graphene oxide (Sigma Aldrich, Steinheim, Germany). 2. Conversion Degree To determine the conversion degree, the materials were subjected to Fourier-transform infrared spectroscopy (FTIR – Vertex 70, Bruker Optik GmbH, Ettlingen, Germany). Specimens (n = 5) were prepared with approximately 0.5 mL of resin infiltrant [ 19 ]. Two readings were obtained: the first from the unpolymerized material; the second from the material 2 minutes after photoactivation with a LED light device (Valo, Ultradent, Salt Lake City, USA) for 40 seconds (Groups I and E) and 80 seconds (GO). The equipment parameters were set at 32 scans and resolution of 4cm − 1 , and the selected bands were 1720 − 1 cm for Icon and 1610cm-1 for experimental infiltrants (E and GO). The degree of conversion was calculated using the Opus v.6 software program (Bruker Optics, GmbH, Germany). 3. Sorption (So) and solubility (Sol) The tests were conducted following the specifications of ISO 4049:2009. Specimens were prepared with dimensions of 5 mm x 1 mm in thickness and photopolymerized with LED light for 40 seconds in groups I and E, and for 80 seconds in the GO group. After that, the volume (mm³) of the samples was obtained with a digital caliper (Mitutoyo, Japan). To define the constant mass values (M1), weighing was performed every 24 hours using high-precision analytical balances (Shimadzu – AUW220D, Tokyo, Japan) until achieve a variation of less than 0.1 mg. Subsequently, after 7 days stored in distilled water in ovens, the same samples were weighed to obtain (M2). They were weighed again every 24 hours to obtain a new constant mass (M3). After obtaining the volume, M1, M2 and M3, sorption and solubility values were calculated. 4. Flexural strength (FS) and elastic modulus (EM) The samples were prepared using silicone matrices (Express XT, 3M ESPE) with a rectangular shape (7x2x1mm). After being deposited into the matrices, the material was polymerized using an LED light source for 40 seconds (Valo, Ultradent) and stored in a dry oven at 37°C for 24 hours. To conduct the three-point bending test (n = 10), first, the dimensions of each specimen were meticulously measured with a digital caliper (Mitutoyo, Tokyo, Japan), then, tests were performed on a universal testing machine (Instron, model 4111, Instron Corp., Canton, MA, USA), operating at a speed of 0.5 mm/min and applying a load of 50 N. The results obtained were analyzed using Bluehill 2 software (Instron Corp., Canton, MA, USA), allowing calculations of elasticity in GPa and flexural strength in MPa. These analyses were based on the physical dimensions of the specimens and the stresses experienced during the tests. 5. Penetration depth Bovine teeth were collected and previously stored in a 0.5% thymol solution. After that, the roots were sectioned, and blocks were obtained using a metallographic cutter (Buehler LTD., Lake Bluff, IL, USA). Twelve enamel blocks (n = 4) were made in a 6x6mm shape. Then, the enamel was flattened using 600, 1200 and 2000 grit sandpaper to standardize the surfaces. At each sandpaper change, the blocks were washed in an ultrasonic bath for 5 minutes. Afterwards, the blocks were polished with a felt disc and diamond paste, followed by another ultrasonic bath. Then, a central area was delimited with 4x4mm, while the remaining areas were isolated with acid-resistant varnish (Colorama, São Paulo, Brazil). The protocol proposed by Moi et al. [ 20 ] was used for the induction of initial caries lesions. The demineralizing solution (DES) was composed of 0.1 M acetate buffer (pH 5.0) containing 1.28 mM Ca, 0.74 mM Pi, and 0.03 µg F/mL (3 mL/mm²), and the remineralizing solution (RE) was composed of 1.5 mM Ca, 0.9 mM Pi, 150 mM KCl, 0.05 µg F/mL, and 0.1 M Tris buffer (pH 7.0) (1.5 mL/mm²). In this protocol, cycling consisted of 20 hours of immersion in the RE solution and 4 hours in the DES solution. This procedure was carried out for seven days (seven cycles), and on the eighth day, the samples remained immersed in the RE solution for 24 hours. The blocks were then infiltrated with resin infiltrants according to the manufacturer's protocol, applying 15% hydrochloric acid for 2 minutes followed by thorough water rinsing. The samples were then immersed in a 0.1% Rhodamine-B solution for 12 hours. Afterwards, the samples were rinsed again, IconDry was applied, followed by gentle air blasting for 30 seconds, and then the application of the resin infiltrant for 3 minutes, with excess removed and followed by photopolymerization (Valo, Ultradent) for 40 seconds. A new application was then carried out for 1 minute, followed by light polymerization. After this process, the samples were stored for 24 hours in an oven, and then sectioned using a metallographic cutter. The slices obtained were manually polished with #600, #1200, and #2000 grit sandpaper until they reached a thickness between 150–200 µm. They were then kept for 12 hours in a 30% hydrogen peroxide solution to remove excess Rhodamine-B. For microscopy preparation, the samples were thoroughly washed in water and immersed in an ethanolic solution of sodium fluorescein for 3 minutes, followed by rinsing with deionized water. The samples were then placed on a glass coverslip containing specific oil for the confocal laser scanning microscope (CLSM) (Leica, TCS NT; Leica Heidelberg, Germany), with lasers of wavelengths 488 and 594 nm and a magnification of 63x selected [ 21 ]. 6. Evaluation of biofilm formation and antimicrobial activity 6.1 Bacterial culture manipulation The Streptococcus mutans UA159 strain (Laboratory of Microbiology and Immunology of the Piracicaba School of Dentistry, State University of Campinas, Piracicaba, Brazil) was used. The strains were maintained in BHI (brain heart infusion - Difco Laboratories, Detroit, USA) culture medium and fed with 20% glycerol at -20°C 9 . The microorganisms were reactivated on BHI plates and incubated at 37°C for 24 hours [ 9 ]. 6.2 Preparation of samples on discs Discs measuring 5 mm in diameter x 2 mm in thickness were prepared for antimicrobial evaluation using an addition silicone matrix made from a prefabricated mold. The samples were polymerized using an LED photopolymerizer (Valo, Ultradent, power density of 1000 mW/cm 2 ) for 40 s on both sides. The discs were then immersed in 10 ml of distilled water and placed in an ultrasonic vibration machine (Branson, Emerson, St. Louis, MO, USA) for 10 minutes to remove residual monomers. For disinfection, these samples were subjected to ultraviolet light for 30 minutes [ 22 ]. 6.3 Anti-biofilm assay Based on a previous study [ 23 ], the discs were distributed in microplates containing bacterial suspension (at 1.5 x 108 CFU/mL). Immediately afterwards, the microplates were incubated at 37°C for 48 hours to form biofilm on the surface of the infiltrating discs. Afterwards, the discs were washed in PBS 1X pH 7.2 buffer (Saline-Phosphate buffer) to remove planktonic bacterial cells and sonicated; the bacterial suspension was diluted and plated on BHI agar. Finally, the S. mutans count in the biofilm was performed. 6.5 MTT Test The MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) test for live and dead cells will measure metabolic activity [ 24 ]. After incubation for 48h, the biofilms were washed with sterile distilled water and then 200 µL/well of MTT dye were added. The 96-well plates were inoculated at 37°C for 1 hour and then the MTT solution was removed. After these steps, 300 µL of DMSO (dimethyl sulfoxide) were added and shaken for 20min at 80rpm in the dark. Then, 200 µL of DMSO were distributed in 96-well plates and subjected to a microplate reader to measure absorbance at OD (540nm). A high absorbance indicates a higher concentration of biofilm, which in turn indicates higher metabolic activity in the biofilm in the material. 6.6 Biofilm analysis by SEM To analyze the initial formation (4 hours) of Streptococcus mutans biofilm on the experimental infiltrant, a scanning electron microscope was used [ 25 ]. A sample of each infiltrant group was placed in a 24-well plate along with 1.5 ml of BHI medium added with 1% sucrose. The media were then inoculated with cultures of the strains in half of the exponential growth phase adjusted to the same absorbance (A550nm of 0.03) and the plates were incubated at 37°C for 4 hours in aerobic agitation (Nova Instrument-Thermo-shaker plate shaker; shaking at 250 rpm). After incubation, the culture medium was removed with a sterile micropipette, and the infiltrant discs were washed with 1.5 ml of 0.9% saline solution (0.9% NaCl) in a shaker (MicroPlate Shaker – MA 562, Marconi Equipamentos, Brazil) for 15 minutes. The washing step was performed three times. Then, the discs were treated with 800 µl of 2.5% glutaraldehyde solution (Sigma-Aldrich) for 30 min at room temperature. The discs were dehydrated by incubating with ethanol solution in increasing concentrations (50–100%) for 15 min for each solution. After dehydration, the discs were dried at room temperature and mounted on stubs to be metallized with gold and analyzed under a scanning electron microscope (Jeol 4 JSM5600 LV 33). Representative images of the specimens were obtained at 1,000X magnification and in duplicate. 