Gel Containing Catechin and Mesoporous Silica Nanoparticles for Protecting Root Dentin Against Erosion: An In Situ Study

Gel Containing Catechin and Mesoporous Silica Nanoparticles for Protecting Root Dentin Against Erosion: An In Situ Study | 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 Gel Containing Catechin and Mesoporous Silica Nanoparticles for Protecting Root Dentin Against Erosion: An In Situ Study Helaine Cajado Alves, Edison Augusto Balreira Gomes, Antonia Flavia Justino Uchoa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3996730/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective This study aimed to evaluate the in situ anti-erosive effect of gels containing epigallocatechin-3-gallate (EGCG) isolated and adsorbed on mesoporous silica nanoparticles (EGCG/MSN) on eroded dentin. Materials and Methods Eleven volunteers participated in this randomized, controlled, cross-over study, which consisted of 4 phases of 5 days. The participants wore an acrylic palatal device containing two dentin blocks treated with one of the gels: placebo (negative control), SnF 2 (0.05% - positive control), EGCG (0.1%), and EGCG/MSN (0.093%). During each phase, the specimens were immersed in citric acid (0.05 M; pH 3.75) for 60 s, 4x/day, followed by treatment with the assigned gel for 60 s. The alterations were evaluated by measuring the percentage of surface hardness loss (%SHL) and through profilometry analysis (wear). Morphological changes were assessed using scanning electron microscopy (SEM). The data were analyzed using ANOVA, followed by Tukey's post-test. Results %SHL did not show a significant difference among the groups (p = 0.067). Regarding surface wear, the mean results in micrometers were: placebo, 0.66 (± 0.38); EGCG, 0.57 (± 0.11); EGCG/MSN, 0.48 (± 0.05); and SnF2, 0.32 (± 0.08). A significant difference was observed only between the SnF 2 group and the placebo and EGCG groups (p = 0.003 and p = 0.046, respectively). However, there was no difference between the SnF 2 and EGCG/MSN groups (p = 0.306). Conclusion EGCG/MSN shows promise as a protective measure in reducing dentin wear under erosive conditions. Clinical Relevance: Gels containing EGCG adsorbed on mesoporous silica nanoparticles have a protective effect against dentin erosion. Dental Erosion Dentin Tissue Metalloproteinase Inhibitors Catechin Drug Release System Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Erosive tooth wear is a complex condition that affects various age groups in the global population [ 1 , 2 ]. It is characterized by the chemical loss of mineralized tooth substance due to exposure to non-bacterial acids [ 3 , 4 ]. The prevalence of dental erosion has increased globally due to changes in lifestyle and dietary habits, including the higher consumption of acidic foods, carbonated drinks, and fresh fruit juices [ 5 , 6 ]. Prevalence studies have reported erosive conditions in young adults ranging from 7.3–33.8% [ 7 – 10 ]. Controlling erosive acid attacks on dental tissues poses a significant challenge in preventing and treating dental erosion in clinical practice [ 10 , 11 ]. While saliva and remineralizing foods have shown to decrease enamel erosion [ 11 , 12 ], measures to reduce tooth wear have been investigated, particularly those that enhance acid resistance or promote remineralization of tooth surfaces. Traditional preventive and control treatments for dental erosion focus on patient education, dietary control, and psychological counseling [ 11 ]. However, some treatments aim to promote mineralization through the use of fluoride. Fluoride treatments involve the formation of calcium fluoride on the tooth surface (in the case of amine fluoride and sodium fluoride), which is easily soluble in acid or metal-rich precipitates formation (in the case of titanium tetrafluoride and fluoride products containing tin) [ 11 ]. Among the fluorides studied for their anti-erosion effects on dentin, those containing tin, a polyvalent metallic cation, have emerged as effective anti-erosion agents [ 12 – 16 ]. Tin forms a protective barrier on the dentin surface due to its strong affinity for mineralized dental tissue. It also acts as an inhibitor of matrix metalloproteinases (MMPs) 2 and 9 [ 17 ]. However, stannous fluoride may cause undesirable effects, such as tooth surface staining and an astringent sensation on the oral mucosa [ 11 , 18 ]. Therefore, there is a need to explore more biocompatible substances to minimize the effects of erosion. The use of MMP inhibitors has emerged as a new approach to the treatment of this condition. Green tea and its main catechin, epigallocatechin-3-gallate (EGCG), fall into this category, as they have shown significant reduction in dentin wear after erosive/abrasive challenges and low toxicity in dental pulp cells [ 19 – 22 ]. They also act as cross-linking agents for collagen fibrils, thereby stabilizing collagen and improving the mechanical properties of dentin while inhibiting proteolytic degradation of the dentin's organic matrix [ 20 ]. In medicine, the use of drug nanocarriers and functional nanoparticles has been extensively explored in cancer. Mesoporous silica nanoparticles (MSNs) are a type of nanocarrier known for their stability, large surface area, and excellent thermal and chemical performance [ 23 – 26 ]. In dentistry, MSNs have been used with nanohydroxyapatite (nHAp), EGCG, calcium oxide, and calcium carbonate, with or without phosphoric acid. In these cases, MSNs act as carriers of these substances to the dentinal tubules [ 27 – 29 ]. Due to challenges in conducting in vivo erosive studies, in situ and in vitro models have been utilized to analyse erosive challenges in dental tissues. In situ studies provide a more realistic evaluation of the effects of erosive agents and substances that protect dental tissues in the oral environment. These studies consider salivary flow, acquired pellicle formation, and routine care [ 30 ]. The present in situ study aims to evaluate the effects of applying a gel containing MMP inhibitors, either EGCG or EGCG/MSN, on extrinsic erosion caused by citric acid. A placebo and SnF2 will be used as control groups. MATERIALS AND METHODS Experimental design The present in situ, randomized, controlled, blind, and crossover study evaluated the effects of applying a gel containing MMP inhibitors, 0.1% EGCG or 0.093% EGCG/MSN, on extrinsic erosion by citric acid, using a placebo and the 0.05% SnF 2 as controls. The experiment was carried out in four phases of five days each, with an interval of two days between the phases. The volunteers used a palatal appliance with two dentin blocks (Fig. 1 ). It was immersed four times/day in a solution of 50 mL of citric acid (0.05 M and pH 3.75) at room temperature for 60 s. After the erosive challenge, the predetermined treatment was applied for 60 s. At the end of each phase, the dentin blocks were removed from the device to perform the following quantitative analyses: hardness and profilometry tests; and new blocks were placed in the device, starting a new phase with a new substance. Thus, the response variables were the loss of surface hardness (%SHL), the wear profile of the dentin structure, and the morphological surface analyzed by SEM. Reagents The following reagents were used: EGCG, hexadecyltrimethyl ammonium bromide (CTAB), ammonium hydroxide (NH 4 OH), tertraethylorthosilicate (TEOS) and ammonium nitrate (NH 4 NO 3 ), SnF 2 , PLURONIC F127® from Sigma-Aldrich (St. Louis, MO, USA). All reagents mentioned are analytical grade and used without prior purification. Synthesis of Mesoporous Silica Nanoparticles (MSNs) The MSNs were prepared according to the previous literature with some modifications (ZOU et al., 2017). 5.73 g of CTAB were dissolved in 280 mL of deionized water under mechanical agitation at 40°C in the initial 15 min and then at room temperature overnight. After this period, 0.5 mL of NH 4 OH and 80 mL of ethanol were added to the system, which remained under stirring for another 1 h. The temperature of the mixture was then increased to 70°C, and then 14.6 mL of TEOS was added dropwise. This system remained under agitation for 2 h. Subsequently, the solid was collected by centrifugation, washed with etanol, and dried at 100°C for 5 h. Surfactant extraction was performed using a NH 4 NO 3 /ethanol mixture (133 mg/50mL) at 60°C, with continuous magnetic stirring for 40 minutes. Finally, the solid was collected by centrifugation, washed three times with deionized water, and dried at 100°C overnight, thus obtaining the MSNs. Synthesis of Epigallocatechin-3-gallate Adsorbed on Mesoporous Silica Nanoparticles (EGCG/MSN) The synthesis of EGCG encapsulated in MSNs (EGCG/MSN) was performed according to the techniques reported in a previous study by Yu et al. (2017) with some modifications. 100 mg of dry MSN was dispersed in an aqueous EGCG solution (1.8 mg/mL) by vigorous horizontal stirring for 72 h in the dark. This process allows EGCG molecules to infiltrate into the pores of the MSNs to achieve maximum charge. The mixture was then centrifuged, washed three times with deionized water and ethanol, and dried under vacuum for 48 h to obtain EGCG/MSN, being stored in the dark at 4°C until use. A calibration curve was used to quantify the drug from a series of reference solutions whose concentrations ranged from 0.1 to 30 ppm, obtaining a linear relationship between the highest absorbance peak and the drug concentration. A UV Vis spectrophotometer (DU® 730; Beckman Coulter, Fullerton, CA, USA) was used to assess and confirm the peak absorbance of EGCG at 274nm. The encapsulation efficiency was calculated at 93%. Gel Synthesis The copolymer PLURONIC F127® (Sigma Chemical Co., St. Louis, MO, USA) was used to prepare placebo and active gels containing the following mass percentages: no active component (placebo), 0.05% of SnF 2 , 0.1% of EGCG, or 37% of EGCG/MSN. The gel was prepared at a concentration of 20% PLURONIC F127®/water. 