Fluoride-Based Validation of a Dynamic Microcosm Biofilm Model for Root Caries-Like Lesions

Fluoride-Based Validation of a Dynamic Microcosm Biofilm Model for Root Caries-Like Lesions | 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 Fluoride-Based Validation of a Dynamic Microcosm Biofilm Model for Root Caries-Like Lesions Giovanna Santos Medeiros Sagardia, Glenda Ávila Marques, Bruna Moraes Kremer, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8484242/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 To assess the effect of distinct fluoride concentrations ([F]) on root dentin carious lesions produced by the MOCS. Materials and Methods Microcosm biofilms were grown for 4 days on bovine root dentin discs from saliva of caries-free donor, under intermittent sucrose flow (5%, 0.25 ml/min, 6 min, 3×/day), and treated with NaF solutions at 0 (control), 450, or 1,350 ppmF, twice daily. Response variables included microbial composition (CFU/mg biofilm), biofilm fluoride concentration (µgF/mg biofilm), and dentin demineralization (% surface hardness change; %SHC). Results Fluoride treatments at 450 ppmF and 1,350 ppmF significantly reduced %SHC compared to 0 ppmF (p ≤ 0.035, Tukey). The %SHC was 64.1 in the control group, and 25.1 and 27.1 in biofilms exposed to 450 ppmF and 1,350 ppmF, respectively (linear regression, R = 0.57, p 0.05). Biofilms exposed to 1,350 ppmF present [F] statistically higher than control group (p < 0.001). Conclusion The MOCS demonstrated an appropriate fluoride remineralization response, supporting its validity as a pre-clinical model for testing anticariogenic agents. Clinical Relevance : This study contributes to scientific advancement in cariology by validating the MOCS as a suitable model for studies aiming at preventing and controlling root caries. Biofilm. Fluorides. Demineralization. Microcosm. Root caries Figures Figure 1 Figure 2 Figure 3 Introduction Dental caries is a biofilm-sugar-dependent disease that results in mineral loss due to dietary-induced pH fluctuations over the biofilm-tooth interface over the life course [ 1 , 2 ]. Carious lesions may develop on any dental surface, including the root ones which are exposed to the oral environment due to gingival recession, periodontal disease, surgery and other factors [ 3 , 4 ]. Due to increasing life expectancy and improved oral health, more adults and elderly individuals are retaining their natural teeth longer, which leads to root exposure posing them to an increased risk for caries development. A recent review on the global burden of untreated caries found that both the prevalence and incidence of root carious lesion increase significantly after the age of 40, affecting 35–40% of older adults [ 5 ], emphasizing the need for targeted prevention strategies, as well as the development therapies to manage root caries lesion in adults and aging populations. Fluoride remains the most widely recognized preventive and therapeutic agent for managing dental caries, including root caries [ 6 ]. Its anti-caries and cariostatic effects are frequency- [ 7 ] and concentration-dependent [ 8 ]. An increase in fluoride levels in saliva and in the solid and fluid phases of the biofilm [ 9 , 10 ] is observed under the use of fluoride-containing products, being this one of the most important contributions of fluoride to reduce demineralization and to activate the remineralization of dental hard tissues. Therefore, it is expected that fluoride exhibits a dose effect in reducing dentin demineralization, especially considering the higher susceptibility of this tissue to the mineral loss [ 11 ]. Laboratory biofilm models for carious lesion development have been employed to test hypotheses prior to clinical investigations [ 12 , 13 , 14 ]. Among them, dynamic microcosm biofilm models are particularly relevant, as they simulate key features of the oral environment, including microbial diversity, pH fluctuations, temperature, salivary flow, and intermittent exposure to dietary carbohydrates [ 15 , 16 ]. To be considered suitable for assessing preventive or therapeutic approaches, validation tests are required to confirm their responsiveness to treatment effects and their translational relevance to clinical condition [ 15 ]. Recently, the Multifunctional Oral Cavity Simulator (MOCS) was established as a dynamic microcosm model able to develop root dentin carious-like lesions [ 16 ], and its microbial composition has already been characterized [ 17 ]. However, its responsiveness to anticariogenic agents, such as fluoride, has not yet been evaluated. Therefore, this study aimed to assess the microbial composition, biofilm fluoride concentration and carious lesion development