7. Statistical analysis Descriptive and exploratory data analyses were carried out. Previous analyses indicated that the data did not meet the assumptions of a classic analysis of variance (ANOVA) with a general linear model. Then, sorption, solubility, elastic modulus and flexural strength data were analyzed with generalized linear models. The degree of conversion data did not fit a known distribution and was therefore analyzed using non-parametric test of Kruskal Wallis and Dunn. All analyzes were performed using R program, with significance level set at 5%. RESULTS 1. Conversion Degree The results of the conversion degree are presented in Table 2 showing that it was significantly higher in the Graphene group with 80 seconds of photoactivation compared to the Graphene group with 40 seconds of photoactivation (p < 0.05). There was no difference among Icon, Experimental and GO 80s or GO 40s. Table 2 Mean (standard deviation) and median (minimum; maximum) of the conversion degree depending on the group. Group Mean (standard deviation) Median (minimum; maximum) Icon (IC) 74,77 (0,45) 74,78 (74,21; 75,29) ab Experimental (E) 72,66 (3,27) 71,95 (69,79; 76,93) ab Graphene (GO) (80 seconds of photoactivation) 82,92 (1,20) 82,50 (82,03; 84,65) a Graphene (GO) (40 seconds of photoactivation) 62,64 (4,88) 62,54 (58,26; 67,23) b p-value p = 0,0045 2. Sorption and solubility As shown in Table 3 , for sorption, Experimental group exhibited higher values than the Icon group but lower than the Graphene group (p < 0.05). Regarding solubility, it can be observed that the Icon and Experimental groups demonstrated lower solubility than the Graphene group (p < 0.05), as depicted in Table 3 . Table 3 Mean (standard deviation) and median (minimum; maximum) of sorption and solubility as a function of group. Variable Group Mean (standard deviation) Median (minimum; maximum) Sorption Icon 5,39x10⁻⁵ (2,25x10⁻⁵) c 5,20x10⁻⁵ (2,02x10⁻⁵; 10,96x10⁻⁵) Experimental 10,89x10⁻⁵ (2,26x10⁻⁵) b 11,37x10⁻⁵ (5,40x10⁻⁵; 12,98x10⁻⁵) Graphene Oxide 13,96x10⁻⁵ (1,20x10⁻⁵) a 14,07x10⁻⁵ (11,66x10⁻⁵; 15,73x10⁻⁵) p-value p < 0,0001 Solubility Icon 1,78x10⁻⁵ (2,13x10⁻⁵) b 0,80x10⁻⁵ (-0,57x10⁻⁵; 6,61x10⁻⁵) Experimental 0,40x10⁻⁵ (8,84x10⁻⁵) b 3,23x10⁻⁵ (-24,61x10⁻⁵; 4,44x10⁻⁵) Graphene Oxide 6,39x10⁻⁵ (3,09x10⁻⁵) a 6,71x10⁻⁵ (1,61x10⁻⁵; 12,05x10⁻⁵) p-value p = 0,0017 3. Flexural strength and elastic modulus The elastic modulus and flexural strength were significantly higher in the Icon group compared to the other two groups (p < 0.05), as shown in Table 4 . There was no difference between Experimental and GO group in both elastic modulus and flexural strength. Table 4 Mean (standard deviation) and median (minimum; maximum) elastic modulus (GPa) and flexural strength (MPa) as a function of group. Variable Group Mean (standard deviation) Median (minimum; maximum) Modulus of elasticity (Automatic Young's) (GPa) Icon 1,09 (0,22) a 1,14 (0,79; 1,41) Experimental 0,34 (0,08) b 0,35 (0,22; 0,45) Graphene Oxide 0,36 (0,11) b 0,40 (0,14; 0,49) p-value p < 0,0001 Flexural strength (MPa) Icon 86,74 (21,82) a 86,66 (42,66; 120,44) Experimental 43,13 (8,39) b 40,28 (29,00; 56,73) Graphene Oxide 47,18 (12,89) b 47,69 (20,54; 68,91) p-value p < 0,0001 4. Penetration depth In the images obtained using confocal microscopy, it was possible to observe superficial deposition of the material and infiltration of the resinous tags in the demineralized area in the Icon (I) (Fig. 1 ) and Graphene Oxide (GO) (Fig. 3 ) groups. In the Experimental group(E) (Fig. 2 ), surface deposition was observed, but the resinous tags were not identified. 5. Anti-biofilm assay Figure 4 shows the approximate number of colonies formed after 48 hours of incubation in each group. The E group exhibited the highest colony formation (1.71 × 10⁷ CFU/ml), while the group with the incorporation of 0.5% GO showed the lowest colony formation (2.22 × 10⁶ CFU/ml), with a statistically significant difference (p < 0.05). 6. MTT metabolic assay Figure 5 shows the metabolic activity of the biofilm, quantified by the MTT assay. The E group exhibited the highest MTT absorbance, indicating higher metabolic activity in the S. mutans biofilms adhered to the discs. The incorporation of 0.5% GO significantly reduced the metabolic activity of the biofilms (p 0.05) 7. Biofilm Analysis by SEM Figures 6 , 7 and 8 shows the initial formation of the Streptococcus mutans biofilm after 4 hours of incubation, observed by scanning electron microscopy (SEM). The I (Fig. 6 ) and E (Fig. 7 ) groups exhibit greater bacterial adhesion, as evidenced by a higher biofilm density. In contrast, the group treated with 0.5% GO (Fig. 8 ) shows reduced S. mutans adhesion, with cleaner surface areas, suggesting an inhibitory effect on biofilm formation. DISCUSSION The incorporation of nanoparticles into resin infiltrants has been widely studied with various results [ 9 – 10 , 17 – 18 , 21 ]. Since it is commercially available from only one company, the development of materials with the same purpose but lower costs and better properties is desired. Graphene oxide is a more reactive particle than regular graphene, also exhibiting potential antibacterial properties against S. mutans [ 26 , 27 ]. Additionally, it is reported to improve the mechanical properties of composite resin when incorporated at concentrations of 0.3% and 0.5%, demonstrating good results in surface microhardness and flexural strength [ 28 ]. The resin infiltrant has a low viscosity, and therefore its texture is similar to dental adhesives. In this context, the study by Lee et al [ 14 ] evaluated the incorporation of graphene oxide into dental adhesives, resulting in increased antimicrobial activity and improved shear strength. This demonstrated that graphene oxide-enriched adhesives are suitable for clinical application, leading to the premise that it would be an interesting material to study in resin infiltrants. The concentration used in this work was based on previous studies, such as Lee et al.[ 29 ], in which the incorporation of graphene oxide into PMMA resulted in superior Vickers hardness at concentrations higher than 0.5% graphene oxide. Similarly, Velo et al. [ 28 ] evaluated concentrations of 0.3% and 0.5% in composite resins. A pilot study conducted in this research found that, due to its very light nature, graphene oxide particles in concentrations greater than 2% represented a very large volume of particles to be incorporated into the resin matrix of the infiltrant. Therefore, we established the concentration at 0.5%. The degree of conversion refers to the amount of free monomers that transform into polymer chains, making it an essential test for new material proposals [ 30 ]. Since the test specimens for sorption, solubility, and flexural strength require a minimum thickness of 1 mm and the resin infiltrant containing graphene oxide exhibits a dark coloration, a photopolymerization time of 80 seconds was established for this group based on a pilot study. However, for comparison purposes, measurements were taken with both 40 seconds and 80 seconds of exposure. As shown in Table 2 , graphene oxide with 80 seconds of photopolymerization achieved the best results compared to the experimental and commercial groups, likely due to its longer exposure to the wavelength of the photopolymerization device, ensuring a higher conversion of monomers. With 40 seconds of activation, the results were lower but did not differ statistically from the experimental and commercial groups, corroborating with Velo [ 28 ], where no difference was found between groups with or without graphene oxide in composite resins. It is important to note that regardless of the time used in this evaluation, all groups presented a degree of conversion above 55%, which is described by Soares [ 31 ] and Silikas [ 32 ] as a potential threshold for resin composites. The sorption and solubility of a resin material indicates its ability to retain liquids and its dissolution capacity. As shown in Table 3 , the commercial group (I) exhibited superior behavior compared to the experimental group (E) in terms of sorption, corroborating the findings of Mathias [ 18 ] and Souza [ 19 ], who also observed higher sorption in the experimental groups. This difference can be attributed to the material's composition, as Icon holds a patent on the product and does not specify all the components, only that it consists of 75% TEGDMA and additives. Nevertheless, both groups I and E were superior to the experimental group containing graphene oxide (GO), which showed higher sorption and solubility. This difference could be mainly due to the presence of nanoparticles in the composition of GO without functionalization, as described by Velo et al [ 28 ]. Therefore, it is possible that the particles and/or the organic matrix of the material dissolved more easily than in the other groups. On the other hand, the methodology of incorporating the particle into the material without treating it is also supported by a study that evaluated the incorporation of graphene into adhesives, such as by Bregnocchi et al [ 15 ]. Graphene is an extremely rigid material, and it is seen as an opportunity for strengthening and stiffening lightweight composites [ 33 ]. However, one of the biggest challenges in working with graphene is its tendency to agglomerate [ 28 ]. This occurs because this particle is highly hydrophilic and is also influenced by Van der Waals forces [ 12 ]. Considering this, according to Yang et al [ 28 ], in graphene-enriched materials, a higher homogeneity of the particle in the polymer matrix increases the chances of positive results in improving physicochemical properties. Thus, its tendency to agglomerate becomes one of the main obstacles limiting its potential to reinforce materials [ 34 – 35 ]. The flexural strength and modulus of elasticity used to assess the material's resistance were superior in group I, while E and GO demonstrated lower values. Generally, the resistance of resin infiltrants tends to be lower compared to resin composites, mainly because they do not have a significant inorganic filler content [ 19 ]. However, this finding does not corroborate the study by Velo et al [ 28 ], which reported superior results in experimental resin composites containing graphene oxide compared to the control. Unlike the findings in resins containing GO, the resistance of this group was similar to base experimental group. This difference can be speculated by two reasons: 1) the methodology employed, as those studies evaluated both surface microhardness and flexural strength, and 2) the coloration of the material. Interestingly, there are no descriptions in the literature about it, but the material exhibits a very strong, dark, and opaque coloration even at low concentrations, which could interfere with the photopolymerization process, directly impacting the material's resistance. A methodological limitation in studies on resin infiltrants is the evaluation of the penetration capacity of particles incorporated into the material. The most used test is confocal microscopy [ 4 , 19 , 36 ], which can access the penetration through lasers and refractive index due to the use of dyes, making it possible to visualize the penetration but not the elements and particles that infiltrated. So, even if it is possible to verify resin tags in GO group, it is not possible to confirm that the graphene oxide particles fully penetrated the enamel microtubules. The commercial group (I) also showed resin tags along its margin, aligning with other studies that also affirm material penetration up to 100 µm [ 4 , 36 ]. In contrast, the base experimental group did not show regularity throughout the sample and/or resin tags, differing from studies such as Cerqueira et al. [ 37 ], which demonstrated material permeation along the enamel microtubules. To halt the progression of caries, it is crucial for the material to penetrate and obliterate the enamel tubules, preventing interaction between the dental substrate and the potentially cariogenic environment [ 4 , 38 ]. The GO group demonstrated an ability to penetrate similarly to the I group, as seen in the images. Thus, the addition of graphene oxide does not interfere with the material's ability to infiltrate dentinal tubules. According to Mao et al.