6 g of PLURONIC F127® were dissolved in 30 mL of deionized water at low temperature (approximately 4 ºC) under magnetic stirring until complete homogenization of the F127. After the mixture was completely dissolved, the temperature was raised to 30°C to form the gel. Ethical aspects and Volunteers selection Before the start of this study, it was approved by the local Ethics Committee (# 2.704.870) and carried out in accordance with the Declaration of Helsinki. The volunteers who participated in the research previously signed a Free and Informed Consent Term and received verbal and written instructions on how to carry out the research procedures. Volunteers were recruited and eleven individuals were selected (01 male and 10 females, between 22 and 41 years old) to participate in the research. All the selected volunteers met the inclusion criteria, which were: good oral hygiene, absence of caries activity and periodontal disease, and absence of erosion lesion, in addition to having stimulated salivary flow > 1 mL/min. The exclusion criteria were the presence of gastroesophageal disorders, pregnant women, lactating women, or those who use a myorelaxant plate, fixed or removable orthodontic appliance in the upper arch, or medications that could alter salivary flow were not included in the research. The sample size was calculated based on the percentage change in surface hardness loss data from a pilot study. The sample of seven volunteers was necessary to provide an α-error of 5%, a power of 80%, and a relevant difference of 10% between groups ( www.openepi.com/menu/oe_menu.htm ). Eleven volunteers were included due to possible losses inherent to in situ studies. Preparation of dentin specimens In this study, freshly extracted, non-carious human third molars were collected after informed consent from the donors were achieved based on a protocol approved by the Ethics Committee of the Federal University of Ceará, Brazil. The selected molars were cleaned and stored in 0.1% thymol solution at 4 ºC were used. About two hundred blocks of human root dentin were cut to size 4x4x2mm, flattened and polished. For this, a double-sided diamond cutting wheel was used under abundant refrigeration (Struers, Minitom, Copenhagen, Denmark). Subsequently, the dentin blocks were placed in an acrylic device and flattened using P1200, P2500, and P4000 abrasive aluminum oxide sandpaper (Buehler, Lake Bluff, IL, USA), coupled to an automatic polisher (Buehler, Automet 250, Lake Bluff, IL, USA) under constant water irrigation and later polished with a felt disc and 1µm diamond paste (Erios, São Paulo, Brazil). During polishing, specimens were sonicated in distilled water for 2 minutes both between the use of sandpaper and at the end of the polishing process to remove residues resulting from these procedures. To standardize the specimens, the initial surface hardness was determined. Five indentations 100 µm apart were made in the center of the specimens using a microhardness tester with Knoop indenter and automatic measuring system (Future Tech Corp, FM-ARS 9000 and FM 100, Tokyo, Japan). A Knoop diamond indenter was used with a load of 10 g applied for 5s [ 21 , 31 ]. Eighty-eight dentin specimens, which had an average hardness of 54.53 ± 5.4 Kg/mm², were selected, packaged, and identified for ethylene oxide sterilization. Then, the specimens were covered with an acid adhesive tape leaving an exposed area of 4 x 2 mm which was subjected to erosion and treatment during the experiment. The covered area was used as a reference for the profilometric analysis. Palatal device preparation A palatal device was made of acrylic resin for each volunteer. Two cavities measuring 5x5x3mm were made in the device, one on the right side and the other on the left. The specimens were randomly assigned using a computer list (Microsoft Excel 2007), fixed with sticky wax, and carefully fitted 1 mm below the device surface level, to avoid abrasion resulting from contact with the tongue. Intraoral phase In the two days before the start of the research, the volunteers used a standard fluoride toothpaste (Colgate ® -1450 ppm fluoride) that was given to them and continued using it until the end of the experiment. Before each experimental phase, the palatal device was used for 12 h to allow equilibrium with the saliva and the formation and maturation of the acquired pellicle. Accordingly, the volunteers’ participation in all stages and testing of all treatments enabled a crossover design. The positions of the specimens were randomly determined according to a computer-generated randomization list. The erosive challenge was performed extraorally 4 times a day at 7 am, 12 pm, 5 pm, and 9 pm during the 5-day experiment [ 32 ]. Research participants removed the device from their mouths and placed it in a container with 50 mL of citric acid solution (0.05M, pH 3.75) for 60 s at room temperature, characterizing erosion [ 21 ]. Then, they were washed in running water for 10 s and excess water was removed with absorbent paper. After the acid challenge, the volunteers applied a thin layer of the gel, which remained in contact with the specimen for 60 s, and carefully removed the excess with absorbent paper (Fig. 1 ). After that the device was reinserted into the mouth. The gels were packaged and coded in syringes to maintain the blindness of the study regarding the volunteers. The volunteers were instructed to avoid eating, drinking (except water), and brushing their teeth while using the device, which was used continuously, including at night, being removed only for meals and oral hygiene, and, on these occasions, the device was kept mostly in plastic boxes. Volunteers were allowed to clean the device with a toothbrush and toothpaste, taking care not to brush the specimens. After each phase, on the morning of the sixth day, the specimens were removed from the device, and stored in humidity under refrigeration until the time of analysis. They were evaluated with respect to the percentage of surface hardness loss (%SHL) and measurement of dentin surface wear. New specimens were inserted into the device, which underwent a new phase of the experiment. Final surface hardness assessment The final surface hardness of each specimen was measured using the same protocol as the initial surface hardness. Five indentations, with 100 µm distance between them, were performed on each specimen in the center of the eroded area. The average of the indentations (final hardness) was used to evaluate the percentage loss of surface hardness (%SHL) according to the following formula: %SHL = [(initial hardness – final hardness) x 100/initial hardness] Assessment of dentin wear The level of dentin wear was determined in relation to the reference area with a profilometer (Hommel Tester, T1000, Hommelwerke GmbH, Germany). Adhesive tape was carefully removed from each dentin block to expose the reference area. Three tracings with a length of 1.5 mm each were performed on each specimen, moving the profilometer pen tip 1.8 µm from the reference surface to the experimental surface. For each specimen, the mean value obtained from the three tracings was calculated. Scanning electron microscopy Two dentin samples from each group were observed under electron microscopy scan for a qualitative analysis. They were immersed in a 2.5% glutaraldehyde fixative solution in a 0.1 mol/L of sodium cacodylate for 24 hours and washed after that with 0.1 mol/L of cacodylate buffer. Then, they were dehydrated with ethanol solutions at concentrations crescents and dried at room temperature for 24 hours in a desiccator [ 33 ]. The specimens were fixed metal stubs and received a gold coating through a metallizer (Hammer VI - sputtering system, Anatech Ltda, Alexandria, USA). After that, as exemplified were admitted by SEM Quanta FEG 450 (FEI Company, Oregon, USA). The voltage of adjustment had an adjustment of 20 kV. The magnification used was 8,000 times Additionally, SEM was employed to determine the morphology and ultrastructure of MSN, (EGCG/MSN) gel was qualitatively evaluated by microscopy to verify its adsorption (Fig. 2 ). Statistical analysis Mean and standard deviation data for wear and hardness loss were calculated. A preliminary, Kolmogorov-Smirnov test was performed on all of the groups to test the normal distribution of errors. Because the values were normally distributed across all groups, one-way ANOVA and Tukey post-hoc tests were applied for the data of wear and hardness loss. Statistical analysis was performed with Statistical Package for Social Sciences -SPSS 20.0 for Windows (SPSS Inc., Chicago IL, USA). The level of significance was set at 5%. RESULTS The values of the means and standard deviation of dentin surface hardness before (initial), after (final) the erosive challenge/treatment, and the percentage of surface hardness loss (%SHL) are described in Table 1 . The results of the % analysis SHL showed no statistical difference (p = 0.067) between the groups. The dentinal wear values (µm) revealed a statistical difference between treatments (p = 0.005). Then, the Tukey post-test