by the MOCS when microcosm biofilms were exposed to distinct fluoride concentrations ([F]). The tested hypothesis was that the inhibition of dentin carious lesions development as well as [F] in biofilms are directly dependent on the exposure to fluoride. Methods Experimental design This study was approved by the Ethics Committee on Human Research at the Federal University of Rio Grande do Sul (UFRGS) under CAAE number 69918823.6.0000.5347. This was an in vitro study using a multifunctional oral cavity simulator (MOCS) as a dynamic microcosm biofilm model. Microcosm biofilms were formed from human saliva (caries-free donor) on bovine root dentin discs for 4 days. The MOCS provided continuous flow regimes of artificial saliva (0.06 ml/min) and intermittent flow of 5% sucrose (0.25 ml/min, three times a day for six minutes). The formed biofilms were treated twice a day with different fluoride solutions (NaF: 0 ppmF - control, 450, and 1,350 ppmF; n = 9 per group). The response variables were: the percentage of surface hardness change (%SHC), microbiological composition of the biofilm through colony-forming unit counts (CFU/mg biofilm) and the biofilm fluoride concentrations. Sample size calculation The sample size was calculated based on the %SHC data obtained from the dynamic biofilm model previously described [ 16 ]. The calculation considered the difference in %SHC observed around restorations made with an anticariogenic material containing S-PRG particles and a non-cariogenic control material without S-PRG particles. The mean difference and standard deviation were estimated as 33.1± 20.5. The Statulator ( https://statulator.com/SampleSize/ss2M.html ) online statistical calculator- was used, assuming a power of 80% and a significance level of 5% [ 18 ]. Considering an expected sample loss of 20%, the final sample size was set at nine specimens per group (n = 9). Root dentin preparation Twenty-seven bovine incisors free of defects were selected. Bovine root dentin discs (6mm in diameter x 2mm in thickness) were prepared using a bench drill and a trephine bur. The pulpal wall was smoothed with #80 grit sandpaper, and the buccal surface was polished with #600, #1200, and #2000 grit sandpaper to remove surface irregularities. All procedures were performed under water cooling. After preparation, the basal and lateral walls, as well as one-third of the surface of the discs were covered with acid-resistant cosmetic nail polish. The baseline surface hardness (SH) of each disc was evaluated through three indentations, each spaced 100 µm apart, made at the center of the dentin disc using a Knoop microhardness indenter with a 25g load applied for 5 seconds (Hardness Tester, HMV 2, Shimadzu, Tokyo, Japan). The values of the three indentations were averaged within each disc. Discs with overall average baseline SH of 45.65 ± 4.26 KHN were selected and randomly distributed into individual support units, totaling 27 samples (n = 9/group). The supports units containing the specimens were sterilized with ethylene oxide (MIC Sterilization services LTDA). Biofilm model The MOCS (Odeme Dental Research, Luzerna-SC, Brazil) consist of a system with three cylindrical chambers on a heating base (37 ± 2ºC), featuring three individual inlets connected to silicone tubes to allow liquid`s flow through the system (artificial saliva, sucrose solution and treatments if needed). Support units are located under each group of inlets. The device is gas supplemented (10% CO 2 , 10% H 2 , and 80% N 2 ). The silicone tubes are originated from artificial saliva and sucrose reservoirs and passed through an infusion system composed of two 10-channel computer-controlled peristaltic pumps (FC 100, Chao Chuing, China), and reached to the specific inlets. The over-flow is directed to a waste reservoir located at the bottom of the chambers (shown in Fig. 1 ). The experimental setup and methodological details have been previously described in [ 16 ]. Inoculation and biofilm growth Saliva (2.5 mL) was collected from a caries-free donor (female, 36 yearsold) who exhibited normal salivary flow (stimulated > 1 mL/min), was systemically healthy, and showed no clinical signs of periodontal disease, and who had not used antimicrobial agents in the past 3 months. Saliva collection was performed under mechanical stimuli (paraffin chewing for 10 minutes; Parafilm "M", American National Can TM, Chicago, Ill., USA). The collected saliva was vortexed and added to 50 ml of sterile DMM (Defined Medium Mucin) artificial saliva to form the microbial inoculum solution. Three mL of this mixture was perfused over the root dentin specimens and remained on the top of dentin discs for 1 hour to allow microbial