[ 27 ], graphene oxide exhibits antimicrobial effects against oral pathogens such as S. Mutans , making it a promising nanomaterial for use in dental materials. In our study, the resin infiltrant containing GO demonstrated superior antibiofilm formation comparing with base experimental, enhancing the possibility that GO was capable to inhibit bacterial growth. This was also supported by Pourhajibagher and Bahador [ 39 ] that incorporated GO into orthodontic adhesives and observed a greater results on CFU counting for 5 and 10% concentration. Bregnocchi et al [ 15 ] also observed in adhesives that the incorporation of 0.2% graphene was able to reduce the CFU count for S. mutans. For the MTT test, an inhibitory effect of GO was also observed compared to experimental E, which is supported by Zhao [ 26 ], who demonstrated that GO concentrations tend to generate a higher amount of reactive oxygen species and exhibit greater antibacterial potential than controls without its incorporation. Recent research suggests that the main antibacterial mechanisms of graphene materials involve the physical disruption of cell membranes, oxidative stress, and cell trapping or wrapping due to their honeycomb conformation, that are functional groups modified considered essential in mediating oxidative stress [ 26 ]. Although SEM micrographs of the biofilm showed that the surface of the experimental infiltrant exhibited a reduction in biofilm formation, the GO group did not differ from the commercial group Icon, indicating that while it exhibited antibacterial activity compared to the experimental base, its effect was not strong enough to differ significantly from the commercial control. New alternatives are always useful for better understanding the mechanisms involved in the use of resinous infiltrants and, therefore, continuing the development of these materials and verifying their performance in different situations and tests are essential. The experimental infiltrant containing graphene oxide showed limitations in relation to commercial control, making it necessary to adapt its incorporation to the resin matrix and verify its behavior in further tests, but seems to be a promising material for infiltrant applications in proximal areas. CONCLUSION Incorporating 0.5% graphene oxide (GO) into the experimental infiltrant significantly increased the degree of conversion when exposed to 80 seconds of photopolymerization. Considering the antibacterial properties, the GO-containing experimental group showed reduced biofilm formation and lower CFU counts for Streptococcus mutans compared to its counterpart without the addition of the nanoparticle. Although the commercial resin infiltrant exhibited superior performance in most mechanical tests, the presence of GO did not compromise the material’s ability to infiltrate initial caries lesions, as demonstrated by confocal laser scanning microscopy. Furthermore, the GO-containing material also presented enhanced antibacterial activity. Declarations ACKNOWLEDGMENTS The authors acknowledge the contribution of Flávia Sammartino Mariano Rodrigues from the Microscopy and Image Center at Piracicaba Dental School (University of Campinas, Piracicaba, São Paulo, Brazil) for the assistance in confocal microscopy analyses. This study was financed by the Fundação de Amparo à Pesquisa de São Paulo (FAPESP) – Process Code 2022/15280-8. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the author(s) used ChatGPT in order to improve the language. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication. References Chen KJ, Gao SS, Duangthip D, Lo ECM, Chu CH. Early childhood caries and oral health care of Hong Kong preschool children. Clin Cosmet Investig Dent. 2019; 17(11):27-35. https://doi.org/10.2147/CCIDE.S190993. 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Mathias C, Gomes RS, Dressano D, Braga RR, Aguiar FHB, Marchi GM. Effect of diphenyliodonium hexafluorophosphate salt on experimental infiltrants containing different diluents. Odontology. 2019 Apr;107(2):202-208. https://doi.org/10.1007/s10266-018-0391-0. Souza AF, Souza MT, Damasceno JE, et al. Effects of the Incorporation of Bioactive Particles on Physical Properties, Bioactivity and Penetration of Resin Enamel Infiltrant. Clin Cosmet Investig Dent. 2023;15:31-43. Published 2023 Mar 9. https://doi.org/10.2147/CCIDE.S398514. Moi GP, Tenuta LM, Cury JA. Anticaries potential of a fluoride mouthrinse evaluated in vitro by validated protocols. Braz Dent J . 2008;19(2):91-96. doi:10.1590/s0103-64402008000200001 Pedreira PR, Damasceno JE, Mathias C, Sinhoreti M, Aguiar F, Marchi GM. Influence of Incorporating Zirconium- and Barium-based Radiopaque Filler Into Experimental and Commercial Infiltrants. Oper Dent. 2021;46(5):566-576. https://doi.org/10.2341/20-020-L. Su M, Yao S, Gu L, Huang Z, Mai S. Antibacterial effect and bond strength of a modified dental adhesive containing the peptide nisin. Peptides . 2018;99:189-194. doi:10.1016/j.peptides.2017.10.003 Ghorbanzadeh R, Hosseinpour Nader A, Salehi-Vaziri A. The effects of bimodal action of photodynamic and photothermal therapy on antimicrobial and shear bond strength properties of orthodontic composite containing nano-graphene oxide. Photodiagnosis Photodyn Ther . 2021;36:102589. doi:10.1016/j.pdpdt.2021.102589 Cheng L, Weir MD, Xu HH, et al. Antibacterial amorphous calcium phosphate nanocomposites with a quaternary ammonium dimethacrylate and silver nanoparticles. Dent Mater . 2012;28(5):561-572. doi:10.1016/j.dental.2012.01.005 Moraes JJ, Stipp RN, Harth-Chu EN, Camargo TM, Höfling JF, Mattos-Graner RO. Two-component system VicRK regulates functions associated with establishment of Streptococcus sanguinis in biofilms. Infect Immun . 2014;82(12):4941-4951. doi:10.1128/IAI.01850-14 Zhao M, Shan T, Wu Q, Gu L. The Antibacterial Effect of Graphene Oxide on Streptococcus mutans. J Nanosci Nanotechnol. 2020 Apr 1;20(4):2095-2103. https://doi.org/10.1166/jnn.2020.17319. Mao M, Zhang W, Huang Z, et al. Graphene Oxide-Copper Nanocomposites Suppress Cariogenic Streptococcus mutans Biofilm Formation. Int J Nanomedicine. 2021;16:7727-7739. Published 2021 Nov 18. https://doi.org/10.2147/IJN.S303521. Velo MMAC, Filho FGN, de Lima Nascimento TR, et al. Enhancing the mechanical properties and providing bioactive potential for graphene oxide/montmorillonite hybrid dental resin composites. Sci Rep. 2022;12(1):10259. https://doi.org/10.1038/s41598-022-13766-1. Lee JH, Jo JK, Kim DA, Patel KD, Kim HW, Lee HH. Nano-graphene oxide incorporated into PMMA resin to prevent microbial adhesion. Dent Mater. 2018;34(4):e63-e72. https://doi.org/10.1016/j.dental.2018.01.019. Borges A, Chase M, Niederberger A, Gonzalez M, Ribeiro A, Pascon F, Zanatta A. A Critical Review on the Conversion Degree of Resin Monomers by Direct Analyses. Braz Dent Sci. 2013, 16. https://doi.org/10.14295/bds.2013.v16i1.845. Soares LE, Liporoni PC, Martin AA. The effect of soft-start polymerization by second generation LEDs on the degree of conversion of resin composite. Oper Dent. 2007 Mar-Apr;32(2):160-5. https://doi.org/10.2341/06-45. Silikas N, Eliades G, Watts DC. Light intensity effects on resin-composite degree of conversion and shrinkage strain Dental Materials. 2000; 16(4) 292-296. https://10.1016/s0109-5641(00)00020-8. Yang, Y., Rigdon, W., Huang, X. & Li, X. Enhancing graphene reinforcing potential in composites by hydrogen passivation induced dispersion. Sci. Rep. 2013; 3: 2086. https://doi.org/10.1038/srep02086. Zhou X, Wu T, Ding K, Hu B, Hou M, Han B. Dispersion of graphene sheets in ionic liquid [bmim][PF6] stabilized by an ionic liquid polymer. Chem Commun (Camb). 2010;46(3):386-388. https://doi.org/10.1039/b914763b. Li D, Müller MB, Gilje S, Kaner RB, Wallace GG. Processable aqueous dispersions of graphene nanosheets. Nat Nanotechnol. 2008;3(2):101-105. https://doi.org/10.1038/nnano.2007.451. Paris S, Soviero VM, Seddig S, Meyer-Lueckel H. Penetration depths of na infiltrant into proximal caries lesions in primary molars after diferente application times in vitro. Int J Paediatr Dent. 2012 Sep;22(5):349-55. https://doi.org/10.1111/j.1365-263X.2011.01204 Cerqueira, GA, et al (2022) Roughness and Microhardness of demineralized enamel treated with resinous infiltrants and subjected to an acid challengue: an in vitro study. The Open Dentistry Journal 17: e187421062302030. https://doi:10.2174/18742106-v17-230223-2022-126. Paris S, Meyer-Lueckel H, Cölfen H, Kielbassa AM. Resin infiltration of artificial enamel caries lesions with experimental light curing resins. Dent Mater J. 2007; 26(4): 582-8. https://doi.org/10.4012/dmj.26.582. Pourhajibagher M, Bahador A. Orthodontic adhesive doped with nano-graphene oxide: physico-mechanical and antimicrobial properties. Folia Med (Plovdiv) . 2021;63(3):413-421. doi:10.3897/folmed.63.e53716 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7011700","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487723378,"identity":"0216c2e2-b071-4f89-ad6b-6df5ed2e89bc","order_by":0,"name":"Jade Laísa Gordilio 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14:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7011700/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7011700/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00784-025-06598-6","type":"published","date":"2025-10-27T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87197423,"identity":"459258a4-438d-4f1f-9668-a73ddd105645","added_by":"auto","created_at":"2025-07-21 12:41:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":201921,"visible":true,"origin":"","legend":"\u003cp\u003ePenetration depth using Confocal Laser Scanning in I group.