was applied to determine the difference between the groups which was observed only for SnF 2 when compared to placebo and EGCG (p = 0.003 and p = 0.046, respectively). Regarding EGCG/MSN, there was also no significant difference between this group and SnF 2 (positive control) (p = 0.306), as shown in Fig. 3 . Table 1 Mean (n = 11;SD) of dentin surface hardness (SH) before (baseline), after the erosive challenge/gel treatments, and percentage of loss (%SHL). GROUPS DENTIN SURFACE HARDNESS Baseline Final %SHL Placebo 56.17(3.54) 39.22(7.55) 29,54 (± 16,24) a SnF 2 56.14(2.17) 43.15(3.56) 23,07 (± 6,52) a EGCG 56.09(2.91) 41.25(3.38) 26,12 (± 8,53) a EGCG/MSN 55.28(3.39) 45.87(6.28) 17,2 (± 8,86) a P-values 0.887 0.052 0.067 Scanning electron microscopy (SEM) images show the patent dentinal tubules in the negative control group (placebo). Whereas the specimen treated with EGCG, EGCG/MSN presents the patent tubules obliterated similarly to the SnF 2 group (Fig. 4 ). Discussion The present study aimed to evaluate the effect of applying a gel containing MMP inhibitors (EGCG, EGCG/MSN) in protecting human root dentin against erosion caused by citric acid. The evaluation was conducted using an in situ model to simulate clinical conditions such as using human dentin substrate, the natural formation of acquired pellicle, and physiological salivary flow [ 30 ]. The quantitative methods selected for analyzing dentin surface alterations after erosive challenges were surface hardness measurement and profilometric analysis. While hardness measurement is commonly used to assess changes in enamel surface in the early stages of erosion [ 34 ], it was chosen in this study because erosion was performed with citric acid (0.05M and pH 3.75), which has a lower erosive potential compared to substances like Coca-Cola (pH 2.6) that are used as extrinsic erosion agents in other studies [ 22 , 35 , 36 ]. However, a limitation of this method is that in heavily eroded dental substrates, where indentation limits are not clearly defined, it can result in imprecise or impossible measurements [ 18 ]. Probably, it may explain the absence of statistical differences between groups. Therefore, profilometric analysis was also performed in this study, as it is a proven method widely used to assess dentin wear after erosive challenges [ 18 , 34 – 38 ]. Furthermore, all products tested presented a gel as a vehicle for the substances to increase contact time with the dentin, enhancing the substantivity of the active ingredient and making clinical use more practical. Because the tested gels shared identical formulations, any observed preventive impact on erosion could be ascribed to the inclusion of active compounds. Different studies using a gel as a vehicle in delivering MMP inhibitors (EGCG and FeSO 4 ) demonstrated effectiveness in reducing tooth wear [ 35 , 39 , 40 ]. Another strategy employed to increase the contact time of the active ingredient with the dentin substrate was the encapsulation of EGCG in MSNs (nanoscale microcapsules), allowing for continuous release of the drug. The use of MSNs for drug encapsulation has shown promising results in Dentistry, including the occlusion of dentinal tubules and the introduction of chlorhexidine in glass ionomer cement [ 26 , 29 , 41 ]. To the best of the authors' knowledge, there are no studies evaluating the use of EGCG/MSN for protecting against dentinal erosion. MMP inhibitors, such as the catechins present in green tea and specifically EGCG, have been shown to be effective in maintaining demineralized organic matrix (DOM) [ 42 ]. EGCG has been identified as an inhibitor of MMPs 2 and 9, with minimum inhibitory concentration values of 6 and 0.8 µM, respectively [ 43 ]. Therefore, the concentration of 0.1% or 4000 µM used in the current study was considered sufficient to inhibit these collagenases, as showed in a previous study [ 31 ]. However, in the present study, the wear was like EGCG gel and negative control. Similarly, other in situ study show that EGCG gel also could not prevent tooth tissue loss after erosive challenges (coke-1 min; 4 times/day/5 days) [ 31 ]. Contrary to the results of the present study, previous studies [ 35 , 39 ] using EGCG gel significantly reduced dentin wear compared to the negative control. This discrepancy could be explained by the fact that the specimens in the current study underwent 12 hours of use to allow the formation of acquired pellicle, which works as a natural protection against the effects of erosion, although it is more effective on enamel than on dentin [ 44 ]. Although employing a validated methodology in this study, subjecting the samples to a more rigorous erosion challenge would enhance the ability to discern potential distinctions among the groups. Others in vitro studies [ 21 , 37 , 45 ] reported that green tea and/or EGCG reduced dentin wear, differing from the negative control. However, it should be noted that in all studies, the treatments were applied for 5 minutes, whereas in the current study, the application was performed for only 1 minute, which could explain the absence of a significant difference. One aspect that seems to influence the action of MMP inhibitors is the timing of their application. In situ studies conducted by Kato et al. (2010a) and Kato et al. (2010b) found that the application of protease inhibitors prior to the erosive challenge almost completely inhibited dentin wear (around 0.05 µm). In the current research, the application was performed for the same duration of time, and after the erosion process, dentin wear was measured as 0.57 µm for EGCG and 0.48 µm for EGCG/MSN. Kato et al. (2009) obtained similar dentin wear values (0.59 µm) in their study when applying EGCG at a concentration of 400 µm for 1 minute after erosion [ 46 ]. It is thus assumed that applying MMP inhibitors before erosion yields better results, possibly due to their interaction with inactive collagenases already present in dentin, preventing their activation during a subsequent pH decrease during the erosive challenge. MMPs require a low pH for activation, followed by medium neutralization, before they can start degrading the DOM [ 47 , 48 ]. It is believed that the treatment with EGCG/MSN was equivalent to SnF 2 in terms of dentin wear due to the characteristics of MSNs, which are resistant to acid challenge [ 49 ], and their ability to promote continuous release of the drug [ 29 , 41 ]. This allows for a longer contact time of catechin with dentin while protecting EGCG from adverse conditions in the oral environment. Furthermore, in the SEM images, it presented a surface pattern like that observed for the group treated with SnF 2 (Fig. 4 ). The anti-erosion effect of tin has been demonstrated in several studies [ 16 , 50 , 51 ], which support the results of the present study SnF 2 (positive control) has recently been identified as an inhibitor of MMPs 2 and 9 [ 17 ] and acts by incorporating it into mineralized tissue, preserving dental organic matrix (DOM) [ 52 ]. When SnF 2 reacts with hydroxyapatite, it forms complex precipitates containing compounds such as Sn 2 OHPO 4 , Sn 3 F 3 PO 4 , Ca(SnF 3 ) 2 , and CaF 2 [ 53 ]. The combined action of these mechanisms explains the better performance of SnF 2 in reducing dentin wear. The results show that the gel containing SnF2 had an excellent performance, reducing dentin loss by approximately 51% compared to the placebo gel. Nevertheless, products containing tin may cause tooth surface discoloration and impart an astringent sensation [ 54 ], thereby restricting their practical application in a clinical setting, which justifies the search for natural products, low cost, easy access, and without side effects. Despite employing a well-established pH-cycling protocol and making an effort to closely mimic clinical conditions, it is crucial to recognize the limitations of this study. These include the inability to assess the effects of protease inhibitors on salivary MMPs and the absence of a comprehensive evaluation of both erosion and abrasion.It is important to note that the protocol used in this research did not directly assess the effect of EGCG as an MMP inhibitor. The reduction in dentin wear promoted by EGCG/MSN can be attributed to the aforementioned mechanisms, as well as the characteristics of the MSNs as previously reported. However, further studies are needed to confirm the inhibitory action of EGCG adsorbed on MSNs on MMPs present in erosively demineralized dentin. CONCLUSION It is concluded that, within the limitations of the present study, the use of EGCG/MSN constitutes a promising protective measure in reducing dentinal erosion. Declarations Acknowledgments The authors express their gratitude to the Central Analítica-UFC for conducting the microscopy measurements, which were supported by Finep-CT-INFRA, CAPES-Pró-Equipamentos, and MCTI-CNPq SisNano2.0. This research also received support from PROAP/PRINT/CAPES and Portal de Periodicos CAPES – Finance Code 001. A. Author Contribution Helaine Cajado Alves: Conception and design of the study, experimental procedures, data collection, data analysis and manuscript drafting. Edison Augusto Balreira Gomes: Experimental procedures and manuscript review. Antonia Flavia Justino Uchoa: Data acquisition, interpretation, statistical evaluation, manuscript review. Nágila Maria Pontes Silva Ricardo: Conceptualization and design of the study, experimental procedures; approval of the final version of the manuscript. Vanara Florêncio Passos: Conceptualization and design of the study, data analysis, manuscript review and correspondent author. Sérgio Lima Santiago: Conceptualization and design of the study, manuscript review, approval of the final version. B. Ethics Approval and Consent to Participate The present study followed the Declaration of Helsinki for the ethical principles of medical research involving human subjects and was approved by our institute’s research ethics committee (approval number (# 2.704.870). Informed written consent was obtained from all subjects and/or their legal guardians. C. Funding Not Applicable D. 