adhesion. After this period, the DMM was perfused at a rate of 0.06 ml/min. The cariogenic challenge was simulated with an intermittent flow of 5% sucrose for 6 minutes, applied 3 times a day with 2-hour intervals, at a flow rate of 0.25 ml/min. The biofilms were formed under anaerobic conditions (80% N 2 , 10% CO 2 , and 10% H 2 ), provided by a gas flow at 0.006 N/mm², applied twice a day for 1 minute during the 4 days of experiment. Biofilm treatment Twenty-four hours after the initial microbial adhesion, the biofilms were treated twice daily (every 12 hours) for 1 minute with 5 mL of NaF solutions at concentrations of 0, 450, and 1,350 ppmF. These solutions were dripped over the biofilms by a syringe inserted into an extra-inlet located above each support units of the cylindrical chambers. Sample collections and analysis Microbial analysis After 4 days of growth, the biofilms formed over the dentin discs were collected. This procedure was done using a sterile loop positioned at the center of each disc and inserted into the biofilm until it reached to the dentin disc surface. The biofilm attached to the loop and to its surrounding was collected and added to pre-weighted microtube for microbial analysis. The remained biofilm was also collected and added to another pre-weighted microtubes for biochemical analysis. The wet weight of biofilms was determined. For microbial analysis, the biofilms were resuspended in one ml of sterile saline solution and the suspension was vortexed, sonicated (20W, 30 s), serially diluted (10 0 -10⁶), and plated in duplicate (20 µL) in Brain Heart Infusion Agar (BHI), in BHI with pH adjusted to 4.8 and in Mitis Salivarius Agar (MSB) (supplemented with 0.2 U of bacitracin per mL) for the cultivation of total microorganisms, total aciduric microorganisms and mutans streptococci, respectively. The agar plates were incubated at 37 o C in anaerobiosis jars for 4 days. Subsequently, the agar plates were observed under a stereoscope and the colony-forming units (CFU) were counted. Results were expressed as CFU (log)/mg of wet biofilm. Fluoride bound to biofilm analysis Biofilm samples were dried for 24 hours in a vacuum desiccator with P₂O₅ and the dry weight was determined. The dried biofilms were resuspended in 100 µL of HCl (per mg/dry-weight). After 3 hours under agitation, an equal volume of TISAB II NaOH was added. The samples were then centrifuged for 10 minutes at 14,000 rpm, and the supernatant was carefully removed and frozen for further analysis. All samples were analyzed using a fluoride ion-specific electrode (Orion Research Inc., Beverly, USA), previously calibrated with standard solutions of known fluoride concentration (from 0.1 ppmF to 16 ppmF) (Orion Research Inc., Beverly, USA), connected to an ion analyzer (Procyon, São Paulo, Brazil). The sample readings were recorded in millivolts (mV) and converted using linear regression from the calibration curve into fluoride concentrations expressed as µmol/L and µg/mg [ 19 , 20 , 21 ]. Dentin carious lesion assessment Dentin discs were cleaned with distilled water and brushed with a soft-bristle toothbrush. The SH was again assessed by placing three indentations at the center of each disc, spaced 100 µm apart from each other and from the baseline indentations. The values of the three indentations were averaged within each disc. Root dentin demineralization was expressed as the percentage of surface hardness change (%SHC) which was calculated using following formula: (SH baseline – SH post treatment) / (SH baseline) x 100 [ 22 ]. Statistical analysis The assumptions of equality of variances and normal distribution of errors were checked for each response variable. If the variable did not fulfill these assumptions, data transformations were applied [ 23 ]. Fluoride concentration data were rank transformed, and One-way ANOVA followed by Tukey's post-hoc test was used for all the outcomes. Additionally, a linear regression was employed to evaluate the relationship between fluoride concentrations and %SHC values. The significance level was set at 5%. All statistical analyses were carried out using Jamovi software (THE JAMOMI PROJECT, 2021; version 1.6). Results In respect to the microbial composition, no effect of any treatment was observed for CFU counts of total microorganisms, mutans streptococci and total aciduric microorganisms (Fig. 2 ). Both fluoridated solutions (450 and 1,350 ppmF) significantly reduced dentin demineralization compared to the control group (p < 0.03)(Fig. 3 a). Biofilms exposed to 1,350 ppmF presented higher fluoride concentration than those exposed to 450 ppmF or to control group (p ≤ 0.02) (Fig. 3 b). Linear regression analysis showed that