\u003c/p\u003e\n\u003cp\u003e* represents superficial deposition and [ represents resinous tags.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/6cb7c9fbb6c61edfc1f330c6.png"},{"id":87197426,"identity":"1d4f5a66-bcd7-4be3-85b5-ada2c42820b9","added_by":"auto","created_at":"2025-07-21 12:41:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":383172,"visible":true,"origin":"","legend":"\u003cp\u003ePenetration depth using Confocal Laser Scanning in E group.\u003c/p\u003e\n\u003cp\u003e* represents superficial deposition and [ represents resinous tags.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/7bd2a4002278283f6044dfcf.png"},{"id":87197428,"identity":"19910b41-9cc8-4e56-b7a4-840716d75460","added_by":"auto","created_at":"2025-07-21 12:41:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":217969,"visible":true,"origin":"","legend":"\u003cp\u003ePenetration depth using Confocal Laser Scanning in GO group.\u003c/p\u003e\n\u003cp\u003e* represents superficial deposition and [ represents resinous tags.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/778f3000a89d267ef1ae1c8c.png"},{"id":87199250,"identity":"276a8702-c2d5-40cf-ae65-12b188fd5cb2","added_by":"auto","created_at":"2025-07-21 12:57:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70538,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical results of colony formation units (CFU/ml).\u003c/p\u003e\n\u003cp\u003eCommercial resin infiltrant Icon (I), Experimental resin infiltrant (E), Containing 0.5% graphene oxide resin infiltrant (GO).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/17e22912b9b37c0176a44755.png"},{"id":87197427,"identity":"d71a3cec-4a1e-437a-8048-791cb5838e4a","added_by":"auto","created_at":"2025-07-21 12:41:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54322,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical results of S. Mutans cell viability (MTT)\u003c/p\u003e\n\u003cp\u003eCommercial resin infiltrant Icon (I), Experimental resin infiltrant (E), Containing 0.5% graphene oxide resin infiltrant (GO).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/08b558c2d19544dab391519a.png"},{"id":87197859,"identity":"9766242a-a133-45c2-862d-53694236973a","added_by":"auto","created_at":"2025-07-21 12:49:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":286049,"visible":true,"origin":"","legend":"\u003cp\u003eMicrograph of S. Mutans biofilm on I group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/2fab67513fb967c1a5203074.png"},{"id":87199251,"identity":"8af6f8c6-82cc-4342-be00-bfbe35d34b13","added_by":"auto","created_at":"2025-07-21 12:57:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":267972,"visible":true,"origin":"","legend":"\u003cp\u003eMicrograph of S. Mutans biofilm on E group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/65a858f9917a96cbae2dbef1.png"},{"id":87199253,"identity":"d27930f8-edcb-4bb6-b85f-0e64f737c135","added_by":"auto","created_at":"2025-07-21 12:57:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":266142,"visible":true,"origin":"","legend":"\u003cp\u003eMicrograph of S. Mutans biofilm on GO group.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/6e6cb5206c763c7dea7cf83a.png"},{"id":95040445,"identity":"5ccc1579-adff-4fbd-8e23-4775fb6d08b0","added_by":"auto","created_at":"2025-11-03 16:08:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2634571,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7011700/v1/3b872d7d-615a-4100-a8ff-161fcb1a54df.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of physicochemical characteristics of experimental resin infiltrant containing graphene oxide","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDental caries is a significant public health issue and remains one of the most prevalent oral health disorders worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This sugar-dependent pathology involves various factors, including diet, dental anatomy, and salivary composition. When cariogenic acids diffuse through the enamel, they dissolve hydroxyapatite crystals, creating micropores within the dental tissue. These micropores, when filled with air or water, exhibit a different refractive index than healthy enamel, resulting in a white, opaque appearance of the lesion, which is clinically visible and serves as the first sign of dental caries [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eResin infiltration was introduced as a microinvasive technique to initial carious lesion treatment. The resin infiltrants are curing materials of low viscosity and high penetration coefficient, being capable of fulfilling the enamel\u0026rsquo;s micropores and consequently obliterating them [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This obliteration stops the interaction between biofilms\u0026rsquo; acids and enamel structure, paralyzing the lesion progression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Resin infiltrant Icon\u0026reg; is the first material in this category, and was released in 2009 by DMG (Hamburg, Germany) and it is composed mainly of triethylene glycol dimethacrylate monomer (TEGDMA) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Since it came to the market, several studies have been carried out considering differents formulations that can improve mechanical and antibacterial properties, to improve and enhance the development of new infiltrants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThus, one proposal is the addition of graphene into the resin infiltrant, that is currently considered the most promising nanomaterial in biomedicine due to its chemical and physical properties, and because it has a wide application in medicine [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Graphene oxide (GO) is formed through the oxidation of graphite and has high biocompatibility [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to its versatility, it has been considered for new biomaterials with better mechanical properties, and it is possible to observe in dentistry several researches into applications of the material, such as in titanium implants, membranes, resin composites, cements and scaffolds [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, a major highlight of GO is its antimicrobial capacity, through the production of oxidative stress and by interfering with the absorption of nutrients, it can combat bacterial growth in the oral cavity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This characteristic is desirable in restorative materials; therefore, GO has become a viable alternative to provide these properties in materials such as resins, adhesives, and cements [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe development of experimental infiltrants with properties beyond the capacity of penetration in demineralized areas are important to improve their use. Properties such as better mechanical performance and possible antibacterial effect are very desirable and, therefore, it is worth considering the use of graphene oxide for this purpose. For this reason, the aim of the study was to evaluate the physochemical properties and antimicrobial capacity of an experimental infiltrant containing 0.5% graphene oxide.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\n\u003ch3\u003e1. Experimental infiltrants formulation\u003c/h3\u003e\n\u003cp\u003eThe manipulation of the experimental infiltrant was formulated with a monomeric base of 75% TEGDMA and 25% BisEMA. Additionally, 0.5% camphorquinone and 1% EDAB were used as photoinitiator systems. In the group containing graphene oxide, 0.5% commercially acquired particles (Sigma-Aldrich, Steinheim, Germany) were incorporated into the experimental base [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The materials were weighed using an analytical balance (Chyo JEX-200, YMC Co Ltd, Tokyo, Japan), and the manipulation was performed in a controlled environment. The manipulated material was homogenized in a Speed Mixer (FlackTeck, INC, USA) at 3000 rpm for 5 minutes and stored under refrigeration at 4\u0026deg;C [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Specifically for manipulation of the resin infiltrant containing graphene oxide, after being homogenized it was taken to an ultrasonic bath for 10 minutes. Prior to the use of experimental infiltrants they were taken to magnetic homogenizer for 30 minutes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The resin infiltrants used in the study are described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGroups division and their composition.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eComposition\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIcon (I)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIcon\u0026reg;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental (E)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75% TEGDMA, 25% BisEMA 0,5% CQ, 1% EDAB\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental containing graphene oxide (GO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75% TEGDMA, 25% BisEMA, 0,5% CQ, 1% EDAB\u0026thinsp;+\u0026thinsp;0,5% Graphene oxide\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eExperimental resin infiltrants: ethoxy bisphenol A glycidyl dimethacrylate (Bis-Ema \u0026ndash; Sigma Aldrich, St. Louis, EUA); triethylene glycol dimethacrylate (TEGDMA - Sigma Aldrich, St. Louis, EUA); camphorquinone (CQ \u0026ndash; Sigma Aldrich, St. Louis, EUA); tertiary amine dimethylaminoethyl benzoate (EDAB \u0026ndash; Sigma Aldrich, St. Louis, EUA); graphene oxide (Sigma Aldrich, Steinheim, Germany).\u003c/p\u003e\n\u003ch3\u003e2. Conversion Degree\u003c/h3\u003e\n\u003cp\u003eTo determine the conversion degree, the materials were subjected to Fourier-transform infrared spectroscopy (FTIR \u0026ndash; Vertex 70, Bruker Optik GmbH, Ettlingen, Germany). Specimens (n\u0026thinsp;=\u0026thinsp;5) were prepared with approximately 0.5 mL of resin infiltrant [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTwo readings were obtained: the first from the unpolymerized material; the second from the material 2 minutes after photoactivation with a LED light device (Valo, Ultradent, Salt Lake City, USA) for 40 seconds (Groups I and E) and 80 seconds (GO). The equipment parameters were set at 32 scans and resolution of 4cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the selected bands were 1720\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm for Icon and 1610cm-1 for experimental infiltrants (E and GO). The degree of conversion was calculated using the Opus v.6 software program (Bruker Optics, GmbH, Germany).