Dentistry, Faculty of
Medicine, Faculty of
Non UBC
Oral Biological and Medical Sciences (OBMS), Department of
DSpace
Coatings 15 (1): 40 (2025)
de Rodríguez, Katleen A. Aguirre
de González, Wendy Y. Escobar
Castañeda Monroy, Vianney
Murphy, Sean
Martínez-Castañón, Gabriel-Alejandro
Bach, Horacio
Niño-Martínez, Nereyda
Research with nanoparticles for the treatment and prevention of dental caries is of special interest given the high prevalence of the disease worldwide. Several studies support the use of nanoparticles associated with materials given their antimicrobial properties and potential demineralization reduction. This study aimed to evaluate the impact of the application of silver nanoparticles (AgNPs) and chitosan gel in combination with commercial fluoride varnish on the remineralization of dental enamel. Ninety-six tooth blocks were macroscopically evaluated via stereomicroscopy, ICDAS II, and laser fluorescence. Enamel blocks were subjected to artificial demineralization and divided into four exposure groups (24, 48, 120, and 168 h), and five different remineralizing agents were applied, namely, FV (fluoride varnish), FV + CG (fluoride varnish + chitosan gel), FV + AgNPs (fluoride varnish + AgNPs), FV + AgNPs + CG (fluoride varnish + AgNPs + chitosan gel), and AgNPs + CG (AgNPs + chitosan gel). Enamel surface changes were evaluated via laser fluorescence, X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. Laser fluorescence results obtained from demineralized blocks and subsequently exposed to remineralizing treatment indicate significant differences. After exposure to remineralizing agents, hydroxyapatite and modified apatite phases were identified mainly in the samples treated with FV + AgNPs + CG in the groups exposed for 24, 48, and 120 h. The FV + AgNPs + CG and AgNPs + CG indicate good performance in terms of the Ca/P ratio in in vitro demineralization compared to the group treated with fluorine varnish.
Academic Editors: Egemen Avcu,Yasemin Yıldıran Avcu andMert GuneyReceived: 9 December 2024Revised: 31 December 2024Accepted: 2 January 2025Published: 3 January 2025Citation: de Rodríguez, K.A.A.; deGonzález, W.Y.E.; Castañeda Monroy,V.; Murphy, S.; Martínez-Castañón,G.-A.; Bach, H.; Niño-Martínez, N.Silver Nanoparticles–ChitosanNanocomposites as ProtectiveCoatings for Dental RemineralizationTreatment: An In Vitro Study. Coatings2025, 15, 40. https://doi.org/10.3390/coatings15010040Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ArticleSilver Nanoparticles–Chitosan Nanocomposites as ProtectiveCoatings for Dental Remineralization Treatment:An In Vitro StudyKatleen A. Aguirre de Rodríguez 1,2 , Wendy Y. Escobar de González 1,2 , Vianney Castañeda Monroy 2,Sean Murphy 3, Gabriel-Alejandro Martínez-Castañón 4,* , Horacio Bach 5 and Nereyda Niño-Martínez 6,*1 Facultad de Estomatología, Zona Universitaria, Av. Dr. Manuel Nava No. 2, San Luis Potosí CP 78290, Mexico;katleen.aguirre@ues.edu.sv (K.A.A.d.R.); wendy.escobar@ues.edu.sv (W.Y.E.d.G.)2 Laboratorio de Biología Molecular, CENSALUD, Universidad de El Salvador (UES),San Salvador CP 1101, El Salvador; vianney.monroy@ues.edu.sv3 Faculty of Dentistry, Department of Oral Biological and Medical Sciences, University of British Columbia,Vancouver, BC V6H 3Z6, Canada; sean.murphy@dentistry.ubc.ca4 Facultad de Estomatología, Universidad Autónoma de San Luis Potosi, Zona Universitaria, Av. Dr. ManuelNava No. 2, San Luis Potosí CP 78290, Mexico5 Faculty of Medicine, Division of Infectious Diseases, University of British Columbia,Vancouver, BC V6H 3Z6, Canada; horacio.bach@ubc.ca6 Facultad de Ciencias, Universidad Autónoma de San Luis Potosi, Av. Parque Chapultepec No. 1570, Privadasdel Pedregal, San Luis Potosí CP 78295, Mexico* Correspondence: mtzcastanon@fciencias.uaslp.mx (G.-A.M.-C.); nereyda.nino@uaslp.mx (N.N.-M.)Abstract: Research with nanoparticles for the treatment and prevention of dental caries is ofspecial interest given the high prevalence of the disease worldwide. Several studies supportthe use of nanoparticles associated with materials given their antimicrobial properties andpotential demineralization reduction. This study aimed to evaluate the impact of the appli-cation of silver nanoparticles (AgNPs) and chitosan gel in combination with commercialfluoride varnish on the remineralization of dental enamel. Ninety-six tooth blocks weremacroscopically evaluated via stereomicroscopy, ICDAS II, and laser fluorescence. Enamelblocks were subjected to artificial demineralization and divided into four exposure groups(24, 48, 120, and 168 h), and five different remineralizing agents were applied, namely,FV (fluoride varnish), FV + CG (fluoride varnish + chitosan gel), FV + AgNPs (fluoridevarnish + AgNPs), FV + AgNPs + CG (fluoride varnish + AgNPs + chitosan gel), andAgNPs + CG (AgNPs + chitosan gel). Enamel surface changes were evaluated via laser fluo-rescence, X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spec-troscopy. Laser fluorescence results obtained from demineralized blocks and subsequentlyexposed to remineralizing treatment indicate significant differences. After exposure to rem-ineralizing agents, hydroxyapatite and modified apatite phases were identified mainly inthe samples treated with FV + AgNPs + CG in the groups exposed for 24, 48, and 120 h. TheFV + AgNPs + CG and AgNPs + CG indicate good performance in terms of the Ca/P ratioin in vitro demineralization compared to the group treated with fluorine varnish.Keywords: enamel remineralization; demineralization; chitosan; silver nanoparticles1. IntroductionDental caries is a highly prevalent disease in the world population. Its progressionfrom the enamel surface to the innermost structures of the tooth depends on multiple factorssuch as cariogenic diet, hygiene habits, time, and salivary pH variations [1]. The onset ofCoatings 2025, 15, 40 https://doi.org/10.3390/coatings15010040Coatings 2025, 15, 40 2 of 15the lesion is related to the acidification of the environment, causing the demineralizationof the teeth, which implies the loss of minerals in the initial stages. This process can bereversed naturally by repair through remineralization, which has the purpose of restoringthe lost minerals. This process occurs at near-neutral physiological pH [2,3].The timely identification of incipient lesions in enamel is of great importance inestablishing preventive treatments and immediate intervention to stop their progression.The use of materials such as varnish fluoride for the management of the remineralizationof dental tissues has been widely studied. Kim