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Yu J, Yang H, Li K, Lei J, Zhou L, Huang C (2016) A novel application of nanohydroxyapatite/mesoporous silica biocomposite on treating dentin hypersensitivity: An in vitro study. Journal of dentistry 50 :21–29 https://doi.org/10.1016/j.jdent.2016.04.005 Yu J, Yang H, Li K, Ren H, Lei J, Huang C (2017) Development of Epigallocatechin-3-gallate-Encapsulated Nanohydroxyapatite/Mesoporous Silica for Therapeutic Management of Dentin Surface. ACS applied materials & interfaces 9 (31):25796–25807. https://doi.org/10.1021/acsami.7b06597 West NX, Davies M, Amaechi BT (2011) In vitro and in situ erosion models for evaluating tooth substance loss. Caries research 45(1):43–52. https://doi.org/10.1159/000325945 DE Moraes MDR, et al. (2021) Protective effect of green tea catechins on eroded human dentin: an in vitro/in situ study. Brazilian oral research . https://doi.org/10.1590/1807-3107bor-2021.vol35.0108 Passos VF, Rodrigues Gerage LK, Lima Santiago S (2017) Magnesium hydroxide-based dentifrice as an anti-erosive agent in an in situ intrinsic erosion model. American journal of dentistry 30 (3):37–141. Toledano M, Yamauti M, Ruiz-Requena ME, Osorio R (2012) A ZnO-doped adhesive reduced collagen degradation favouring dentine remineralization. Journal of dentistry 40 (9):756–765. https://doi.org/10.1016/j.jdent.2012.05.007 Shellis RP, Ganss C, Ren Y, Zero DT, Lussi A (2011) Methodology and models in erosion research: discussion and conclusions. Caries research 45(1) :69–77. https://doi.org/10.1159/000325971 Kato MT, Leite AL, Hannas AR, Buzalaf MA (2010)a Gels containing MMP inhibitors prevent dental erosion in situ. Journal of dental research 89 (5):468–472. https://doi.org/10.1177/0022034510363248 Magalhães AC, Wiegand A, Rios D, Hannas A, Attin T, Buzalaf MA (2009). Chlorhexidine and green tea extract reduce dentin erosion and abrasion in situ. Journal of dentistry , 37(12):994–998. https://doi.org/10.1016/j.jdent.2009.08.007 Passos VF, et al. (2018) Active compounds and derivatives of camellia sinensis responding to erosive attacks on dentin. Brazilian oral research . https://doi.org/10.1590/1807-3107bor-2018.vol32.0040. Bueno TL, et al. (2022) Evaluation of Proanthocyanidin-based dentifrices on dentin-wear after erosion and dental abrasion - In situ study. Journal of clinical and experimental dentistry 14(4):e366–e370. https://doi.org/10.4317/jced.59071 Kato MT, et al. (2010)b Effect of iron on matrix metalloproteinase inhibition and on the prevention of dentine erosion. Caries research 44 (3):309–316. https://doi.org/10.1159/000315932 Kato MT, et al. (2021) Dentifrices or gels containing MMP inhibitors prevent dentine loss: in situ studies. Clinical oral investigations 25 (4):2183–2190. https://doi.org/10.1007/s00784-020-03530-y Yan H, Yang H, Li K, Yu J, Huang, C (2017) Effects of Chlorhexidine-Encapsulated Mesoporous Silica Nanoparticles on the Anti-Biofilm and Mechanical Properties of Glass Ionomer Cement. Molecules (Basel, Switzerland) 22 (7):1225. https://doi.org/10.3390/molecules22071225 Kato MT, et al. (2012). Impact of protease inhibitors on dentin matrix degradation by collagenase. Journal of dental research , 91 (12):1119–1123. https://doi.org/10.1177/0022034512455801 Demeule M, Brossard M, Pagé M, Gingras D, Béliveau R (2000) Matrix metalloproteinase inhibition by green tea catechins. Biochimica et biophysica acta 1478(1):51–60. https://doi.org/10.1016/s0167-4838(00)00009-1 Wiegand A, Bliggenstorfer S, Magalhaes AC, Sener B, Attin T (2008) Impact of the in situ formed salivary pellicle on enamel and dentine erosion induced by different acids. Acta odontologica Scandinavica 66 (4):225–230. https://doi.org/10.1080/00016350802183401 Leal IC, Rabelo CS, Viana ÍEL, Scaramucci T, Santiago SL, Passos VF (2021) Hesperidin reduces dentin wear after erosion and erosion/abrasion cycling in vitro. Archives of oral biology 129 :105208. https://doi.org/10.1016/j.archoralbio.2021.105208 Kato MT, Magalhães AC, Rios D, Hannas AR, Attin T, Buzalaf MA (2009) Protective effect of green tea on dentin erosion and abrasion. Journal of applied oral science 17(6):560–564. https://doi.org/10.1590/s1678-77572009000600004 Buzalaf MA, Kato MT, Hannas AR (2012) The role of matrix metalloproteinases in dental erosion. Advances in dental research 24 (2)72–76. https://doi.org/10.1177/0022034512455029 Chaussain-Miller C, Fioretti F, Goldberg M, Menashi S (2006) The role of matrix metalloproteinases (MMPs) in human caries. Journal of dental research 85(1):22–32. https://doi.org/10.1177/154405910608500104 Li S, Liu M, Sun L. (2011) Preparation of acid-resisting ultramarine blue by novel two-step silica coating process. Industrial & Engineering Chemistry Research, 50:7326–7331. http//dx.doi.org/10.1021/ie200343k Schlueter N, et al. (2020) Terminology of Erosive Tooth Wear: Consensus Report of a Workshop Organized by the ORCA and the Cariology Research Group of the IADR. Caries research 54 (1):2–6. https://doi.org/10.1159/000503308 West NX, He T, Zou Y, DiGennaro J, Biesbrock A, Davies M. (2021) Bioavailable gluconate chelated stannous fluoride toothpaste meta-analyses: Effects on dentine hypersensitivity and enamel erosion. Journal of dentistry . https://doi.org/10.1016/j.jdent.2020.103566 Ganss C, Hardt M, Lussi A, Cocks AK, Klimek J, Schlueter N (2010) Mechanism of action of tin-containing fluoride solutions as anti-erosive agents in dentine - an in vitro tin-uptake, tissue loss, and scanning electron microscopy study. European journal of oral sciences 118 (4):376–384. https://doi.org/10.1111/j.1600-0722.2010.00742.x Babcock FD, King JC, Jordan TH (1978) The reaction of stannous fluoride and hydroxyapatite. Journal of dental research 57(9-10):933–938. https://doi.org/10.1177/00220345780570092301. Ellingsen JE, Eriksen HM, Rölla G (1982) Extrinsic dental stain caused by stannous fluoride. Scandinavian Journal of Dental Research , 90(1):9-13. https://doi: 10.1111/j.1600-0722.1982.tb01518.x.. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-3996730","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276600696,"identity":"6206e38d-f443-4782-a860-7a447406d926","order_by":0,"name":"Helaine Cajado Alves","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Helaine","middleName":"Cajado","lastName":"Alves","suffix":""},{"id":276600697,"identity":"e3363a60-3d11-4b06-ae55-6d4207d4ff14","order_by":1,"name":"Edison Augusto Balreira Gomes","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Edison","middleName":"Augusto Balreira","lastName":"Gomes","suffix":""},{"id":276600698,"identity":"a08fb86d-6593-4428-88c9-cbf196c6b697","order_by":2,"name":"Antonia Flavia Justino Uchoa","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Antonia","middleName":"Flavia Justino","lastName":"Uchoa","suffix":""},{"id":276600699,"identity":"76caa717-9294-434f-b593-bf9b0eeb9529","order_by":3,"name":"Nágila Maria Pontes Silva Ricardo","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Nágila","middleName":"Maria Pontes Silva","lastName":"Ricardo","suffix":""},{"id":276600700,"identity":"9fca6914-2ab7-40b9-8d94-75ef4c4a1477","order_by":4,"name":"Vanara Florêncio Passos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYNCDjw0gkrHxANE6GGc2MEgAqQbitTDzgrUwMODVYnD87MHPFTV35OX7Dx/+bLvDpk63/TDQlhqbaJxazuQlS5459sxww420NOncM2kSZmcSgVqOpeU24NAi2ZBjINnAdphxgwSPGXNu22EJswNALYwNh3Fr6X9j/LPh32H7+f1njD9bgrScf4hfC79EjplkY9thoMk5BtKMIC03CNjCL/HGzLKx71kyyC+SvW1pkttuAG1JwOMXNv4c45sN3+7YzgeG2IefbTb8ZufTHz74UGODUwsUHEDjJ+BXjk3LKBgFo2AUjAIkAADRMWYGZmlTqgAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":true,"prefix":"","firstName":"Vanara","middleName":"Florêncio","lastName":"Passos","suffix":""},{"id":276600701,"identity":"8767048e-3ceb-4a2c-9d7e-d198a8d34b68","order_by":5,"name":"Sérgio Lima Santiago","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Sérgio","middleName":"Lima","lastName":"Santiago","suffix":""}],"badges":[],"createdAt":"2024-02-28 13:14:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3996730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3996730/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52195215,"identity":"37399fc1-2031-4ae3-bea8-b1f8b9efe829","added_by":"auto","created_at":"2024-03-07 19:52:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":560415,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental Design.\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-3996730/v1/ed72f01b7f47c5f709f57e6d.png"},{"id":52195201,"identity":"41930efe-b6cd-4fb2-ae7d-f382521acd3e","added_by":"auto","created_at":"2024-03-07 19:52:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":658766,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs (x150.000) of mesoporous silica nanoparticles with EGCG (average size 164 nm).