\u003c/p\u003e\n\u003ch3\u003e3. Sorption (So) and solubility (Sol)\u003c/h3\u003e\n\u003cp\u003eThe tests were conducted following the specifications of ISO 4049:2009. Specimens were prepared with dimensions of 5 mm x 1 mm in thickness and photopolymerized with LED light for 40 seconds in groups I and E, and for 80 seconds in the GO group. After that, the volume (mm\u0026sup3;) of the samples was obtained with a digital caliper (Mitutoyo, Japan).\u003c/p\u003e\u003cp\u003eTo define the constant mass values (M1), weighing was performed every 24 hours using high-precision analytical balances (Shimadzu \u0026ndash; AUW220D, Tokyo, Japan) until achieve a variation of less than 0.1 mg. Subsequently, after 7 days stored in distilled water in ovens, the same samples were weighed to obtain (M2). They were weighed again every 24 hours to obtain a new constant mass (M3). After obtaining the volume, M1, M2 and M3, sorption and solubility values were calculated.\u003c/p\u003e\n\u003ch3\u003e4. Flexural strength (FS) and elastic modulus (EM)\u003c/h3\u003e\n\u003cp\u003eThe samples were prepared using silicone matrices (Express XT, 3M ESPE) with a rectangular shape (7x2x1mm). After being deposited into the matrices, the material was polymerized using an LED light source for 40 seconds (Valo, Ultradent) and stored in a dry oven at 37\u0026deg;C for 24 hours.\u003c/p\u003e\u003cp\u003eTo conduct the three-point bending test (n\u0026thinsp;=\u0026thinsp;10), first, the dimensions of each specimen were meticulously measured with a digital caliper (Mitutoyo, Tokyo, Japan), then, tests were performed on a universal testing machine (Instron, model 4111, Instron Corp., Canton, MA, USA), operating at a speed of 0.5 mm/min and applying a load of 50 N.\u003c/p\u003e\u003cp\u003eThe results obtained were analyzed using Bluehill 2 software (Instron Corp., Canton, MA, USA), allowing calculations of elasticity in GPa and flexural strength in MPa. These analyses were based on the physical dimensions of the specimens and the stresses experienced during the tests.\u003c/p\u003e\n\u003ch3\u003e5. Penetration depth\u003c/h3\u003e\n\u003cp\u003eBovine teeth were collected and previously stored in a 0.5% thymol solution. After that, the roots were sectioned, and blocks were obtained using a metallographic cutter (Buehler LTD., Lake Bluff, IL, USA). Twelve enamel blocks (n\u0026thinsp;=\u0026thinsp;4) were made in a 6x6mm shape. Then, the enamel was flattened using 600, 1200 and 2000 grit sandpaper to standardize the surfaces. At each sandpaper change, the blocks were washed in an ultrasonic bath for 5 minutes. Afterwards, the blocks were polished with a felt disc and diamond paste, followed by another ultrasonic bath. Then, a central area was delimited with 4x4mm, while the remaining areas were isolated with acid-resistant varnish (Colorama, S\u0026atilde;o Paulo, Brazil).\u003c/p\u003e\u003cp\u003eThe protocol proposed by Moi et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was used for the induction of initial caries lesions. The demineralizing solution (DES) was composed of 0.1 M acetate buffer (pH 5.0) containing 1.28 mM Ca, 0.74 mM Pi, and 0.03 \u0026micro;g F/mL (3 mL/mm\u0026sup2;), and the remineralizing solution (RE) was composed of 1.5 mM Ca, 0.9 mM Pi, 150 mM KCl, 0.05 \u0026micro;g F/mL, and 0.1 M Tris buffer (pH 7.0) (1.5 mL/mm\u0026sup2;). In this protocol, cycling consisted of 20 hours of immersion in the RE solution and 4 hours in the DES solution. This procedure was carried out for seven days (seven cycles), and on the eighth day, the samples remained immersed in the RE solution for 24 hours.\u003c/p\u003e\u003cp\u003eThe blocks were then infiltrated with resin infiltrants according to the manufacturer's protocol, applying 15% hydrochloric acid for 2 minutes followed by thorough water rinsing. The samples were then immersed in a 0.1% Rhodamine-B solution for 12 hours. Afterwards, the samples were rinsed again, IconDry was applied, followed by gentle air blasting for 30 seconds, and then the application of the resin infiltrant for 3 minutes, with excess removed and followed by photopolymerization (Valo, Ultradent) for 40 seconds. A new application was then carried out for 1 minute, followed by light polymerization.\u003c/p\u003e\u003cp\u003eAfter this process, the samples were stored for 24 hours in an oven, and then sectioned using a metallographic cutter. The slices obtained were manually polished with #600, #1200, and #2000 grit sandpaper until they reached a thickness between 150\u0026ndash;200 \u0026micro;m. They were then kept for 12 hours in a 30% hydrogen peroxide solution to remove excess Rhodamine-B.\u003c/p\u003e\u003cp\u003eFor microscopy preparation, the samples were thoroughly washed in water and immersed in an ethanolic solution of sodium fluorescein for 3 minutes, followed by rinsing with deionized water. The samples were then placed on a glass coverslip containing specific oil for the confocal laser scanning microscope (CLSM) (Leica, TCS NT; Leica Heidelberg, Germany), with lasers of wavelengths 488 and 594 nm and a magnification of 63x selected [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003e6. Evaluation of biofilm formation and antimicrobial activity\u003c/h3\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e6.1 Bacterial culture manipulation\u003c/h2\u003e\u003cp\u003eThe Streptococcus mutans UA159 strain (Laboratory of Microbiology and Immunology of the Piracicaba School of Dentistry, State University of Campinas, Piracicaba, Brazil) was used. The strains were maintained in BHI (brain heart infusion - Difco Laboratories, Detroit, USA) culture medium and fed with 20% glycerol at -20\u0026deg;C\u003csup\u003e9\u003c/sup\u003e. The microorganisms were reactivated on BHI plates and incubated at 37\u0026deg;C for 24 hours [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e6.2 Preparation of samples on discs\u003c/h2\u003e\u003cp\u003eDiscs measuring 5 mm in diameter x 2 mm in thickness were prepared for antimicrobial evaluation using an addition silicone matrix made from a prefabricated mold. The samples were polymerized using an LED photopolymerizer (Valo, Ultradent, power density of 1000 mW/cm\u003csup\u003e2\u003c/sup\u003e) for 40 s on both sides. The discs were then immersed in 10 ml of distilled water and placed in an ultrasonic vibration machine (Branson, Emerson, St. Louis, MO, USA) for 10 minutes to remove residual monomers. For disinfection, these samples were subjected to ultraviolet light for 30 minutes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e6.3 Anti-biofilm assay\u003c/h2\u003e\u003cp\u003eBased on a previous study [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the discs were distributed in microplates containing bacterial suspension (at 1.5 x 108 CFU/mL). Immediately afterwards, the microplates were incubated at 37\u0026deg;C for 48 hours to form biofilm on the surface of the infiltrating discs. Afterwards, the discs were washed in PBS 1X pH 7.2 buffer (Saline-Phosphate buffer) to remove planktonic bacterial cells and sonicated; the bacterial suspension was diluted and plated on BHI agar. Finally, the S. mutans count in the biofilm was performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e6.5 MTT Test\u003c/h2\u003e\u003cp\u003eThe MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) test for live and dead cells will measure metabolic activity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. After incubation for 48h, the biofilms were washed with sterile distilled water and then 200 \u0026micro;L/well of MTT dye were added. The 96-well plates were inoculated at 37\u0026deg;C for 1 hour and then the MTT solution was removed. After these steps, 300 \u0026micro;L of DMSO (dimethyl sulfoxide) were added and shaken for 20min at 80rpm in the dark. Then, 200 \u0026micro;L of DMSO were distributed in 96-well plates and subjected to a microplate reader to measure absorbance at OD (540nm). A high absorbance indicates a higher concentration of biofilm, which in turn indicates higher metabolic activity in the biofilm in the material.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e6.6 Biofilm analysis by SEM\u003c/h2\u003e\u003cp\u003eTo analyze the initial formation (4 hours) of Streptococcus mutans biofilm on the experimental infiltrant, a scanning electron microscope was used [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A sample of each infiltrant group was placed in a 24-well plate along with 1.5 ml of BHI medium added with 1% sucrose. The media were then inoculated with cultures of the strains in half of the exponential growth phase adjusted to the same absorbance (A550nm of 0.03) and the plates were incubated at 37\u0026deg;C for 4 hours in aerobic agitation (Nova Instrument-Thermo-shaker plate shaker; shaking at 250 rpm). After incubation, the culture medium was removed with a sterile micropipette, and the infiltrant discs were washed with 1.5 ml of 0.9% saline solution (0.9% NaCl) in a shaker (MicroPlate Shaker \u0026ndash; MA 562, Marconi Equipamentos, Brazil) for 15 minutes. The washing step was performed three times. Then, the discs were treated with 800 \u0026micro;l of 2.5% glutaraldehyde solution (Sigma-Aldrich) for 30 min at room temperature. The discs were dehydrated by incubating with ethanol solution in increasing concentrations (50\u0026ndash;100%) for 15 min for each solution. After dehydration, the discs were dried at room temperature and mounted on stubs to be metallized with gold and analyzed under a scanning electron microscope (Jeol 4 JSM5600 LV 33). Representative images of the specimens were obtained at 1,000X magnification and in duplicate.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e7. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eDescriptive and exploratory data analyses were carried out. Previous analyses indicated that the data did not meet the assumptions of a classic analysis of variance (ANOVA) with a general linear model. Then, sorption, solubility, elastic modulus and flexural strength data were analyzed with generalized linear models. The degree of conversion data did not fit a known distribution and was therefore analyzed using non-parametric test of Kruskal Wallis and Dunn. All analyzes were performed using R program, with significance level set at 5%.\u003c/p\u003e"},{"header":"RESULTS","content":"\n\u003ch3\u003e1. Conversion Degree\u003c/h3\u003e\n\u003cp\u003eThe results of the conversion degree are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showing that it was significantly higher in the Graphene group with 80 seconds of photoactivation compared to the Graphene group with 40 seconds of photoactivation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no difference among Icon, Experimental and GO 80s or GO 40s.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean (standard deviation) and median (minimum; maximum) of the conversion degree depending on the group.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean (standard deviation)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMedian (minimum; maximum)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIcon (IC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74,77 (0,45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e74,78 (74,21; 75,29) ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental (E)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72,66 (3,27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71,95 (69,79; 76,93) ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGraphene (GO) (80 seconds of photoactivation)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e82,92 (1,20)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82,50 (82,03; 84,65) a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGraphene (GO) (40 seconds of photoactivation)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62,64 (4,88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62,54 (58,26; 67,23) b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0,0045\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e2. Sorption and solubility\u003c/h3\u003e\n\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, for sorption, Experimental group exhibited higher values than the Icon group but lower than the Graphene group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Regarding solubility, it can be observed that the Icon and Experimental groups demonstrated lower solubility than the Graphene group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as depicted in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean (standard deviation) and median (minimum; maximum) of sorption and solubility as a function of group.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMean (standard deviation)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMedian (minimum; maximum)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eIcon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5,39x10⁻⁵ (2,25x10⁻⁵) c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5,20x10⁻⁵ (2,02x10⁻⁵; 10,96x10⁻⁵)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eExperimental\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10,89x10⁻⁵ (2,26x10⁻⁵) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11,37x10⁻⁵ (5,40x10⁻⁵; 12,98x10⁻⁵)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eGraphene Oxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13,96x10⁻⁵ (1,20x10⁻⁵) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14,07x10⁻⁵ (11,66x10⁻⁵; 15,73x10⁻⁵)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0,0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSolubility\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eIcon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,78x10⁻⁵ (2,13x10⁻⁵) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0,80x10⁻⁵ (-0,57x10⁻⁵; 6,61x10⁻⁵)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eExperimental\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0,40x10⁻⁵ (8,84x10⁻⁵) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3,23x10⁻⁵ (-24,61x10⁻⁵; 4,44x10⁻⁵)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eGraphene Oxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,39x10⁻⁵ (3,09x10⁻⁵) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6,71x10⁻⁵ (1,61x10⁻⁵; 12,05x10⁻⁵)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0,0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e3. Flexural strength and elastic modulus\u003c/h3\u003e\n\u003cp\u003eThe elastic modulus and flexural strength were significantly higher in the Icon group compared to the other two groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. There was no difference between Experimental and GO group in both elastic modulus and flexural strength.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean (standard deviation) and median (minimum; maximum) elastic modulus (GPa) and flexural strength (MPa) as a function of group.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean (standard deviation)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMedian (minimum; maximum)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eModulus of elasticity (Automatic Young's) (GPa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIcon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,09 (0,22) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,14 (0,79; 1,41)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExperimental\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,34 (0,08) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0,35 (0,22; 0,45)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGraphene Oxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,36 (0,11) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0,40 (0,14; 0,49)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0,0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFlexural strength (MPa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIcon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e86,74 (21,82) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86,66 (42,66; 120,44)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExperimental\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43,13 (8,39) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40,28 (29,00; 56,73)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGraphene Oxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47,18 (12,89) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47,69 (20,54; 68,91)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0,0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e4. Penetration depth\u003c/h3\u003e\n\u003cp\u003eIn the images obtained using confocal microscopy, it was possible to observe superficial deposition of the material and infiltration of the resinous tags in the demineralized area in the Icon (I) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and Graphene Oxide (GO) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) groups. In the Experimental group(E) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), surface deposition was observed, but the resinous tags were not identified.\u003c/p\u003e\n\u003ch3\u003e5. Anti-biofilm assay\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e shows the approximate number of colonies formed after 48 hours of incubation in each group. The E group exhibited the highest colony formation (1.71 \u0026times; 10⁷ CFU/ml), while the group with the incorporation of 0.5% GO showed the lowest colony formation (2.22 \u0026times; 10⁶ CFU/ml), with a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003ch3\u003e6. MTT metabolic assay\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the metabolic activity of the biofilm, quantified by the MTT assay. The E group exhibited the highest MTT absorbance, indicating higher metabolic activity in the S. mutans biofilms adhered to the discs. The incorporation of 0.5% GO significantly reduced the metabolic activity of the biofilms (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The I group (commercial control) showed no significant difference compared to the other groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05)\u003c/p\u003e\n\u003ch3\u003e7. Biofilm Analysis by SEM\u003c/h3\u003e\n\u003cp\u003eFigures \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e shows the initial formation of the Streptococcus mutans biofilm after 4 hours of incubation, observed by scanning electron microscopy (SEM). The I (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) and E (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) groups exhibit greater bacterial adhesion, as evidenced by a higher biofilm density. In contrast, the group treated with 0.5% GO (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) shows reduced S. mutans adhesion, with cleaner surface areas, suggesting an inhibitory effect on biofilm formation.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe incorporation of nanoparticles into resin infiltrants has been widely studied with various results [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Since it is commercially available from only one company, the development of materials with the same purpose but lower costs and better properties is desired.\u003c/p\u003e\u003cp\u003eGraphene oxide is a more reactive particle than regular graphene, also exhibiting potential antibacterial properties against \u003cem\u003eS. mutans\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, it is reported to improve the mechanical properties of composite resin when incorporated at concentrations of 0.3% and 0.5%, demonstrating good results in surface microhardness and flexural strength [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe resin infiltrant has a low viscosity, and therefore its texture is similar to dental adhesives. In this context, the study by Lee et al [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] evaluated the incorporation of graphene oxide into dental adhesives, resulting in increased antimicrobial activity and improved shear strength. This demonstrated that graphene oxide-enriched adhesives are suitable for clinical application, leading to the premise that it would be an interesting material to study in resin infiltrants.