et al. (2021) compared the demineralizationresistance and remineralization effects of various fluoride-releasing restorative commercialmaterials under clinical conditions and found that alkasite restorative material is a superiorfluoride-releasing restorative material in stimulating demineralization and remineralizationin an enamel [3]. However, in a different study, Chhatwani et al. (2021) found that theeffectiveness of fluoride varnishes is compromised and limited due to its permanencein the oral environment, which stresses the materials under different temperature andpH conditions [4]. It has also been reported that the use of fluoride varnish (FV) couldproduce sensitivity and a burning sensation in some patients and, in regions where thelevels of fluoride in water are high, the use of this varnish is limited; Parisay et al. (2024)explain that excessive fluoride consumption can result in symptoms like nausea, vomiting,diarrhea, escalated salivation, abdominal discomfort, muscle frailty, and spasms. Fluoridevarnish is not recommended for patients with ulcerative gingivitis, gingivitis stomatitis,or asthma [5]. Silver diamine fluoride has also been reported as a material which inhibitsdemineralization, promoting the remineralization of both enamel and dentin, but stainingon the areas where it is applied limits its use [6,7].On the other hand, nanotechnology and its application in dentistry have allowed forthe identification of some characteristics, properties, and benefits in the management oforal diseases such as dental caries [7]; silver nanoparticles (AgNPs) have been extensivelyused due to their antibacterial properties with sustained activity [8]. Pichaiaukrit et al.(2019) and Ghafar et al. (2020) studied combinations of chitosan with calcium fluorideand sodium fluoride nanoparticles, trying to obtain a material with a sustained release offluoride for the treatment of incipient lesions of dental caries [9,10]. Hydroxyapatite NPshave been used in toothpaste [11] and other dentifrices to promote remineralization, buttheir abrasive effect on enamel still causes concerns [12]. In another in vitro study, Surijaet al. (2018) found satisfactory results on the effects of chitosan; it inhibits the enameldemineralization process in vitro by increasing the environmental pH and reducing thesolubility of the enamel [13].This study aimed to evaluate the impact of the application of AgNPs and chitosangel (CG) in combination with a commercial FV on the remineralization of dental enamelthrough macroscopic evaluation, ICDAS II, laser fluorescence (DIAGNOdent pen), scanningelectron microscopy (SEM), and X-ray diffraction (XRD). The following null hypothesisis proposed: There are no differences in the remineralizing effect between the proposedFV + AgNPs + CG treatment and the conventional protocol with FV for initial lesions onsmooth surfaces.2. Materials and Methods2.1. ParticipantsThe protocol of this study was approved by the Research Ethics Committee of theFaculty of Dentistry of the University of El Salvador with the following document number:CEI-FOUES/2022/003. Participants provided verbal consent, and the recruitment of thepatients ranged from 1 April 2022 until 3 March 2023. The 61 healthy teeth used in thisCoatings 2025, 15, 40 3 of 15study were extracted for orthodontic reasons without any relation to this protocol. Thepatients were informed, and they donated their pieces for this study with verbal consent.2.2. Preparation and Characterization of Chitosan Gel with AgNPsFor the preparation of CG, a published protocol was used [14]. In brief, 2 mL of aceticacid was added to 47.8 mL of a dispersion of AgNPs (0.1% by weight concentration). Tothis mixture, 3.5 wt.% chitosan was added, together with 0.2 mL of glycerol. The mixturewas stirred under the same conditions mentioned above until the gel was formed.2.3. Sample PreparationThe sample consisted of 122 blocks of 61 healthy teeth extracted for orthodonticreasons, preserved in chloramine-T at −17 ◦C in the Biobank of Dental Organs (BBOD) ofthe Research Center of the School of Dentistry, University of El Salvador. Previously, softtissue and calculus residues were removed from the teeth and cleaned with a low-speedmicromotor Lynx Classic motor MTI Dental, USA, and pumice stone. A coronal cut of theteeth was made with a diamond disc at low speed, and tooth blocks of vestibular and/orlingual surface were obtained with a proportion of 4 mm long× 3 mm wide, correspondingto the middle third of the oral surface. Previous studies on in vitro remineralization wereconsidered to determine the dimensions of the samples [15–17]. The thickness of the blockswas approximately 2 mm. This was considered to specifically evaluate the middle third ofthe teeth given the structural characteristics and the thickness of the enamel identified inthis location. The surface of the sample blocks was not polished. The blocks were stored in5 mL beakers with deionized water.2.4. Macroscopic EvaluationThe blocks were macroscopically evaluated using a stereomicroscope (Leica EZ4E,Leica Microsystems, Balgach, Switzerland), and a vestibular and/or lingual surface pho-tographic record was/were taken. The visual examination was performed according tothe criteria of the International Caries Detection and Assessment System ICDAS II andcoded as follows: 0—healthy tooth surface; 1—caries lesion in the outer half of the enamel;and 2—caries lesion in the inner half of the enamel. Two previously calibrated researchersperformed macroscopic evaluations. Weighted Kappa was calculated to evaluate agree-ment between examiners. High intra-examiner agreement was obtained for macroscopicevaluation/ICDAS II (Kappa 1.00) and macroscopic evaluation/DIAGNOdent pen (Kappa0.967). The investigators individually evaluated each of the blocks while the surface waswet. Ninety-six enamel blocks, which met the characteristics of healthy enamel accordingto published evaluations, were selected [18].2.5. Laser Fluorescence MeasurementThe DIAGNOdent pen 2190 (DIAGNOdent pen, Kavo, Biberach, Germany) was usedaccording to the manufacturer’s recommendations; this is a device which measures thedepth of the caries lesion and gives a unitless numerical value. A cylindrical sapphiretip was fitted for smooth surfaces and calibrated using a ceramic reference block. Foreach measurement, the device was zeroed; the sapphire tip was moved over the selectedevaluation sites at a 90-degree angle, as indicated by the manufacturer. Evaluations werecompleted by two examiners. Each examiner made two measurements for each test