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3996730/v1/0eef2020a0a343ab50263d81.png"},{"id":52195211,"identity":"8adb21a1-f048-4c87-a6de-73c337c71efc","added_by":"auto","created_at":"2024-03-07 19:52:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46112,"visible":true,"origin":"","legend":"\u003cp\u003eMean values of wear (µm). Vertical bars and lines denote wear differences in the groups studied and the standard deviations, respectively. Different letters denote statistically significant differences identified via Tukey test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3996730/v1/2b2a2090c0dcff49364552fa.png"},{"id":52195192,"identity":"3f098f8e-a58c-4018-88ac-e78d08e62c9d","added_by":"auto","created_at":"2024-03-07 19:52:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":873254,"visible":true,"origin":"","legend":"\u003cp\u003eSEM (x8000): (a) placebo, (b) EGCG, (c) SnF2, (d) EGCG/MSN.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3996730/v1/3cf2ed694d39c4b08f3e0c4e.png"},{"id":52350082,"identity":"20a57784-182d-4013-8877-99e22c6e77d5","added_by":"auto","created_at":"2024-03-09 17:47:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2713642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3996730/v1/a4d48a65-60c4-4eca-b51e-079022a95727.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gel Containing Catechin and Mesoporous Silica Nanoparticles for Protecting Root Dentin Against Erosion: An In Situ Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eErosive tooth wear is a complex condition that affects various age groups in the global population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is characterized by the chemical loss of mineralized tooth substance due to exposure to non-bacterial acids [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The prevalence of dental erosion has increased globally due to changes in lifestyle and dietary habits, including the higher consumption of acidic foods, carbonated drinks, and fresh fruit juices [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Prevalence studies have reported erosive conditions in young adults ranging from 7.3\u0026ndash;33.8% [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eControlling erosive acid attacks on dental tissues poses a significant challenge in preventing and treating dental erosion in clinical practice [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While saliva and remineralizing foods have shown to decrease enamel erosion [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], measures to reduce tooth wear have been investigated, particularly those that enhance acid resistance or promote remineralization of tooth surfaces. Traditional preventive and control treatments for dental erosion focus on patient education, dietary control, and psychological counseling [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, some treatments aim to promote mineralization through the use of fluoride. Fluoride treatments involve the formation of calcium fluoride on the tooth surface (in the case of amine fluoride and sodium fluoride), which is easily soluble in acid or metal-rich precipitates formation (in the case of titanium tetrafluoride and fluoride products containing tin) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the fluorides studied for their anti-erosion effects on dentin, those containing tin, a polyvalent metallic cation, have emerged as effective anti-erosion agents [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Tin forms a protective barrier on the dentin surface due to its strong affinity for mineralized dental tissue. It also acts as an inhibitor of matrix metalloproteinases (MMPs) 2 and 9 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, stannous fluoride may cause undesirable effects, such as tooth surface staining and an astringent sensation on the oral mucosa [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, there is a need to explore more biocompatible substances to minimize the effects of erosion. The use of MMP inhibitors has emerged as a new approach to the treatment of this condition. Green tea and its main catechin, epigallocatechin-3-gallate (EGCG), fall into this category, as they have shown significant reduction in dentin wear after erosive/abrasive challenges and low toxicity in dental pulp cells [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. They also act as cross-linking agents for collagen fibrils, thereby stabilizing collagen and improving the mechanical properties of dentin while inhibiting proteolytic degradation of the dentin's organic matrix [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn medicine, the use of drug nanocarriers and functional nanoparticles has been extensively explored in cancer. Mesoporous silica nanoparticles (MSNs) are a type of nanocarrier known for their stability, large surface area, and excellent thermal and chemical performance [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In dentistry, MSNs have been used with nanohydroxyapatite (nHAp), EGCG, calcium oxide, and calcium carbonate, with or without phosphoric acid. In these cases, MSNs act as carriers of these substances to the dentinal tubules [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Due to challenges in conducting in vivo erosive studies, in situ and in vitro models have been utilized to analyse erosive challenges in dental tissues. In situ studies provide a more realistic evaluation of the effects of erosive agents and substances that protect dental tissues in the oral environment. These studies consider salivary flow, acquired pellicle formation, and routine care [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present in situ study aims to evaluate the effects of applying a gel containing MMP inhibitors, either EGCG or EGCG/MSN, on extrinsic erosion caused by citric acid. A placebo and SnF2 will be used as control groups.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eThe present in situ, randomized, controlled, blind, and crossover study evaluated the effects of applying a gel containing MMP inhibitors, 0.1% EGCG or 0.093% EGCG/MSN, on extrinsic erosion by citric acid, using a placebo and the 0.05% SnF\u003csub\u003e2\u003c/sub\u003e as controls. The experiment was carried out in four phases of five days each, with an interval of two days between the phases. The volunteers used a palatal appliance with two dentin blocks (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e). It was immersed four times/day in a solution of 50 mL of citric acid (0.05 M and pH 3.75) at room temperature for 60 s. After the erosive challenge, the predetermined treatment was applied for 60 s. At the end of each phase, the dentin blocks were removed from the device to perform the following quantitative analyses: hardness and profilometry tests; and new blocks were placed in the device, starting a new phase with a new substance. Thus, the response variables were the loss of surface hardness (%SHL), the wear profile of the dentin structure, and the morphological surface analyzed by SEM.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eThe following reagents were used: EGCG, hexadecyltrimethyl ammonium bromide (CTAB), ammonium hydroxide (NH\u003csub\u003e4\u003c/sub\u003eOH), tertraethylorthosilicate (TEOS) and ammonium nitrate (NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e), SnF\u003csub\u003e2\u003c/sub\u003e, PLURONIC F127\u0026reg; from Sigma-Aldrich (St. Louis, MO, USA). All reagents mentioned are analytical grade and used without prior purification.\u003c/p\u003e\n\u003ch3\u003eSynthesis of Mesoporous Silica Nanoparticles (MSNs)\u003c/h3\u003e\n\u003cp\u003eThe MSNs were prepared according to the previous literature with some modifications (ZOU et al., 2017). 5.73 g of CTAB were dissolved in 280 mL of deionized water under mechanical agitation at 40\u0026deg;C in the initial 15 min and then at room temperature overnight. After this period, 0.5 mL of NH\u003csub\u003e4\u003c/sub\u003eOH and 80 mL of ethanol were added to the system, which remained under stirring for another 1 h. The temperature of the mixture was then increased to 70\u0026deg;C, and then 14.6 mL of TEOS was added dropwise. This system remained under agitation for 2 h. Subsequently, the solid was collected by centrifugation, washed with etanol, and dried at 100\u0026deg;C for 5 h. Surfactant extraction was performed using a NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e/ethanol mixture (133 mg/50mL) at 60\u0026deg;C, with continuous magnetic stirring for 40 minutes. Finally, the solid was collected by centrifugation, washed three times with deionized water, and dried at 100\u0026deg;C overnight, thus obtaining the MSNs.\u003c/p\u003e\n\u003ch3\u003eSynthesis of Epigallocatechin-3-gallate Adsorbed on Mesoporous Silica Nanoparticles (EGCG/MSN)\u003c/h3\u003e\n\u003cp\u003eThe synthesis of EGCG encapsulated in MSNs (EGCG/MSN) was performed according to the techniques reported in a previous study by Yu et al. (2017) with some modifications. 100 mg of dry MSN was dispersed in an aqueous EGCG solution (1.8 mg/mL) by vigorous horizontal stirring for 72 h in the dark. This process allows EGCG molecules to infiltrate into the pores of the MSNs to achieve maximum charge. The mixture was then centrifuged, washed three times with deionized water and ethanol, and dried under vacuum for 48 h to obtain EGCG/MSN, being stored in the dark at 4\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003eA calibration curve was used to quantify the drug from a series of reference solutions whose concentrations ranged from 0.1 to 30 ppm, obtaining a linear relationship between the highest absorbance peak and the drug concentration. A UV Vis spectrophotometer (DU\u0026reg; 730; Beckman Coulter, Fullerton, CA, USA) was used to assess and confirm the peak absorbance of EGCG at 274nm. The encapsulation efficiency was calculated at 93%.