\u003c/p\u003e\u003cp\u003eThe concentration used in this work was based on previous studies, such as Lee et al.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], in which the incorporation of graphene oxide into PMMA resulted in superior Vickers hardness at concentrations higher than 0.5% graphene oxide. Similarly, Velo et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] evaluated concentrations of 0.3% and 0.5% in composite resins. A pilot study conducted in this research found that, due to its very light nature, graphene oxide particles in concentrations greater than 2% represented a very large volume of particles to be incorporated into the resin matrix of the infiltrant. Therefore, we established the concentration at 0.5%.\u003c/p\u003e\u003cp\u003eThe degree of conversion refers to the amount of free monomers that transform into polymer chains, making it an essential test for new material proposals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Since the test specimens for sorption, solubility, and flexural strength require a minimum thickness of 1 mm and the resin infiltrant containing graphene oxide exhibits a dark coloration, a photopolymerization time of 80 seconds was established for this group based on a pilot study. However, for comparison purposes, measurements were taken with both 40 seconds and 80 seconds of exposure.\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, graphene oxide with 80 seconds of photopolymerization achieved the best results compared to the experimental and commercial groups, likely due to its longer exposure to the wavelength of the photopolymerization device, ensuring a higher conversion of monomers. With 40 seconds of activation, the results were lower but did not differ statistically from the experimental and commercial groups, corroborating with Velo [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], where no difference was found between groups with or without graphene oxide in composite resins. It is important to note that regardless of the time used in this evaluation, all groups presented a degree of conversion above 55%, which is described by Soares [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Silikas [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] as a potential threshold for resin composites.\u003c/p\u003e\u003cp\u003eThe sorption and solubility of a resin material indicates its ability to retain liquids and its dissolution capacity. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the commercial group (I) exhibited superior behavior compared to the experimental group (E) in terms of sorption, corroborating the findings of Mathias [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and Souza [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], who also observed higher sorption in the experimental groups. This difference can be attributed to the material's composition, as Icon holds a patent on the product and does not specify all the components, only that it consists of 75% TEGDMA and additives. Nevertheless, both groups I and E were superior to the experimental group containing graphene oxide (GO), which showed higher sorption and solubility. This difference could be mainly due to the presence of nanoparticles in the composition of GO without functionalization, as described by Velo et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, it is possible that the particles and/or the organic matrix of the material dissolved more easily than in the other groups. On the other hand, the methodology of incorporating the particle into the material without treating it is also supported by a study that evaluated the incorporation of graphene into adhesives, such as by Bregnocchi et al [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGraphene is an extremely rigid material, and it is seen as an opportunity for strengthening and stiffening lightweight composites [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, one of the biggest challenges in working with graphene is its tendency to agglomerate [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This occurs because this particle is highly hydrophilic and is also influenced by Van der Waals forces [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Considering this, according to Yang et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], in graphene-enriched materials, a higher homogeneity of the particle in the polymer matrix increases the chances of positive results in improving physicochemical properties. Thus, its tendency to agglomerate becomes one of the main obstacles limiting its potential to reinforce materials [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe flexural strength and modulus of elasticity used to assess the material's resistance were superior in group I, while E and GO demonstrated lower values. Generally, the resistance of resin infiltrants tends to be lower compared to resin composites, mainly because they do not have a significant inorganic filler content [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, this finding does not corroborate the study by Velo et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which reported superior results in experimental resin composites containing graphene oxide compared to the control.\u003c/p\u003e\u003cp\u003eUnlike the findings in resins containing GO, the resistance of this group was similar to base experimental group. This difference can be speculated by two reasons: 1) the methodology employed, as those studies evaluated both surface microhardness and flexural strength, and 2) the coloration of the material. Interestingly, there are no descriptions in the literature about it, but the material exhibits a very strong, dark, and opaque coloration even at low concentrations, which could interfere with the photopolymerization process, directly impacting the material's resistance.\u003c/p\u003e\u003cp\u003eA methodological limitation in studies on resin infiltrants is the evaluation of the penetration capacity of particles incorporated into the material. The most used test is confocal microscopy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which can access the penetration through lasers and refractive index due to the use of dyes, making it possible to visualize the penetration but not the elements and particles that infiltrated. So, even if it is possible to verify resin tags in GO group, it is not possible to confirm that the graphene oxide particles fully penetrated the enamel microtubules. The commercial group (I) also showed resin tags along its margin, aligning with other studies that also affirm material penetration up to 100 \u0026micro;m [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, the base experimental group did not show regularity throughout the sample and/or resin tags, differing from studies such as Cerqueira et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which demonstrated material permeation along the enamel microtubules.\u003c/p\u003e\u003cp\u003eTo halt the progression of caries, it is crucial for the material to penetrate and obliterate the enamel tubules, preventing interaction between the dental substrate and the potentially cariogenic environment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The GO group demonstrated an ability to penetrate similarly to the I group, as seen in the images. Thus, the addition of graphene oxide does not interfere with the material's ability to infiltrate dentinal tubules.\u003c/p\u003e\u003cp\u003eAccording to Mao et al.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], graphene oxide exhibits antimicrobial effects against oral pathogens such as \u003cem\u003eS. Mutans\u003c/em\u003e, making it a promising nanomaterial for use in dental materials. In our study, the resin infiltrant containing GO demonstrated superior antibiofilm formation comparing with base experimental, enhancing the possibility that GO was capable to inhibit bacterial growth. This was also supported by Pourhajibagher and Bahador [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] that incorporated GO into orthodontic adhesives and observed a greater results on CFU counting for 5 and 10% concentration. Bregnocchi et al [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] also observed in adhesives that the incorporation of 0.2% graphene was able to reduce the CFU count for \u003cem\u003eS. mutans.\u003c/em\u003e For the MTT test, an inhibitory effect of GO was also observed compared to experimental E, which is supported by Zhao [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], who demonstrated that GO concentrations tend to generate a higher amount of reactive oxygen species and exhibit greater antibacterial potential than controls without its incorporation.\u003c/p\u003e\u003cp\u003eRecent research suggests that the main antibacterial mechanisms of graphene materials involve the physical disruption of cell membranes, oxidative stress, and cell trapping or wrapping due to their honeycomb conformation, that are functional groups modified considered essential in mediating oxidative stress [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although SEM micrographs of the biofilm showed that the surface of the experimental infiltrant exhibited a reduction in biofilm formation, the GO group did not differ from the commercial group Icon, indicating that while it exhibited antibacterial activity compared to the experimental base, its effect was not strong enough to differ significantly from the commercial control.\u003c/p\u003e\u003cp\u003eNew alternatives are always useful for better understanding the mechanisms involved in the use of resinous infiltrants and, therefore, continuing the development of these materials and verifying their performance in different situations and tests are essential. The experimental infiltrant containing graphene oxide showed limitations in relation to commercial control, making it necessary to adapt its incorporation to the resin matrix and verify its behavior in further tests, but seems to be a promising material for infiltrant applications in proximal areas.