site,and the mean values were recorded (reported as initial evaluation in Table 1). The manufac-turer’s suggested cut-off points were used to interpret the measurements and were codedas follows: 0–12 healthy tooth surfaces; 13–24 superficial caries in enamel; >25 lesions inthe inner half of enamel [18].Coatings 2025, 15, 40 4 of 15Table 1. DIAGNOdent pen scores were obtained at different exposure times and different remineral-ization treatments (n = 16/group), expressed as mean ± SD.Initial a After bExposition TimeGroup 24 h 48 h 120 h 168 hn Mean SD Mean SD p * Mean SD Mean SD Mean SD Mean SD p **1 16 6.44 1.79 66.00 29.49<0.0561.75 ** 16.03 ** 81.5 ** 21.76 ** 61.25 ** 32.36 ** 36.50 ** 11.21 **<0.052 16 6.94 2.41 64.56 32.77 46.75 ** 12.04 ** 48.25 35.38 80.5 ** 37.00 ** 34.75 ** 11.35 **3 16 7.25 2.44 65.06 30.55 47.00 ** 6.22 ** 77.50 ** 28.44 ** 12.5 4.43 37.00 ** 22.14 **4 16 8.31 2.55 76.75 21.92 81.75 ** 8.06 ** 36.25 11.12 30.5 13.77 33.50 ** 1.91 **5 16 7.06 2.29 70.50 29.35 14.25 6.95 10.50 3.70 27.75 12.04 12.25 5.686 16 6.00 1.41 61.44 29.83 28.25 12.04 13.50 1.73 7.5 1.73 4.75 1.50a Evaluation with DIAGNOdent pen; b after demineralization; * associations evaluated via Student’s t-testp < 0.05. ** Bonferroni correction p < 0.05. Group 1, FV; fluoride varnish; 2, fluoride varnish + chitosangel; 3, fluoride varnish + AgNPs; 4, fluoride varnish + AgNPs + chitosan gel; 5, AgNPs + chitosan gel;6, artificial saliva.2.6. Artificial Demineralization of the Sample SurfacesThe vestibular and/or lingual surfaces of the blocks were demineralized following theapplication of 35% phosphoric acid 3M, ESPE, St. Paul, MN, USA, for 1 min (Figure 1B,D).The samples were washed with deionized water for acid removal and evaluated againusing the DIAGNOdent pen (Table 1) to establish differences between measurements andan adequate demineralization process [19].Coatings 2025, 15, x FOR PEER REVIEW 4 of 16 evaluation sites at a 90-degree angle, as indicated by the manufacturer. Evaluations were completed by two examiners. Each examiner made two measurements for each test site, and the mean values were recorded (reported as initial evaluation in Table 1). The manu-facturer’s suggested cut-off points were used to interpret the measurements and were coded as follows: 0–12 healthy tooth surfaces; 13–24 superficial caries in enamel; >25 le-sions in the inner half of enamel [18]. Table 1. DIAGNOdent pen scores were obtained at different exposure times and different reminer-alization treatments (n = 16/group), expressed as mean ± SD. Initial a After b Exposition Time Group 24 h 48 h 120 h 168 h n Mean SD Mean SD p * Mean SD Mean SD Mean SD Mean SD p ** 1 16 6.44 1.79 66.00 29.49 <0.05 61.75 ** 16.03 ** 81.5 ** 21.76 ** 61.25 ** 32.36 ** 36.50 ** 11.21 ** <0.05 2 16 6.94 2.41 64.56 32.77 46.75 ** 12.04 ** 48.25 35.38 80.5 ** 37.00 ** 34.75 ** 11.35 ** 3 16 7.25 2.44 65.06 30.55 47.00 ** 6.22 ** 77.50 ** 28.44 ** 12.5 4.43 37.00 ** 22.14 ** 4 16 8.31 2.55 76.75 21.92 81.75 ** 8.06 ** 36.25 11.12 30.5 13.77 33.50 ** 1.91 ** 5 16 7.06 2.29 70.50 29.35 14.25 6.95 10.50 3.70 27.75 12.04 12.25 5.68 6 16 6.00 1.41 61.44 29.83 28.25 12.04 13.50 1.73 7.5 1.73 4.75 1.50 a Evaluation with DIAGNOdent pen; b after demineralization; * associations evaluated via Student’s t-test p < 0.05. ** Bonferroni correction p < 0.05. Group 1, FV; fluoride varnish; 2, fluoride varnish + chitosan gel; 3, fluoride varni h + AgNPs; 4, fluoride varnish + AgNPs + chitosan gel; 5, AgNPs + chitosan gel; 6, artificial saliva. 2.6. Artificial Demineralization of the Sample Surfaces The vestibular and/or lingual surfaces of the blocks were demineralized following the application of 35% phosphoric acid 3M, ESPE, St. Paul, MN, USA, for 1 min (Figure 1B,D). The samples were washed with deionized water for acid removal and evaluated again using the DIAGNOdent pen (Table 1) to establish differences between measure-ments and an adequate demineralization process [19]. (A) (B) Coatings 2025, 15, x FOR PEER REVIEW 5 of 16 (C) (D) Figure 1. Macroscopic evaluation of vestibular/lingual surfaces by stereomicroscopy: (A) smooth and (B) continuous surfaces without white spots in healthy enamel; (C) smooth and (D) continuous surfaces showing porous opaque demineralized enamel. 2.7. Application of Remineralizing Agents The samples were divided into six different groups (n = 16 for each group) according to the remineralizing agent: (1) FV (Clinpro, 3M ESPE, St. Paul, MN, USA); (2) FV + CG; (3) FV + AgNPs; (4) FV + AgNPs + CG; (5) AgNPs + CG; and (6) artificial saliva (Kim Hidrat, Spain) composed of Xylitol 1%, potassium thiocynate, potassium chloride, sodium chlo-ride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium saccharin, hydrogenated castor oil, sodium methylparaben, sodium propylparaben, bon-oprol, menthol, aroma, citric acid, and water. For the preparation of the different treat-ment combinations, a ratio of 900 µL:20 µL FV–composite was used. The application vol-ume was 1000 microliters, with an FV ratio of 90% and 2% compound. This ratio was considered based on a previous study on the bacteriological efficacy of chitosan gel and silver nanoparticles [14,20]. A thin layer of remineralizing agent was applied using a microbrush on the demin-eralized enamel surfaces. The remineralizing agent applications were completed accord-ing to the manufacturer’s directions. After application, the samples were allowed to stand for 5 min and then immersed in a commercial artificial saliva solution with a pH of 7.2 and kept in an incubator at 37 °C for the exposure time. Four exposure times were consid-ered (24, 48, 120, and 168 h), and treatment was applied every 24 h. At the end of the exposure time, the varnish was carefully removed, and the samples were washed with deionized water. After the application of remineralizing agents, the samples were rinsed with deionized water, removing excess material with a scalpel blade. Group 6 was im-mersed in the artificial saliva solution without any treatment. The pH value of the artificial saliva solution for the groups was 7.2, and the solution was renewed daily. Evaluations with a DIAGNOdent pen 2190 were performed at the end of each exposure time to record the surface changes. 