\u003c/p\u003e\n\u003ch3\u003eGel Synthesis\u003c/h3\u003e\n\u003cp\u003eThe copolymer PLURONIC F127\u0026reg; (Sigma Chemical Co., St. Louis, MO, USA) was used to prepare placebo and active gels containing the following mass percentages: no active component (placebo), 0.05% of SnF\u003csub\u003e2\u003c/sub\u003e, 0.1% of EGCG, or 37% of EGCG/MSN. The gel was prepared at a concentration of 20% PLURONIC F127\u0026reg;/water. 6 g of PLURONIC F127\u0026reg; were dissolved in 30 mL of deionized water at low temperature (approximately 4 \u0026ordm;C) under magnetic stirring until complete homogenization of the F127. After the mixture was completely dissolved, the temperature was raised to 30\u0026deg;C to form the gel.\u003c/p\u003e\n\u003ch3\u003eEthical aspects and Volunteers selection\u003c/h3\u003e\n\u003cp\u003eBefore the start of this study, it was approved by the local Ethics Committee (# 2.704.870) and carried out in accordance with the Declaration of Helsinki. The volunteers who participated in the research previously signed a Free and Informed Consent Term and received verbal and written instructions on how to carry out the research procedures.\u003c/p\u003e\n\u003cp\u003eVolunteers were recruited and eleven individuals were selected (01 male and 10 females, between 22 and 41 years old) to participate in the research. All the selected volunteers met the inclusion criteria, which were: good oral hygiene, absence of caries activity and periodontal disease, and absence of erosion lesion, in addition to having stimulated salivary flow\u0026thinsp;\u0026gt;\u0026thinsp;1 mL/min. The exclusion criteria were the presence of gastroesophageal disorders, pregnant women, lactating women, or those who use a myorelaxant plate, fixed or removable orthodontic appliance in the upper arch, or medications that could alter salivary flow were not included in the research. The sample size was calculated based on the percentage change in surface hardness loss data from a pilot study. The sample of seven volunteers was necessary to provide an \u0026alpha;-error of 5%, a power of 80%, and a relevant difference of 10% between groups (\u003cspan\u003e\u003cspan\u003ewww.openepi.com/menu/oe_menu.htm\u003c/span\u003e\u003c/span\u003e). Eleven volunteers were included due to possible losses inherent to in situ studies.\u003c/p\u003e\n\u003ch3\u003ePreparation of dentin specimens\u003c/h3\u003e\n\u003cp\u003eIn this study, freshly extracted, non-carious human third molars were collected after informed consent from the donors were achieved based on a protocol approved by the Ethics Committee of the Federal University of Cear\u0026aacute;, Brazil. The selected molars were cleaned and stored in 0.1% thymol solution at 4 \u0026ordm;C were used. About two hundred blocks of human root dentin were cut to size 4x4x2mm, flattened and polished. For this, a double-sided diamond cutting wheel was used under abundant refrigeration (Struers, Minitom, Copenhagen, Denmark). Subsequently, the dentin blocks were placed in an acrylic device and flattened using P1200, P2500, and P4000 abrasive aluminum oxide sandpaper (Buehler, Lake Bluff, IL, USA), coupled to an automatic polisher (Buehler, Automet 250, Lake Bluff, IL, USA) under constant water irrigation and later polished with a felt disc and 1\u0026micro;m diamond paste (Erios, S\u0026atilde;o Paulo, Brazil).\u003c/p\u003e\n\u003cp\u003eDuring polishing, specimens were sonicated in distilled water for 2 minutes both between the use of sandpaper and at the end of the polishing process to remove residues resulting from these procedures. To standardize the specimens, the initial surface hardness was determined. Five indentations 100 \u0026micro;m apart were made in the center of the specimens using a microhardness tester with Knoop indenter and automatic measuring system (Future Tech Corp, FM-ARS 9000 and FM 100, Tokyo, Japan). A Knoop diamond indenter was used with a load of 10 g applied for 5s [\u003cspan\u003e21\u003c/span\u003e, \u003cspan\u003e31\u003c/span\u003e]. Eighty-eight dentin specimens, which had an average hardness of 54.53\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 Kg/mm\u0026sup2;, were selected, packaged, and identified for ethylene oxide sterilization. Then, the specimens were covered with an acid adhesive tape leaving an exposed area of 4 x 2 mm which was subjected to erosion and treatment during the experiment. The covered area was used as a reference for the profilometric analysis.\u003c/p\u003e\n\u003ch3\u003ePalatal device preparation\u003c/h3\u003e\n\u003cp\u003eA palatal device was made of acrylic resin for each volunteer. Two cavities measuring 5x5x3mm were made in the device, one on the right side and the other on the left. The specimens were randomly assigned using a computer list (Microsoft Excel 2007), fixed with sticky wax, and carefully fitted 1 mm below the device surface level, to avoid abrasion resulting from contact with the tongue.\u003c/p\u003e\n\u003ch3\u003eIntraoral phase\u003c/h3\u003e\n\u003cp\u003eIn the two days before the start of the research, the volunteers used a standard fluoride toothpaste (Colgate \u0026reg; -1450 ppm fluoride) that was given to them and continued using it until the end of the experiment. Before each experimental phase, the palatal device was used for 12 h to allow equilibrium with the saliva and the formation and maturation of the acquired pellicle. Accordingly, the volunteers\u0026rsquo; participation in all stages and testing of all treatments enabled a crossover design. The positions of the specimens were randomly determined according to a computer-generated randomization list. The erosive challenge was performed extraorally 4 times a day at 7 am, 12 pm, 5 pm, and 9 pm during the 5-day experiment [\u003cspan\u003e32\u003c/span\u003e]. Research participants removed the device from their mouths and placed it in a container with 50 mL of citric acid solution (0.05M, pH 3.75) for 60 s at room temperature, characterizing erosion [\u003cspan\u003e21\u003c/span\u003e]. Then, they were washed in running water for 10 s and excess water was removed with absorbent paper. After the acid challenge, the volunteers applied a thin layer of the gel, which remained in contact with the specimen for 60 s, and carefully removed the excess with absorbent paper (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e). After that the device was reinserted into the mouth. The gels were packaged and coded in syringes to maintain the blindness of the study regarding the volunteers.\u003c/p\u003e\n\u003cp\u003eThe volunteers were instructed to avoid eating, drinking (except water), and brushing their teeth while using the device, which was used continuously, including at night, being removed only for meals and oral hygiene, and, on these occasions, the device was kept mostly in plastic boxes. Volunteers were allowed to clean the device with a toothbrush and toothpaste, taking care not to brush the specimens.\u003c/p\u003e\n\u003cp\u003eAfter each phase, on the morning of the sixth day, the specimens were removed from the device, and stored in humidity under refrigeration until the time of analysis. They were evaluated with respect to the percentage of surface hardness loss (%SHL) and measurement of dentin surface wear. New specimens were inserted into the device, which underwent a new phase of the experiment.\u003c/p\u003e\n\u003ch3\u003eFinal surface hardness assessment\u003c/h3\u003e\n\u003cp\u003eThe final surface hardness of each specimen was measured using the same protocol as the initial surface hardness. Five indentations, with 100 \u0026micro;m distance between them, were performed on each specimen in the center of the eroded area. The average of the indentations (final hardness) was used to evaluate the percentage loss of surface hardness (%SHL) according to the following formula:\u003c/p\u003e\n\u003cp\u003e%SHL = [(initial hardness \u0026ndash; final hardness) x 100/initial hardness]\u003c/p\u003e\n\u003ch3\u003eAssessment of dentin wear\u003c/h3\u003e\n\u003cp\u003eThe level of dentin wear was determined in relation to the reference area with a profilometer (Hommel Tester, T1000, Hommelwerke GmbH, Germany). Adhesive tape was carefully removed from each dentin block to expose the reference area. Three tracings with a length of 1.5 mm each were performed on each specimen, moving the profilometer pen tip 1.8 \u0026micro;m from the reference surface to the experimental surface. For each specimen, the mean value obtained from the three tracings was calculated.\u003c/p\u003e\n\u003ch3\u003eScanning electron microscopy\u003c/h3\u003e\n\u003cp\u003eTwo dentin samples from each group were observed under electron microscopy scan for a qualitative analysis. They were immersed in a 2.5% glutaraldehyde fixative solution in a 0.1 mol/L of sodium cacodylate for 24 hours and washed after that with 0.1 mol/L of cacodylate buffer. Then, they were dehydrated with ethanol solutions at concentrations crescents and dried at room temperature for 24 hours in a desiccator [\u003cspan\u003e33\u003c/span\u003e]. The specimens were fixed metal stubs and received a gold coating through a metallizer (Hammer VI - sputtering system, Anatech Ltda, Alexandria, USA). After that, as exemplified were admitted by SEM Quanta FEG 450 (FEI Company, Oregon, USA). The voltage of adjustment had an adjustment of 20 kV. The magnification used was 8,000 times Additionally, SEM was employed to determine the morphology and ultrastructure of MSN, (EGCG/MSN) gel was qualitatively evaluated by microscopy to verify its adsorption (Fig. \u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eMean and standard deviation data for wear and hardness loss were calculated. A preliminary, Kolmogorov-Smirnov test was performed on all of the groups to test the normal distribution of errors. Because the values were normally distributed across all groups, one-way ANOVA and Tukey post-hoc tests were applied for the data of wear and hardness loss. Statistical analysis was performed with Statistical Package for Social Sciences -SPSS 20.0 for Windows (SPSS Inc., Chicago IL, USA). The level of significance was set at 5%.