the exposure to 450 ppmF and to 1,350 ppmF significantly reduced the %SHC by 25.1% (p = 0.013) and by 27.1% (p = 0.006), respectively, compared to the control solution (Table 1 ). Table 1 Linear regression analysis for the effect of fluoride concentrations in %SHC Predictor Effect 95% Confidence Interval p-value Lower Upper Intercept ᵃ 64.10 50.9 77.3 < 0.001 450 ppm F -25.1 -44.4 -5.89 0.013 1350 ppm F -27.10 -45.8 -8.44 0.006 a reference: 0 ppmF. R = 0.57 Discussion In vitro biofilm models have been used to investigate pathogenic mechanisms involved in the carious lesion development and progression, as well as to assess the effects of anticariogenic strategies [ 24 ]. Despite their widespread application, relatively few models have been validated regarding their response to fluoride in carious lesion development [ 25 ]. In this study, the MOCS, a dynamic microcosm biofilm model, presented an appropriate fluoride remineralization response. Therefore, the hypotheses proposed in this study were accepted, and the results support the suitability of the MOCS as a validated preclinical model for investigating root caries lesions and anticariogenic properties. The %SHC data showed that both 450 ppmF and 1,350 ppmF treatments significantly reduced demineralization compared with the control group, with a greater reduction observed at 1,350 ppmF. Linear regression analysis confirmed that fluoride solutions decreased SHC by 25.1% and 27.1% in the 450 ppmF and 1,350 ppmF groups, respectively. These findings demonstrate the fluoride effect in reducing demineralization, and thus validate the response ofthe lesions induced in the MOCS to fluoride concentrations. The microbiological analysis revealed no significant differences in the counts of total microorganisms, mutans streptococci, or total acidurics among the treatment groups. While fluoride is widely recognized for its effects on de- and remineralization, its antimicrobial properties remain less consistent and not fully understood. Most studies examining fluoride’s antimicrobial effects have focused on individual species rather than complex microbial communities, with limited data available on fluoride’s broader impact on the oral microbiome [ 25 , 26 ]. Koopman et al. (2015) [ 27 ], for example, found that fluoride mouthwash (100ppm - AmF and 150ppm - SnF2) used in adolescents induced only minor and transient changes in the microbiota. Similarly, Rosin-Grget et al. (2013) [ 28 ] observed minimal effects of fluoride mouthwashes on oral microbiota composition. These findings are consistent with the present study, which also found no significant microbiological shifts in a microcosm biofilm model following fluoride exposure. These findings reinforce that fluoride's primary anticaries mechanism is physicochemical—reducing demineralization and enhancing remineralization—rather than antimicrobial effect [ 29 , 30 ]. Regarding biofil fluoride retention, statistically significant differences were observed among the groups. Biofilms formed under 1,350 ppmF treatment exhibited higher fluoride concentrations than those exposed to 450 ppmF or no fluoride (0 ppmF), demonstrating greater fluoride uptake with increased treatment concentration. These findings align with previous results where higher fluoride exposure enhances biofilm fluoride content and contributes to reduced demineralization [ 31 , 32 ]. However, no significant difference in fluoride concentration was found between the control and 450 ppmF groups, which may be attributed to the use of a saliva-derived inoculum from a donor with prior fluoride exposure through toothbrushing with fluoridated dentifrice and drinking of fluoridated water (0.7 ppmF), potentially influencing baseline fluoride levels. It is also important to consider the behavior of fluoride in contact with dentin substrate. It is known that dentin is more reactive to fluoride than enamel [ 33 ]. At lower concentrations, fluoride may be quickly incorporated into the dentin surface, reducing the amount available to accumulate in the biofilm. In contrast, at higher concentrations, there may be sufficient fluoride to both react with the dentin and be retained in the biofilm, which could explain the observed differences in biofilm fluoride concentrations and similar mineral protection. Additionally, it is important to note that no control for biofilm thickness was performed in this study, which limits the interpretation of fluoride penetration and its action in deeper biofilm layers. Future research should address this limitation and further refine the model to better evaluate subtle differences in fluoride effect, including potential dose-response behaviors. Conclusion In conclusion, the