2.8. X-Ray Diffraction For the analysis of the crystal structure of tooth enamel, a Rigaku miniFlex 300/600 System X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) using CuKα radiation was used. The operating conditions were as follows: 600 W X-ray tube; wavelength of λ = 0.154056 nm at 40 kV. Diffraction spectra were acquired in the range of 10–80 degrees. Thirty-two tooth blocks were evaluated which, were pulverized with mortar, obtaining 30 mg; this content was dehydrated using ethanol and placed in the sample holder for the respective analysis in the X-ray diffractor. i r 1. acrosco ic evaluation of vestibular/li l rf stere i r s : ( ) s ootand (B) continuous surfaces ithout hite spots in healthy ena el; (C) s ooth and ( ) continuoussurfaces showing porous opaque demineralized enamel.2.7. Application of Remineralizing AgentsThe samples were divided into six different groups (n = 16 for each group) accordingto the remineralizing agent: (1) FV (Clinpro, 3M ESPE, St. Paul, MN, USA); (2) FV + CG;(3) FV + AgNPs; (4) FV + AgNPs + CG; (5) AgNPs + CG; and (6) artificial saliva (KimCoatings 2025, 15, 40 5 of 15Hidrat, Spain) composed of Xylitol 1%, potassium thiocynate, potassium chloride, sodiumchloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodiumsaccharin, hydrogenated castor oil, sodium methylparaben, sodium propylparaben, bono-prol, menthol, aroma, citric acid, and water. For the preparation of the different treatmentcombinations, a ratio of 900 µL:20 µL FV–composite was used. The application volumewas 1000 microliters, with an FV ratio of 90% and 2% compound. This ratio was consid-ered based on a previous study on the bacteriological efficacy of chitosan gel and silvernanoparticles [14,20].A thin layer of remineralizing agent was applied using a microbrush on the deminer-alized enamel surfaces. The remineralizing agent applications were completed accordingto the manufacturer’s directions. After application, the samples were allowed to stand for5 min and then immersed in a commercial artificial saliva solution with a pH of 7.2 and keptin an incubator at 37 ◦C for the exposure time. Four exposure times were considered (24, 48,120, and 168 h), and treatment was applied every 24 h. At the end of the exposure time, thevarnish was carefully removed, and the samples were washed with deionized water. Afterthe application of remineralizing agents, the samples were rinsed with deionized water,removing excess material with a scalpel blade. Group 6 was immersed in the artificialsaliva solution without any treatment. The pH value of the artificial saliva solution for thegroups was 7.2, and the solution was renewed daily. Evaluations with a DIAGNOdent pen2190 were performed at the end of each exposure time to record the surface changes.2.8. X-Ray DiffractionFor the analysis of the crystal structure of tooth enamel, a Rigaku miniFlex 300/600System X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) using CuKα radiationwas used. The operating conditions were as follows: 600 W X-ray tube; wavelength ofλ = 0.154056 nm at 40 kV. Diffraction spectra were acquired in the range of 10–80 degrees.Thirty-two tooth blocks were evaluated which, were pulverized with mortar, obtaining30 mg; this content was dehydrated using ethanol and placed in the sample holder for therespective analysis in the X-ray diffractor.2.9. Scanning Electron Microscopy and Energy-Dispersive X-Ray AnalysesScanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX)were performed at the Centre for High-Throughput Phenogenomics (University of BritishColumbia) on an FEI Helios Nanolab 650 SEM/FIB equipped with an EDAX OctanePro EDX detector (FEI Company, Hillsboro, OR, USA). SEM was used to characterizemorphological changes in enamel surfaces after the remineralization procedures. Surfacemorphology images of the enamel samples were acquired using a 1 kV electron beamwith accelerating voltage. Compositional information was obtained via EDX analysis todetermine the presence of calcium and phosphorous and their relative concentrations inthe sample surfaces. The EDX analyses were performed using a 10 kV electron beam withaccelerating voltage.2.10. Statistical AnalysisThe data obtained were subjected to statistical analysis using SPSS V25 software.Kolmogorov–Smirnov was used to identify the normality of the data. Data analysis wasperformed using the Student’s T and Bonferroni correction test. The Bonferroni correctiontest was used to analyze the comparison of means between laser fluorescence assessments.The significance level was set at p < 0.05, with a 95% confidence interval.Coatings 2025, 15, 40 6 of 153. Results3.1. Macroscopic EvaluationA total of 122 blocks of vestibular/lingual surfaces were examined in this study, and96 of them were included in this study after the microscope examination. Selected imagesof the blocks were evaluated for healthy enamel characteristics (smooth and continuoussurface without white spots) via stereomicroscopy and ICDAS II system assessments(Figure 1A,B). In contrast, images of enamel after the demineralization process are shownin Figure 1C,D. The demineralization process was confirmed using fluorescence.3.2. Laser FluorescenceAfter different times of each treatment, the assessments obtained by DIAGNOdent pen(demineralized/post-treatment) were compared, and we observed that the laser fluores-cence values decreased significantly after 168 h of applying the remineralization process inall treatment groups. Table 1 indicates the average values obtained with laser fluorescencebefore and after demineralization and after the application of remineralizing agents. Signif-icant differences (p < 0.05) were obtained between the initial evaluations and after artificialdemineralization. Likewise, the comparison of means between the exposure groups was es-tablished, being significant for the FV groups (24, 48, 120, and 168 h), FV+ CG (24, 120, and168 h), FV + AgNPs (24, 48, and 168 h), and FV + AgNPs + CG (24 and 168 h). The lowestvalue was achieved using the FV + AgNPs + CG treatment, with a reduction from 72.5 to4.75. The means values obtained with laser fluorescence before and after demineralizationwere recorded (Table 1). The individual values obtained for the group at 48 and 120 h arescattered around the mean, which represents the variation in the data obtained.3.3. X-Ray DiffractionFor X-ray diffraction (XRD) evaluation, the results were analyzed in SmartLab StudioII and OriginPro software, Version 2023b (OriginLab Corporation, Northampton, MA,USA). Refinement was performed via the Rietveld method. Lattice parameters indicatingthe presence of hydroxyapatite and fluorapatite were observed in the samples exposed toFV + CG + AgNPs at 24, 48, and 120 h. The phases identified in the diffractogram (Figure 2)indicate the presence of apatite in healthy enamel and calcium hydroxide phosphate indemineralized enamel. Table 2 indicates the