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIncorporating 0.5% graphene oxide (GO) into the experimental infiltrant significantly increased the degree of conversion when exposed to 80 seconds of photopolymerization. Considering the antibacterial properties, the GO-containing experimental group showed reduced biofilm formation and lower CFU counts for \u003cem\u003eStreptococcus mutans\u003c/em\u003e compared to its counterpart without the addition of the nanoparticle. Although the commercial resin infiltrant exhibited superior performance in most mechanical tests, the presence of GO did not compromise the material\u0026rsquo;s ability to infiltrate initial caries lesions, as demonstrated by confocal laser scanning microscopy. Furthermore, the GO-containing material also presented enhanced antibacterial activity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the contribution of Fl\u0026aacute;via Sammartino Mariano Rodrigues from the Microscopy and Image Center at Piracicaba Dental School (University of Campinas, Piracicaba, S\u0026atilde;o Paulo, Brazil) for the assistance in confocal microscopy analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was financed by the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa de S\u0026atilde;o Paulo (FAPESP) \u0026ndash; Process Code 2022/15280-8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the author(s) used ChatGPT in order to improve the language. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChen KJ, Gao SS, Duangthip D, Lo ECM, Chu CH. Early childhood caries and oral health care of Hong Kong preschool children. Clin Cosmet Investig Dent. 2019; 17(11):27-35. https://doi.org/10.2147/CCIDE.S190993.\u003c/li\u003e\n \u003cli\u003eGiray FE, Durhan MA, Haznedaroglu E, Durmus B, Kalyoncu IO, Tanboga I. Resin infiltration technique and fluoride varnish on white spot lesions in children: Preliminary findings of a randomized clinical trial. Niger J Clin Pract 2018; 21:1564-9. https://doi.org/10.4103/njcp.njcp_209_18.\u003c/li\u003e\n \u003cli\u003eMeyer-Lueckel H, Paris S. Progression of artificial enamel caries lesions after infiltration with experimental light curing resins. 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Anticaries potential of a fluoride mouthrinse evaluated in vitro by validated protocols. \u003cem\u003eBraz Dent J\u003c/em\u003e. 2008;19(2):91-96. doi:10.1590/s0103-64402008000200001\u003c/li\u003e\n \u003cli\u003ePedreira PR, Damasceno JE, Mathias C, Sinhoreti M, Aguiar F, Marchi GM. Influence of Incorporating Zirconium- and Barium-based Radiopaque Filler Into Experimental and Commercial Infiltrants. Oper Dent. 2021;46(5):566-576. https://doi.org/10.2341/20-020-L.\u003c/li\u003e\n \u003cli\u003eSu M, Yao S, Gu L, Huang Z, Mai S. Antibacterial effect and bond strength of a modified dental adhesive containing the peptide nisin. \u003cem\u003ePeptides\u003c/em\u003e. 2018;99:189-194. doi:10.1016/j.peptides.2017.10.003\u003c/li\u003e\n \u003cli\u003eGhorbanzadeh R, Hosseinpour Nader A, Salehi-Vaziri A. 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The Antibacterial Effect of Graphene Oxide on Streptococcus mutans. J Nanosci Nanotechnol. 2020 Apr 1;20(4):2095-2103. https://doi.org/10.1166/jnn.2020.17319.\u003c/li\u003e\n \u003cli\u003eMao M, Zhang W, Huang Z, et al. Graphene Oxide-Copper Nanocomposites Suppress Cariogenic Streptococcus mutans Biofilm Formation. Int J Nanomedicine. 2021;16:7727-7739. Published 2021 Nov 18. https://doi.org/10.2147/IJN.S303521.\u003c/li\u003e\n \u003cli\u003eVelo MMAC, Filho FGN, de Lima Nascimento TR, et al. Enhancing the mechanical properties and providing bioactive potential for graphene oxide/montmorillonite hybrid dental resin composites. Sci Rep. 2022;12(1):10259. https://doi.org/10.1038/s41598-022-13766-1.\u003c/li\u003e\n \u003cli\u003eLee JH, Jo JK, Kim DA, Patel KD, Kim HW, Lee HH. Nano-graphene oxide incorporated into PMMA resin to prevent microbial adhesion. Dent Mater. 2018;34(4):e63-e72. https://doi.org/10.1016/j.dental.2018.01.019.\u003c/li\u003e\n \u003cli\u003eBorges A, Chase M, Niederberger A, Gonzalez M, Ribeiro A, Pascon F, Zanatta A. A Critical Review on the Conversion Degree of Resin Monomers by Direct Analyses. Braz Dent Sci. 2013, 16. https://doi.org/10.14295/bds.2013.v16i1.845.\u003c/li\u003e\n \u003cli\u003eSoares LE, Liporoni PC, Martin AA. The effect of soft-start polymerization by second generation LEDs on the degree of conversion of resin composite. Oper Dent. 2007 Mar-Apr;32(2):160-5. https://doi.org/10.2341/06-45.\u003c/li\u003e\n \u003cli\u003eSilikas N, Eliades G, Watts DC. Light intensity effects on resin-composite degree of conversion and shrinkage strain Dental Materials. 2000; 16(4) 292-296. https://10.1016/s0109-5641(00)00020-8.\u003c/li\u003e\n \u003cli\u003eYang, Y., Rigdon, W., Huang, X. \u0026amp; Li, X. Enhancing graphene reinforcing potential in composites by hydrogen passivation induced dispersion. Sci. Rep. 2013; 3: 2086. https://doi.org/10.1038/srep02086.\u003c/li\u003e\n \u003cli\u003eZhou X, Wu T, Ding K, Hu B, Hou M, Han B. Dispersion of graphene sheets in ionic liquid [bmim][PF6] stabilized by an ionic liquid polymer. Chem Commun (Camb). 2010;46(3):386-388. https://doi.org/10.1039/b914763b.\u003c/li\u003e\n \u003cli\u003eLi D, Müller MB, Gilje S, Kaner RB, Wallace GG. Processable aqueous dispersions of graphene nanosheets. Nat Nanotechnol. 2008;3(2):101-105. https://doi.org/10.1038/nnano.2007.451.\u003c/li\u003e\n \u003cli\u003eParis S, Soviero VM, Seddig S, Meyer-Lueckel H. Penetration depths of na infiltrant into proximal caries lesions in primary molars after diferente application times in vitro. Int J Paediatr Dent. 2012 Sep;22(5):349-55. https://doi.org/10.1111/j.1365-263X.2011.01204\u003c/li\u003e\n \u003cli\u003eCerqueira, GA, et al (2022) Roughness and Microhardness of demineralized enamel treated with resinous infiltrants and subjected to an acid challengue: an in vitro study. The Open Dentistry Journal 17: e187421062302030. https://doi:10.2174/18742106-v17-230223-2022-126.\u003c/li\u003e\n \u003cli\u003eParis S, Meyer-Lueckel H, Cölfen H, Kielbassa AM. Resin infiltration of artificial enamel caries lesions with experimental light curing resins. Dent Mater J. 2007; 26(4): 582-8. https://doi.org/10.4012/dmj.26.582.\u003c/li\u003e\n \u003cli\u003ePourhajibagher M, Bahador A. Orthodontic adhesive doped with nano-graphene oxide: physico-mechanical and antimicrobial properties. \u003cem\u003eFolia Med (Plovdiv)\u003c/em\u003e. 2021;63(3):413-421. doi:10.3897/folmed.63.e53716\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Dentistry, Dental Caries, Dental Enamel, Graphene Oxide","lastPublishedDoi":"10.21203/rs.3.rs-7011700/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7011700/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aimed to evaluate the influence of 0.5% graphene oxide (GO) incorporated into an experimental resin infiltrant on its physicochemical and antibacterial properties compared to Commercial Icon (IC) and Experimental (E) groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroups E and GO were manipulated, and specimens were prepared according to test requirements. Fourier-transform infrared spectroscopy (FTIR) was used to assess the degree of conversion (DC) (n = 5) before and after photoactivation. Sorption (So) and solubility (Sol) (n = 10) were evaluated after 7 days of water storage. Three-point bending tests determined the elastic modulus (EM) and flexural strength (FS) (n = 10). Initial carious lesions were induced in bovine enamel and analyzed through Confocal Laser Scanning Microscopy (CLSM) for penetration depth (n = 3). Antibacterial activity was assessed using antibiofilm assay (CFU), MTT metabolic test, and biofilm analysis via Scanning Electron Microscopy (SEM). Generalized linear models were applied for So, Sol, EM, and FS, while Kruskal-Wallis and Dunn’s tests analyzed DC, CFU, and MTT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGO differed from IC and E in DC after 80s photoactivation, exhibiting superior results. GO showed higher So and Sol values. IC demonstrated the best EM and FS. CLSM confirmed enamel infiltration for GO and IC. E showed the highest CFU, while GO had the lowest. E exhibited the highest MTT absorbance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIncorporating GO into the experimental infiltrant is feasible, demonstrating good infiltration and antibacterial activity. Further studies are needed to optimize its properties.\u003c/p\u003e\n\u003cp\u003eClinical relevance: Infiltrants with graphene oxide and commercial formulations both demonstrated satisfactory antibacterial and infiltration performance.\u003c/p\u003e","manuscriptTitle":"Evaluation of physicochemical characteristics of experimental resin infiltrant containing graphene oxide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 12:41:00","doi":"10.21203/rs.3.rs-7011700/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-30T00:35:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-28T08:01:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T22:36:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163063711932653278888947490077046384722","date":"2025-07-19T06:21:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287517163276175186329822748318797260935","date":"2025-07-17T12:09:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-17T05:40:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-01T06:50:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-01T06:49:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Oral Investigations","date":"2025-06-30T14:28:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e3704c8b-20eb-4c55-8ee3-ace1e1f0e20c","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:04:33+00:00","versionOfRecord":{"articleIdentity":"rs-7011700","link":"https://doi.org/10.1007/s00784-025-06598-6","journal":{"identity":"clinical-oral-investigations","isVorOnly":false,"title":"Clinical Oral Investigations"},"publishedOn":"2025-10-27 15:57:38","publishedOnDateReadable":"October 27th, 2025"},"versionCreatedAt":"2025-07-21 12:41:00","video":"","vorDoi":"10.1007/s00784-025-06598-6","vorDoiUrl":"https://doi.org/10.1007/s00784-025-06598-6","workflowStages":[]},"version":"v1","identity":"rs-7011700","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7011700","identity":"rs-7011700","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}