MOCS, as a dynamic biofilm model, was able to produce dentin root carious lesions whose demineralization was reduced by fluoride, indicating its potential as validated pre-clinical model for testing anticariogenic agents. Declarations Statement of Ethics This study was approved by the Research Committee of the School of Dentistry and the Ethics Committee on Human Research at the Federal University of Rio Grande do Sul (UFRGS) under CAAE number 69918823.6.0000.5347. Written informed consent was obtained from the volunteer who provided the saliva sample. Conflict of Interest Statement The authors declare a conflict of interest with Odeme Dental Research. The system was received by UFRGS (T.T.M., R.A.A., and L.N.H.) to be assembled and tested as a laboratory cavity simulator. Generative Artificial Intelligence (AI) GPT-4.0 mini was used to improve grammar and fluency in the text originally generated by the authors. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the publication. Declarations Competing Interests The authors declare a conflict of interest with Odeme Dental Research. The system was received by UFRGS (T.T.M., R.A.A., and L.N.H.) to be assembled and tested as a laboratory cavity simulator. Funding Sources This study was supported by Brazilian research agency FAPERGS (Foundation for the Support of Research in the State of Rio Grande do Sul; application ARD 10/2020). Author Contribution T.T.M., and R.A.A. conceived and designed the study. G.S, G.A.M, B.MK, L.M.C carried out the study and and G.S and L.NH collected the data. T.T.M and G.S analysed the data and written the draft of the paper. All authors reviewed and approved the final manuscript. Acknowledgement The authors thank the technicians Luiza Weber Mercado and Douglas Fernando Paixão for their support during the laboratory procedures at LABIM (Laboratory of Biochemical and Microbiology of Dentistry School - UFRGS). Their assistance was important for the successful execution of this study. Data Availability All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author. References Machiulskiene V, Campus G, Carvalho JC, Dige I, Ekstrand KR, Jablonski-Momeni A et al (2020) Terminology of dental caries and dental caries management: consensus report of a workshop organized by ORCA and Cariology Research Group of IADR. Caries Res 54(1):7–14 Selwitz RH, Ismail AI, Pitts NB (2007) Dental caries. Lancet. ;369(9555):51–9. Available from: https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(07)60031-2/abstract Takahashi N, Nyvad B (2016) Ecological hypothesis of dentin and root caries. 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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-8484242","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":570112821,"identity":"0b8d9635-b1a6-4ff8-bdb1-2627239364c5","order_by":0,"name":"Giovanna Santos Medeiros Sagardia","email":"data:image/png;base64,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","orcid":"","institution":"Federal University of Rio Grande do Sul","correspondingAuthor":true,"prefix":"","firstName":"Giovanna","middleName":"Santos Medeiros","lastName":"Sagardia","suffix":""},{"id":570112823,"identity":"6bbaddb1-d748-4c0c-ae73-6b731cadb76e","order_by":1,"name":"Glenda Ávila Marques","email":"","orcid":"","institution":"Federal University of Rio Grande do 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Sul","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"Naomi","lastName":"Hashizume","suffix":""},{"id":570112827,"identity":"3b62cdb5-147f-4192-a265-447b5cec63d5","order_by":5,"name":"Rodrigo Alex Arthur","email":"","orcid":"","institution":"Federal University of Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"Alex","lastName":"Arthur","suffix":""},{"id":570112828,"identity":"88b4970e-86e1-4212-b4ae-245bf1441da8","order_by":6,"name":"Tamires Timm Maske","email":"","orcid":"","institution":"Federal University of Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Tamires","middleName":"Timm","lastName":"Maske","suffix":""}],"badges":[],"createdAt":"2025-12-30 21:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8484242/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8484242/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99705230,"identity":"d27f85b1-32a0-4ebf-bb0e-7e4ccd091213","added_by":"auto","created_at":"2026-01-07 12:29:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":275409,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptMOCS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/2bcc58649458f4e076e12438.docx"},{"id":99705225,"identity":"2ac1c45d-add9-4e62-a384-0149209eaf07","added_by":"auto","created_at":"2026-01-07 