crystalline phases and crystallographic param-eters identified in the samples through refinement via the Rietvelt method. The phases ofapatite and modified hydroxyapatite are mainly identified, highlighting the identificationof fluorapatite in the FV + AgNPs + CG group at 24 h of exposure. The crystallographic axes(a, b, and c) are also indicated, which indicate that the structures evaluated are anisotropicin the “c” direction, which means that the properties and arrangement of the atoms variesin this direction with a hexagonal configuration.3.4. Scanning Electron Microscopy and EDX AnalysisSurface analysis using scanning electron microscopy showed a characteristic smoothsurface in healthy enamel (Figure 3) but not in the artificial saliva group (Figure 4), whereeroded prismatic enamel and interprismatic enamel protrusions are identified as a result ofthe demineralization process. The concentration of the crystalline structure of the enamelshows variations in all groups with respect to normal enamel, with an increase concerningthe eroded enamel of the control group. In the groups to which the remineralizing agentwas applied at 120 h and 168 h (Figures 4 and 5), morphological changes were observed onthe surface where the presence of small crystals was identified in all the groups; however,they do not have a homogeneous distribution.Coatings 2025, 15, 40 7 of 15Coatings 2025, 15, x FOR PEER REVIEW 7 of 16 2) indicate the presence of apatite in healthy enamel and calcium hydroxide phosphate in demineralized enamel. Table 2 indicates the crystalline phases and crystallographic pa-rameters identified in the samples through refinement via the Rietvelt method. The phases of apatite and modified hydroxyapatite are mainly identified, highlighting the identifica-tion of fluorapatite in the FV + AgNPs + CG group at 24 h of exposure. The crystallographic axes (a, b, and c) are also indicated, which indicate that the structures evaluated are ani-sotropic in the “c” direction, which means that the properties and arrangement of the at-oms varies in this direction with a hexagonal configuration. Figure 2. X-ray diffraction spectra: (a) healthy enamel; (b) enamel demineralized for 1 min with 35% phosphoric acid; (c) FV + CG + AgNPs-24 h; (d) FV + CG + AgNPs-48 h; (e) FV + CG + AgNPs-120 h. Table 2. Identified crystalline phase. Exposition Time Treatment Phase a/Å * b/Å * c/Å * Healthy enamel Apatite 9.43 9.43 6.87 Demineralized enamel Calcium phosphate hydroxide 9.45 9.45 6.89 24 h Fluoride varnish Carbonate Hidroxiapatite 9.44 9.44 6.88 Fluoride varnish + chitosan gel Apatite 9.43 9.43 6.88 Fluoride varnish + NpAg + chitosan gel Apatite Fluorapatite 9.45 9.45 9.45 9.45 6.89 6.88 NpAg + chitosan gel Apatite 9.43 9.43 6.87 48 h Fluoride varnish Apatite 9.45 9.45 6.88 Fluoride varnish + chitosan gel Apatite 9.45 9.45 6.88 Fluoride varnish + NpAg + chitosan gel Hidroxiapatite 9.43 9.45 6.87 NpAg + chitosan gel Apatite 9.43 9.43 6.87 120 h Fluoride varnish Apatite 9.42 9.42 6.86 Fluoride varnish + chitosan gel Apatite 9.42 9.42 6.87 Fluoride varnish + NpAg + chitosan gel Apatite Hidroxiapatite 9.42 9.42 9.42 9.42 6.86 6.85 NpAg + chitosan gel Apatite Potasium, calcium 9.47 3.88 9.47 3.88 6.89 16.63 168 h Fluoride varnish Carbonate Hidroxiapatite 9.44 9.44 6.87 Fluoride varnish + chitosan gel Apatite 9.43 9.43 6.86 i r . -ra diffracti s ectr : (a) healthy ena el; (b) ena el de i erali e f r 1 in ith 35g - ; g s- .Table 2. Identified crystalline phase.Exposition Time Treatment Phase a/Å * b/Å * c/Å *Healthy enamel Apatite 9.43 9.43 6.87Demineralized enamel Calcium phosphate hydroxide 9.45 9.45 6.8924 hFluoride varnish Carbonate Hidroxiapatite 9.44 9.44 6.88Fluoride varnish + chitosan gel Apatite 9.43 9.43 6.88Fluoride varnish + NpAg + chitosan gel ApatiteFluorapatite9.459.459.459.456.896.88NpAg + chitosan gel Apatite 9.43 9.43 6.8748 hFluoride varnish Apatite 9.45 9.45 6.88Fluoride varnish + chitosan gel Apatite 9.45 9.45 6.88Fluoride varnish + NpAg + chitosan gel Hidroxiapatite 9.43 9.45 6.87NpA chitosan gel Apatite 9.43 9.43 6.87120 hFluoride varnish Apatite 9.42 9.42 6.86Fluoride varnish + chitosan gel Apatite 9.42 9.42 6.87Fluoride varnish + NpAg + chitosan gel ApatiteHidroxiapatite9.429.429.429.426.866.85NpAg + chitosan gel ApatitePotasium, calcium9.473.889.473.886.8916.63168 hFluoride varnish Carbonate Hidroxiapatite 9.44 9.44 6.87Fluoride varnish + chitosan gel Apatite 9.43 9.43 6.86Fluoride varnish + NpAg + chitosan gel Apatite 9.42 9.42 6.86NpAg + chitosan gel Apatite 9.45 9.45 6.88* lattice parameters.The EDX elemental analysis (Figure S1 in Supplementary File) confirmed that thesurface consisted of calcium, phosphoru , carbon, and oxygen. Within the comparisonof the groups exposed at 120 h and 168 h, he results of h present study show that afterremineralization, the carbon and oxygen conten increased in all exposed groups, exceptin the control group. F content was identified in the FV group (0.92 weight %) and thecombination of FV + CG + AgNPs group (0.91) at 120 h and in the groups exposed to 168 hFV + CG + AgNPs and CG gel + AgNPs at 0.19 and 3.5, respectively, with higher F contentin the samples combined with CG + AgNPs for the 120 h and 168 h groups. The detectionlimit of the technique could be the reason why there were groups where FV was used but Fwas not detected. The Ca/P ratio was higher in the samples treated with FV + CG + AgNPs,CG, and AgNPs in both exposure groups. The values closer to normal enamel indicate thatremineralizing treatment achieves a great percentage recovery of enamel mineralization.Coatings 2025, 15, 40 8 of 15Coatings 2025, 15, x FOR PEER REVIEW 8 of 16 Fluoride varnish + NpAg + chitosan gel Apatite 9.42 9.42 6.86 NpAg + chitosan gel Apatite 9.45 9.45 6.88 * lattice parameters. 3.4. Scanning Electron Microscopy and EDX Analysis Surface analysis using scanning electron microscopy showed a characteristic smooth surface in healthy enamel (Figure 3) but not in the artificial saliva group (Figure 4), where eroded prismatic enamel and interprismatic enamel protrusions are identified as a result of the demineralization process. The concentration of the crystalline structure of the enamel shows variations in all groups with respect to normal enamel, with an increase concerning the eroded enamel of the control group. In the groups to which the remineral-izing agent was applied at 120 h and 168 h (Figures 4 and 5), morphological changes were observed on the surface where the presence of small crystals was identified in all the groups; however, they do not have a homogeneous distribution. Figure 3. Scanning electron microscopy images of healthy enamel. (a) (b) Figure 3. Scanni g electron micros opy images of healthy enamel.Coatings 2025, 15, x FOR PEER REVIEW 8 of 16 Fluoride varnish + NpAg + chito an gel Apatite 9.42 9.42 6.86 NpAg + chitosan gel Apatite 9.45 9.45 6.88 * lattice parameters. 