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe values of the means and standard deviation of dentin surface hardness before (initial), after (final) the erosive challenge/treatment, and the percentage of surface hardness loss (%SHL) are described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results of the % analysis SHL showed no statistical difference (p\u0026thinsp;=\u0026thinsp;0.067) between the groups. The dentinal wear values (\u0026micro;m) revealed a statistical difference between treatments (p\u0026thinsp;=\u0026thinsp;0.005). Then, the Tukey post-test was applied to determine the difference between the groups which was observed only for SnF\u003csub\u003e2\u003c/sub\u003e when compared to placebo and EGCG (p\u0026thinsp;=\u0026thinsp;0.003 and p\u0026thinsp;=\u0026thinsp;0.046, respectively). Regarding EGCG/MSN, there was also no significant difference between this group and SnF\u003csub\u003e2\u003c/sub\u003e (positive control) (p\u0026thinsp;=\u0026thinsp;0.306), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\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\u003eMean (n\u0026thinsp;=\u0026thinsp;11;SD) of dentin surface hardness (SH) before (baseline), after the erosive challenge/gel treatments, and percentage of loss (%SHL).\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGROUPS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eDENTIN SURFACE HARDNESS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaseline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%SHL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePlacebo\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56.17(3.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.22(7.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29,54 (\u0026plusmn;\u0026thinsp;16,24) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSnF\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56.14(2.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.15(3.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23,07 (\u0026plusmn;\u0026thinsp;6,52) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEGCG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56.09(2.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41.25(3.38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26,12 (\u0026plusmn;\u0026thinsp;8,53)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEGCG/MSN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55.28(3.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45.87(6.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17,2 (\u0026plusmn;\u0026thinsp;8,86)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP-values\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.887\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.052\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.067\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) images show the patent dentinal tubules in the negative control group (placebo). Whereas the specimen treated with EGCG, EGCG/MSN presents the patent tubules obliterated similarly to the SnF\u003csub\u003e2\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study aimed to evaluate the effect of applying a gel containing MMP inhibitors (EGCG, EGCG/MSN) in protecting human root dentin against erosion caused by citric acid. The evaluation was conducted using an \u003cem\u003ein situ\u003c/em\u003e model to simulate clinical conditions such as using human dentin substrate, the natural formation of acquired pellicle, and physiological salivary flow [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe quantitative methods selected for analyzing dentin surface alterations after erosive challenges were surface hardness measurement and profilometric analysis. While hardness measurement is commonly used to assess changes in enamel surface in the early stages of erosion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], it was chosen in this study because erosion was performed with citric acid (0.05M and pH 3.75), which has a lower erosive potential compared to substances like Coca-Cola (pH 2.6) that are used as extrinsic erosion agents in other studies [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, a limitation of this method is that in heavily eroded dental substrates, where indentation limits are not clearly defined, it can result in imprecise or impossible measurements [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Probably, it may explain the absence of statistical differences between groups. Therefore, profilometric analysis was also performed in this study, as it is a proven method widely used to assess dentin wear after erosive challenges [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, all products tested presented a gel as a vehicle for the substances to increase contact time with the dentin, enhancing the substantivity of the active ingredient and making clinical use more practical. Because the tested gels shared identical formulations, any observed preventive impact on erosion could be ascribed to the inclusion of active compounds. Different studies using a gel as a vehicle in delivering MMP inhibitors (EGCG and FeSO\u003csub\u003e4\u003c/sub\u003e) demonstrated effectiveness in reducing tooth wear [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Another strategy employed to increase the contact time of the active ingredient with the dentin substrate was the encapsulation of EGCG in MSNs (nanoscale microcapsules), allowing for continuous release of the drug. The use of MSNs for drug encapsulation has shown promising results in Dentistry, including the occlusion of dentinal tubules and the introduction of chlorhexidine in glass ionomer cement [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To the best of the authors' knowledge, there are no studies evaluating the use of EGCG/MSN for protecting against dentinal erosion.\u003c/p\u003e \u003cp\u003eMMP inhibitors, such as the catechins present in green tea and specifically EGCG, have been shown to be effective in maintaining demineralized organic matrix (DOM) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. EGCG has been identified as an inhibitor of MMPs 2 and 9, with minimum inhibitory concentration values of 6 and 0.8 \u0026micro;M, respectively [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, the concentration of 0.1% or 4000 \u0026micro;M used in the current study was considered sufficient to inhibit these collagenases, as showed in a previous study [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, in the present study, the wear was like EGCG gel and negative control. Similarly, other in situ study show that EGCG gel also could not prevent tooth tissue loss after erosive challenges (coke-1 min; 4 times/day/5 days) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Contrary to the results of the present study, previous studies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] using EGCG gel significantly reduced dentin wear compared to the negative control. This discrepancy could be explained by the fact that the specimens in the current study underwent 12 hours of use to allow the formation of acquired pellicle, which works as a natural protection against the effects of erosion, although it is more effective on enamel than on dentin [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Although employing a validated methodology in this study, subjecting the samples to a more rigorous erosion challenge would enhance the ability to discern potential distinctions among the groups. Others in vitro studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] reported that green tea and/or EGCG reduced dentin wear, differing from the negative control. However, it should be noted that in all studies, the treatments were applied for 5 minutes, whereas in the current study, the application was performed for only 1 minute, which could explain the absence of a significant difference.