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12:29:02","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32452,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/10f6eb1bbae31aea6b4706f2.png"},{"id":99705232,"identity":"bda81d1b-14a2-4039-a306-e86a12f56bce","added_by":"auto","created_at":"2026-01-07 12:29:02","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9919,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/c6f6150f4f0ab6546c0796d4.png"},{"id":99796736,"identity":"1332ecbc-62e4-45d2-9463-10c92dc3dca9","added_by":"auto","created_at":"2026-01-08 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12:29:03","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89100,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/481e7a8ba7ffb489465576c9.html"},{"id":99797507,"identity":"0ec95f0d-7d9c-4917-a146-1406489fb318","added_by":"auto","created_at":"2026-01-08 13:45:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":153297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultifunctional Oral Cavity Simulator (MOCS).\u003c/strong\u003e Cylindrical chambers containing specimen holders are positioned over a heating unit. Computer-controlled peristaltic pumps regulate the flow of artificial saliva and sucrose solution. Silicone tubes connect the reservoirs to the chambers, allowing the solutions to flow over the specimens before being directed to waste.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/d75bd4b57d145fe14a04ee11.png"},{"id":99705227,"identity":"cf7011e4-f016-4628-93d9-b9faa176a3c9","added_by":"auto","created_at":"2026-01-07 12:29:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35851,"visible":true,"origin":"","legend":"\u003cp\u003eCFU (log)/mg counts (means ±sd) for total microorganisms (A), mutans streptococci (B) and total aciduric microorganisms (C), according to different treatments. Bars followed by similar letters are not statistically different among the groups evaluated (p \u0026gt; 0.05, Tukey).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/a45871a754bf862db5119fec.png"},{"id":99797400,"identity":"8a80c6a7-f95d-44f4-a666-e2b7bf55782c","added_by":"auto","created_at":"2026-01-08 13:45:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34853,"visible":true,"origin":"","legend":"\u003cp\u003e%SHC (A) and biofilm fluoride concentrations (B) (µg/mg wet weight) (means ± sd) according to different treatments. Bars followed by different letters are statistically different (p \u0026lt; 0.05, Tukey). Bars represent means and horizontal lines represent standard deviation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/05ac50b80acb9126069d818a.png"},{"id":101511726,"identity":"741bfdbb-9099-4826-a7a2-696920b82493","added_by":"auto","created_at":"2026-01-30 15:14:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":872460,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8484242/v1/7b5581dc-dee8-46e6-80c4-99cdf71f07e0.pdf"}],"financialInterests":"Competing interest reported. The authors declare a conflict of interest with Odeme Dental Research. The system was received by UFRGS (T.T.M., R.A.A., and L.N.H.) to be assembled and tested as a laboratory cavity simulator.","formattedTitle":"Fluoride-Based Validation of a Dynamic Microcosm Biofilm Model for Root Caries-Like Lesions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDental caries is a biofilm-sugar-dependent disease that results in mineral loss due to dietary-induced pH fluctuations over the biofilm-tooth interface over the life course [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Carious lesions may develop on any dental surface, including the root ones which are exposed to the oral environment due to gingival recession, periodontal disease, surgery and other factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Due to increasing life expectancy and improved oral health, more adults and elderly individuals are retaining their natural teeth longer, which leads to root exposure posing them to an increased risk for caries development. A recent review on the global burden of untreated caries found that both the prevalence and incidence of root carious lesion increase significantly after the age of 40, affecting 35\u0026ndash;40% of older adults [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], emphasizing the need for targeted prevention strategies, as well as the development therapies to manage root caries lesion in adults and aging populations.