3.4. Scanning Electron Microscopy and EDX Analysis Surface analysis using s an ing electron microscopy showed a characteristic smooth surface in healthy enamel (Figure 3) but not in the artificial saliva group (F gure 4), where eroded prismatic enamel and interprismatic enamel otrusions are identified as a result of the demineralization process. The c centration of the ryst lline structure of the enamel shows variations in all groups with respect to normal enamel, with an increase concerning the eroded namel of the control group. In the g ups to whic the remineral-izing agent was applied at 120 h and 168 h (Figures 4 and 5), morphological changes were observed on the surface where the p esence of small crystals was identified in all the groups; however, they do n t have a omogeneous distribution. Figure 3. Scanning elect on microscopy images of healthy enamel. (a) (b) Coatings 2025, 15, x FOR PEER REVIEW 9 of 16 (c) (d) Figure 4. Scanning electron microscopy images of the enamel surface. Exposure to (a) FV, (b) FV + CG + AgNPs, (c) CG + AgNPs, and (d) artificial saliva after 120 h of exposure. (a) (b) (c) (d) Figure 5. Scanning electron microscopy images of enamel surface. Exposure to (a) FV, (b) FV + CG + AgNPs, (c) CG + AgNPs, and (d) artificial saliva after 168 h of exposure. The EDX elemental analysis (Figure S1 in Supplementary File) confirmed that the surface consisted of calcium, phosphorus, carbon, and oxygen. Within the comparison of the groups exposed at 120 h and 168 h, the results of the present study show that after remineralization, the carbon and oxygen content increased in all exposed groups, except in the control group. F content was identified in the FV group (0.92 weight %) and the combination of FV + CG + AgNPs group (0.91) at 120 h and in the groups exposed to 168 h FV + CG + AgNPs and CG gel + AgNPs at 0.19 and 3.5, respectively, with higher F con-tent in the samples combined with CG + AgNPs for the 120 h and 168 h groups. The de-tection limit of the technique could be the reason why there were groups where FV was used but F was not detected. The Ca/P ratio was higher in the samples treated with FV + CG + AgNPs, CG, and AgNPs in both exposure groups. The values closer to normal Figure 4. Scanning electron microscopy images of the enamel face. Exposure to (a) FV, (b) FV +CG + AgNPs, (c) CG + AgNPs, and (d) artificial saliva after 120 h of exposure.Coatings 2025, 15, 40 9 of 15Coatings 2025, 15, x FOR PEER REVIEW 9 of 16 (c) (d) Figure 4. Scanning electron microscopy images of the enamel surface. Exposure to (a) FV, (b) FV + CG + AgNPs, (c) CG + AgNPs, and (d) artificial saliva after 120 h of exposure. (a) (b) (c) (d) Figure 5. Scanning electron microscopy images of enamel surface. Exposure to (a) FV, (b) FV + CG + AgNPs, (c) CG + AgNPs, and (d) artificial saliva after 168 h of exposure. The EDX elemental analysis (Figure S1 in Supplementary File) confirmed that the surface consisted of calcium, phosphorus, carbon, and oxygen. Within the comparison of the groups exposed at 120 h and 168 h, the results of the present study show that after remineralization, the carbon and oxygen content increased in all exposed groups, except in the control group. F content was identified in the FV group (0.92 weight %) and the combination of FV + CG + AgNPs group (0.91) at 120 h and in the groups exposed to 168 h FV + CG + AgNPs and CG gel + AgNPs at 0.19 and 3.5, respectively, with higher F con-tent in the samples combined with CG + AgNPs for the 120 h and 168 h groups. The de-tection limit of the technique could be the reason why there were groups where FV was used but F was not detected. The Ca/P ratio was higher in the samples treated with FV + CG + AgNPs, CG, and AgNPs in both exposure groups. The values closer to normal Figure 5. Scanning electron microscopy images of namel rface. Exposure to (a) FV, (b) FV + CG +AgNPs, (c) CG + AgNPs, and (d) artificial saliva after 168 h of exposure.4. DiscussionRemineralization is a natural process by which lost minerals are redeposited in theenamel. However, this process is often not sufficient to counteract the continuous loss ofminerals, as conditions may vary according to oral conditions. In this regard, biomaterialssuch as AgNPs and CG play an important role in the remineralization process due to theircharacteristics and properties [12–14,18,19]. AgNPs have a broad spectrum of antibacterialproperties which can inactivate enzymes and prevent DNA replication in bacteria, therebyreducing the risk of caries [20,21]. Moreover, s veral studies indicate t at chit san productsenhance remineralization in artificial lesions in vitro [22–24]. Changes in the surface ofdemineralized enamel indicate a egative charge due to the leaching of Ca++, and thepositive charge left from the chitosan favors adhesion to the demineralized surface [25].Diagnostic evaluation using the DIAGOdent pen laser is a complementary methodfor the diagnosis of lesions on the smooth surface of the enamel. Likewise, it is capable ofdetecting in vitro changes in the surface of dental enamel after exposure to a demineralizingagent. DIAGNOdent pen titrations obtained pre- and post-artificial demineralizationindicate significant differences. This is congruent with the XRD analysis and SEM analysisshowing important variations among the samples evaluated.X-ray diffraction results indicate changes related to significant mineral loss and sur-face changes in the artificially demineralized samples, indicating the presence of calciumhydroxide phosphate. This denotes variations in the composition of the enamel surface inan in vitro context. This allows us to establish that laser fluorescence is capable of detectingin vitro changes in the surface of dental enamel after exposure to a demineralizing agent.Coatings 2025, 15, 40 10 of 15The identified differences between initial and post-demineralization values were measuredwith the DIAGNOdent pen on the smooth surface of permanent teeth [25]. However, slightvariations were measured with the DIAGNOdent pen on the enamel surface after exposureto different acid solutions [26].The mechanism of remineralization under in vitro conditions has been studied else-where, but there is still no agreement among researchers. According to Enax