\u003c/p\u003e \u003cp\u003eOne aspect that seems to influence the action of MMP inhibitors is the timing of their application. In situ studies conducted by Kato et al. (2010a) and Kato et al. (2010b) found that the application of protease inhibitors prior to the erosive challenge almost completely inhibited dentin wear (around 0.05 \u0026micro;m). In the current research, the application was performed for the same duration of time, and after the erosion process, dentin wear was measured as 0.57 \u0026micro;m for EGCG and 0.48 \u0026micro;m for EGCG/MSN. Kato et al. (2009) obtained similar dentin wear values (0.59 \u0026micro;m) in their study when applying EGCG at a concentration of 400 \u0026micro;m for 1 minute after erosion [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. It is thus assumed that applying MMP inhibitors before erosion yields better results, possibly due to their interaction with inactive collagenases already present in dentin, preventing their activation during a subsequent pH decrease during the erosive challenge. MMPs require a low pH for activation, followed by medium neutralization, before they can start degrading the DOM [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is believed that the treatment with EGCG/MSN was equivalent to SnF\u003csub\u003e2\u003c/sub\u003e in terms of dentin wear due to the characteristics of MSNs, which are resistant to acid challenge [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and their ability to promote continuous release of the drug [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This allows for a longer contact time of catechin with dentin while protecting EGCG from adverse conditions in the oral environment. Furthermore, in the SEM images, it presented a surface pattern like that observed for the group treated with SnF\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe anti-erosion effect of tin has been demonstrated in several studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], which support the results of the present study SnF\u003csub\u003e2\u003c/sub\u003e (positive control) has recently been identified as an inhibitor of MMPs 2 and 9 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and acts by incorporating it into mineralized tissue, preserving dental organic matrix (DOM) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. When SnF\u003csub\u003e2\u003c/sub\u003e reacts with hydroxyapatite, it forms complex precipitates containing compounds such as Sn\u003csub\u003e2\u003c/sub\u003eOHPO\u003csub\u003e4\u003c/sub\u003e, Sn\u003csub\u003e3\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, Ca(SnF\u003csub\u003e3\u003c/sub\u003e)\u003csup\u003e2\u003c/sup\u003e, and CaF\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The combined action of these mechanisms explains the better performance of SnF\u003csub\u003e2\u003c/sub\u003e in reducing dentin wear. The results show that the gel containing SnF2 had an excellent performance, reducing dentin loss by approximately 51% compared to the placebo gel. Nevertheless, products containing tin may cause tooth surface discoloration and impart an astringent sensation [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], thereby restricting their practical application in a clinical setting, which justifies the search for natural products, low cost, easy access, and without side effects.\u003c/p\u003e \u003cp\u003eDespite employing a well-established pH-cycling protocol and making an effort to closely mimic clinical conditions, it is crucial to recognize the limitations of this study. These include the inability to assess the effects of protease inhibitors on salivary MMPs and the absence of a comprehensive evaluation of both erosion and abrasion.It is important to note that the protocol used in this research did not directly assess the effect of EGCG as an MMP inhibitor. The reduction in dentin wear promoted by EGCG/MSN can be attributed to the aforementioned mechanisms, as well as the characteristics of the MSNs as previously reported. However, further studies are needed to confirm the inhibitory action of EGCG adsorbed on MSNs on MMPs present in erosively demineralized dentin.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIt is concluded that, within the limitations of the present study, the use of EGCG/MSN constitutes a promising protective measure in reducing dentinal erosion.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eThe authors express their gratitude to the Central Anal\u0026iacute;tica-UFC for conducting the microscopy measurements, which were supported by Finep-CT-INFRA, CAPES-Pr\u0026oacute;-Equipamentos, and MCTI-CNPq SisNano2.0. This research also received support from PROAP/PRINT/CAPES and Portal de Periodicos CAPES \u0026ndash; Finance Code 001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Author Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHelaine Cajado Alves: Conception and design of the study, experimental procedures, data collection, data analysis and manuscript drafting.\u003c/p\u003e\n\u003cp\u003eEdison Augusto Balreira Gomes: Experimental procedures and manuscript review.\u003c/p\u003e\n\u003cp\u003eAntonia Flavia Justino Uchoa: Data acquisition, interpretation, statistical evaluation, manuscript review.\u003c/p\u003e\n\u003cp\u003eN\u0026aacute;gila Maria Pontes Silva Ricardo: Conceptualization and design of the study, experimental procedures; approval of the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003eVanara Flor\u0026ecirc;ncio Passos: Conceptualization and design of the study, data analysis, manuscript review and correspondent author.\u003c/p\u003e\n\u003cp\u003eS\u0026eacute;rgio Lima Santiago: Conceptualization and design of the study, manuscript review, approval of the final version.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eB. Ethics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study followed the Declaration of Helsinki for the ethical principles of medical research involving human subjects and was approved by our institute\u0026rsquo;s research ethics committee (approval number (# 2.704.870).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed written consent was obtained from all subjects and/or their legal guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. Conflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeven AJ, Ashley M (2023) Epidemiology, aetiology and prevention of tooth wear. \u003cem\u003eBritish dental journal\u003c/em\u003e 234(6):439\u0026ndash;444. https://doi.org/10.1038/s41415-023-5624-0\u003c/li\u003e\n\u003cli\u003eNijakowski K, Jankowski J, Gruszczyński D, Surdacka A (2023) Eating Disorders and Dental Erosion: A Systematic Review. \u003cem\u003eJournal of clinical medicine\u003c/em\u003e\u003cem\u003e12\u003c/em\u003e(19):6161. https://doi.org/10.3390/jcm12196161\u003c/li\u003e\n\u003cli\u003eJaeggi, T, Lussi A (2014) Prevalence, incidence and distribution of erosion. \u003cem\u003eMonographs in oral science\u003c/em\u003e 25:55\u0026ndash;73. https://doi.org/10.1159/000360973\u003c/li\u003e\n\u003cli\u003eSchlueter N, Amaechi BT, Bartlett D, Buzalaf MAR, Carvalho TS, Ganss C, Hara AT, Huysmans, MDNJM, Lussi A, Moazzez R, Vieira AR, West NX, Wiegand A, Young A, Lippert F (2020) Terminology of Erosive Tooth Wear: Consensus Report of a Workshop Organized by the ORCA and the Cariology Research Group of the IADR. \u003cem\u003eCaries research\u003c/em\u003e\u003cem\u003e54\u003c/em\u003e(1):2\u0026ndash;6. https://doi.org/10.1159/000503308\u003c/li\u003e\n\u003cli\u003eChan AS, Tran TTK, Hsu YH, Liu SYS, Kroon J (2020) A systematic review of dietary acids and habits on dental erosion in adolescents. \u003cem\u003eInternational journal of paediatric dentistry\u003c/em\u003e\u003cem\u003e30\u003c/em\u003e(6): 713\u0026ndash;733. https://doi.org/10.1111/ipd.12643\u003c/li\u003e\n\u003cli\u003eLUSSI A, et al. 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The participants wore an acrylic palatal device containing two dentin blocks treated with one of the gels: placebo (negative control), SnF\u003csub\u003e2\u003c/sub\u003e (0.05% - positive control), EGCG (0.1%), and EGCG/MSN (0.093%). During each phase, the specimens were immersed in citric acid (0.05 M; pH 3.75) for 60 s, 4x/day, followed by treatment with the assigned gel for 60 s. The alterations were evaluated by measuring the percentage of surface hardness loss (%SHL) and through profilometry analysis (wear). Morphological changes were assessed using scanning electron microscopy (SEM). The data were analyzed using ANOVA, followed by Tukey's post-test.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e%SHL did not show a significant difference among the groups (p\u0026thinsp;=\u0026thinsp;0.067). Regarding surface wear, the mean results in micrometers were: placebo, 0.66 (\u0026plusmn;\u0026thinsp;0.38); EGCG, 0.57 (\u0026plusmn;\u0026thinsp;0.11); EGCG/MSN, 0.48 (\u0026plusmn;\u0026thinsp;0.05); and SnF2, 0.32 (\u0026plusmn;\u0026thinsp;0.08). A significant difference was observed only between the SnF\u003csub\u003e2\u003c/sub\u003e group and the placebo and EGCG groups (p\u0026thinsp;=\u0026thinsp;0.003 and p\u0026thinsp;=\u0026thinsp;0.046, respectively). However, there was no difference between the SnF\u003csub\u003e2\u003c/sub\u003e and EGCG/MSN groups (p\u0026thinsp;=\u0026thinsp;0.306).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eEGCG/MSN shows promise as a protective measure in reducing dentin wear under erosive conditions.\u003c/p\u003e\u003ch2\u003eClinical Relevance:\u003c/h2\u003e \u003cp\u003eGels containing EGCG adsorbed on mesoporous silica nanoparticles have a protective effect against dentin erosion.\u003c/p\u003e","manuscriptTitle":"Gel Containing Catechin and Mesoporous Silica Nanoparticles for Protecting Root Dentin Against Erosion: An In Situ Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 19:51:26","doi":"10.21203/rs.3.rs-3996730/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b16d8750-4a8f-4fac-ad24-3153c89e0b53","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-09T17:46:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 19:51:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3996730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3996730","identity":"rs-3996730","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}