et al. (2024),“. . .It has been conclusively shown by such in vitro crystallization studies under well-defined conditions that additives can influence nucleation and crystal growth of calciumphosphate even at low concentrations. . .” [27]. In our research, we found that the pres-ence of silver nanoparticles promotes a different sort of compound in the crystallizationproducts (see DRX results) due to a heterogeneous precipitation process, and also, thegreat affinity of silver to fluoride is well known, and the presence of silver fluoride inlow concentrations—not detectable via XRD—can influence the remineralization process.Research in this area is also needed.For this present study, the comparison between combinations of remineralizing treat-ments, considering measurements of demineralized surface and post-treatment with theDIAGNOdent pen, indicate significant differences. Additionally, an increase in means isidentified for the FV, FV + AgNPs, FV + GC, and FV + CG + AgNPs groups at 24 and68h post-exposure, compared to values recorded post-desmineralization. The evaluationsstand out for groups containing CG (FV + CG and FV + CG + AgNPs) compared to theconventional FV treatment group; this could be related to CG’s adhesion capability on theenamel surface. Other studies identified differences in the means obtained in the groupswhere chitosan was incorporated, showing significantly lower levels compared to otherremineralizing agents [16,28]. Similar conclusions were identified with the same system ingroups of enamel blocks exposed to remineralizing treatment [29].The results related to the exposure time show significant differences in the four exposedgroups. It is noteworthy that the XRD results identify crystalline phases of hydroxyapatitein the FV + CG + AgNPs treatment for 120 h groups, congruent with remineralization. Thecontrol group, in which only artificial saliva was used, shows remineralizing potential,indicating a decrease in the measurements obtained in this group.The results of the commercial FV show a predictable behavior concerning its reminer-alizing effects; however, the means obtained after the application of the treatment groupsthat included chitosan indicate significant variations with respect to the measurements indemineralized samples.The samples processed in XRD indicate important variations in the composition andphases identified in the samples submitted to artificial demineralization compared tohealthy samples. This allows us to establish that the variations identified by the post-remineralization evaluations allow us to identify phases that coincide with a remineraliza-tion process.Other studies reported similar characteristics in the diffraction spectra obtained inhealthy enamel and enamel exposed to F [30–33]. During the evaluation, a loss of crys-tallinity in demineralized enamel was recorded. Although the samples exposed to F denotecharacteristic apatite peaks, they indicate that the apatite crystal lattice was recovered tosome extent. However, samples exposed to nanocomplexes of phosphorylated chitosanand amorphous calcium phosphate (Pchi-ACP) showed narrower and sharper peaks in thediffractogram. Similar results were reported with the use of mesoporous bioactive glassesloaded with amorphous calcium phosphate to treat white spot lesions [34]. Diffraction anal-ysis indicated that remineralized deposits in all four groups showed a diffraction patternsimilar to that of normal enamel and shared a crystal structure similar to hydroxyapatite.Likewise, as a result of the refinement through the Rietveld method, variations in theCoatings 2025, 15, 40 11 of 15crystallographic parameters were identified, registering the presence of fluorapatite andhydroxyapatite in the group of blocks exposed to FV + CG + AgNPs at 48 h and 120 h ofexposure, which is congruent with remineralization on the enamel surface.On the other hand, previous studies have used SEM-EDX analysis to study the changesassociated with the demineralization–remineralization of dental enamel [3,28,35–37]. In thecurrent study, enamel remineralization included the evaluation of samples on EDX, identi-fying the weight percentage of different minerals such as calcium and phosphate becausethey represent the main composition of hydroxyapatite. Ca/P titrations are importantindicators of the effects of agents on enamel remineralization [38]. For example, changesin the Ca/P ratio were monitored for samples exposed at 120 h and 168 h. The resultsindicate that remineralizing agents such as FV, combined with CG and AgNPs, improvedenamel remineralization. It is considered that the remineralization process is related to thereplacement of minerals, mainly through the redeposition of calcium and phosphate in theenamel [3]. Research on the release of F and its applications in the remineralization processindicates the capacity of F release after an initial application followed by continuous diffu-sion of F, which allows us to determine that it is one of the treatments with better resultsfor the management of white spot lesions in enamel; however, its effect decreases afterseven days of treatment [4,38]. On the other hand, other minerals or compounds besides Caand P could have a great influence on the remineralization process; for example, chitosan,which, due to its high nitrogen content, represents an excellent vehicle for transportingions such as Ca and P for biomineralization. Likewise, chitosan adheres to negativelycharged surfaces such as those of demineralized enamel [30,39–41]. The results of thisstudy indicate a higher Ca/P ratio in the groups in which commercial FV was combinedwith CG and AgNPs.Table 3 shows the results of some studies concerning remineralization using nanopar-ticles and chitosan in recent years. As we can see, there is active research seeking a coatingmaterial with sustained activity without the complications of the use of fluoride, as westated in the introduction to this paper.Table 3. Comparison of findings on remineralization using nanoparticles and chitosan in selected articles.Author(s) Aim Bioactive Materials Main Results Ref.Deokar et al.To evaluate and compare the efficacy ofthe remineralizing potential ofacidulated phosphate fluoride gel,chitosan nanoparticles, and silverdiamine fluoride on the microhardnessof artificial carious lesions created onextracted teeth.Chitosan nanoparticles, silverdiamine fluoride, and acidulatedphosphate fluoride gel• Chitosan nanoparticles showed the highestincrease in remineralization followed bySDF and APF gel[42]Hanafy et al.Chitosan was employed as a novelbiomimetic mineralization model torepair damaged enamel to compare itsperformance with that of bioinspiredzinc-doped nanohydroxyapatite.Chitosan–hydrogel and azinc-doped nanohydroxyapatite• Marked reduction in the Ca/P ratio andthe lack of the characteristichydroxyapatite diffraction peaks weredetected for demineralized enamel.• The remineralization stage revealedevident recovery of the mineral contentswith apparent distinctive XRD patterns ofhydroxyapatite in both groups.• SEM analysis showed the absence ofetched enamel porosity with the formationof a newly formed rod-like apatite layer.[43]Mus¸at et al.To induce the biomimeticremineralization of acid-etched humanenamel in artificial saliva containingfluoride under agarose and novelchitosan–agarose hydrogels actionwithout a toxic crosslinking agent forchitosan.Chitosan (CS) and agarose (A) ina biopolymer-based hydrogel• The values for the CS-A series are closer tothe theoretical value of the carbonatedhydroxyapatite of natural enamel; those forthe series-A indicate the formation ofcalcium deficient phosphates.[40]Coatings 2025, 15, 40 12 of 15Table 3. Cont.Author(s) Aim Bioactive Materials Main Results Ref.Magalhães et al.Synthesize, characterize, and determinethe effects of a ChNPs suspension onhuman enamel after cariogenicchallenge via pH cycling.Chitosan nanoparticles• ChNPs suspension minimized humanenamel demineralization after a cariogenicchallenge.[44]Yamakami et al.Evaluate the effects of an experimentalchitosan/casein gel on enameldemineralization/remineralization inan environment with a high cariogenicchallenge.Chitosan/casein gel• Chitosan seems to be the major componentresponsible for reducing the volume loss ofdemineralized enamel.[16]Xu et al.Review the types, properties, andpotential uses of non-metallicnanomaterials systematically formanaging dental caries.Biological organic nanomaterials,synthetic organic nanomaterials,carbon-based nanomaterials,and selenium nanomaterials• Chitosan nanoparticles have shown aremineralization ability on demineralizedenamel.[45]Wahied et al.Evaluate the effect of chitosannanoparticles on the remineralizationof the demineralized enamel surfaceafter being added tonano-hydroxyapatite and nano-calciumphosphate materials.Nano-hydroxyapatite andnano-calcium phosphate• Addition of chitosan nanoparticles had asignificant effect on the remineralization ofthe demineralized enamel surface.• The nano NHA + chitosan group had thehighest significant value, while theNβ-TCP group exhibited the lowestsignificant value.[46]Zhang et al.The purpose of this literature review isto provide an overview of currentbioactive materials for cariesmanagement.Bioactive materials for cariesmanagement• Combination of AgNPs and NaF ornano-silver fluoride does not cause blackstaining and metallic taste, unlike SDF.• AgNPs’ properties suggest their potentialas an effective anticaries agent withoutblack staining.[47]Finally, some questions about the biocompatibility of silver nanoparticles in dentistryhave been raised by researchers; in previous studies, we have evaluated the biocompatibilityof these gallic acid stabilized-silver nanoparticles, finding cell viability percentages higherthan 95% when tested against human dermal fibroblast, and we have also reported thatAgNPs concentrations less than 4 µg/mL showed a cytotoxic effect that resulted in a deathrate of 13.8% or less. We also found that AgNPs at 500 ppm results in a viability above 75%in human fibroblasts. In an in vivo study, it was found that silver nanoparticles suspensionsadministrated to Wistar rats did not generate alterations among clinical characteristics, andclinical chemistry and hematology values showed slight alterations; however, at the end ofthe treatment, most were inside the normal concentration [48].Considering that the present study was carried out in a laboratory environment, someof the limitations can be attributed to the difficulty of imitating the in vivo characteristicsof saliva in the oral cavity. It is necessary to continue the evaluations assessing the micro-hardness of the enamel after exposure to remineralizing agents. Likewise, based on theresults, in vivo evaluations could be considered.5. ConclusionsThe results of the present in vitro study suggest that the combination of FV + CG + AgNPsand CG + AgNPs could induce changes in the surface of the dental enamel related toremineralization, extending its effect up to 168 h compared to commercial FV. Althoughall the tested materials produce a remineralization effect, it is concluded that they do itat different proportions, producing different remineralization products as observed inX-ray diffraction analysis. Future research could include atomic force microscopy andmicrohardness analysis to confirm these results. The main contributions of this work areas follows:• The changes evaluated on the enamel surface after applying FV + CG + AgNPs arerelated to the remineralization process, extending its effect up to 168 h compared to FV.• The absence of pigmentation on the enamel surface is also highlighted.Coatings 2025, 15, 40 13 of 15Although these results are remarkable, further investigation into the variations in themicrohardness of the enamel surface after exposure to the treatment is needed. Additionally,its clinical evaluation should be considered, considering the variations in salivary pHin vivo.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/coatings15010040/s1, Figure S1. EDX results for all groups at120 h and 168 h of treatment.Author Contributions: Conceptualization, N.N.-M. and G.-A.M.-C.; formal analysis, K.A.A.d.R.;funding acquisition, N.N.-M.; investigation, K.A.A.d.R., W.Y.E.d.G., V.C.M., S.M., N.N.-M. and G.-A.M.-C.; methodology, K.A.A.d.R., W.Y.E.d.G., V.C.M., N.N.-M. and G.-A.M.-C.; writing—originaldraft preparation, K.A.A.d.R., N.N.-M. and G.-A.M.-C.; writing—review and editing, H.B. andG.-A.M.-C. All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: This study was conducted in accordance with the Declarationof Helsinki and approved by Research Ethics Committee of the Faculty of Dentistry of the Universityof El Salvador with the following document number: CEI-FOUES/2022/003.Informed Consent Statement: Informed consent was obtained from all subjects involved in thisstudy. 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enamel remineralization
demineralization
chitosan
silver nanoparticles
Silver Nanoparticles–Chitosan Nanocomposites as Protective Coatings for Dental Remineralization Treatment: An In Vitro Study
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