\u003c/p\u003e \u003cp\u003eFluoride remains the most widely recognized preventive and therapeutic agent for managing dental caries, including root caries [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Its anti-caries and cariostatic effects are frequency- [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and concentration-dependent [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. An increase in fluoride levels in saliva and in the solid and fluid phases of the biofilm [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] is observed under the use of fluoride-containing products, being this one of the most important contributions of fluoride to reduce demineralization and to activate the remineralization of dental hard tissues. Therefore, it is expected that fluoride exhibits a dose effect in reducing dentin demineralization, especially considering the higher susceptibility of this tissue to the mineral loss [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLaboratory biofilm models for carious lesion development have been employed to test hypotheses prior to clinical investigations [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Among them, dynamic microcosm biofilm models are particularly relevant, as they simulate key features of the oral environment, including microbial diversity, pH fluctuations, temperature, salivary flow, and intermittent exposure to dietary carbohydrates [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To be considered suitable for assessing preventive or therapeutic approaches, validation tests are required to confirm their responsiveness to treatment effects and their translational relevance to clinical condition [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, the Multifunctional Oral Cavity Simulator (MOCS) was established as a dynamic microcosm model able to develop root dentin carious-like lesions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and its microbial composition has already been characterized [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, its responsiveness to anticariogenic agents, such as fluoride, has not yet been evaluated. Therefore, this study aimed to assess the microbial composition, biofilm fluoride concentration and carious lesion development by the MOCS when microcosm biofilms were exposed to distinct fluoride concentrations ([F]). The tested hypothesis was that the inhibition of dentin carious lesions development as well as [F] in biofilms are directly dependent on the exposure to fluoride.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThis study was approved by the Ethics Committee on Human Research at the Federal University of Rio Grande do Sul (UFRGS) under CAAE number 69918823.6.0000.5347. This was an in vitro study using a multifunctional oral cavity simulator (MOCS) as a dynamic microcosm biofilm model. Microcosm biofilms were formed from human saliva (caries-free donor) on bovine root dentin discs for 4 days. The MOCS provided continuous flow regimes of artificial saliva (0.06 ml/min) and intermittent flow of 5% sucrose (0.25 ml/min, three times a day for six minutes). The formed biofilms were treated twice a day with different fluoride solutions (NaF: 0 ppmF - control, 450, and 1,350 ppmF; n\u0026thinsp;=\u0026thinsp;9 per group). The response variables were: the percentage of surface hardness change (%SHC), microbiological composition of the biofilm through colony-forming unit counts (CFU/mg biofilm) and the biofilm fluoride concentrations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample size calculation\u003c/h3\u003e\n\u003cp\u003eThe sample size was calculated based on the %SHC data obtained from the dynamic biofilm model previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The calculation considered the difference in %SHC observed around restorations made with an anticariogenic material containing S-PRG particles and a non-cariogenic control material without S-PRG particles. The mean difference and standard deviation were estimated as 33.1\u0026plusmn; 20.5. The Statulator (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://statulator.com/SampleSize/ss2M.html\u003c/span\u003e\u003cspan address=\"https://statulator.com/SampleSize/ss2M.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) online statistical calculator- was used, assuming a power of 80% and a significance level of 5% [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Considering an expected sample loss of 20%, the final sample size was set at nine specimens per group (n\u0026thinsp;=\u0026thinsp;9).\u003c/p\u003e\n\u003ch3\u003eRoot dentin preparation\u003c/h3\u003e\n\u003cp\u003eTwenty-seven bovine incisors free of defects were selected. Bovine root dentin discs (6mm in diameter x 2mm in thickness) were prepared using a bench drill and a trephine bur. The pulpal wall was smoothed with #80 grit sandpaper, and the buccal surface was polished with #600, #1200, and #2000 grit sandpaper to remove surface irregularities. All procedures were performed under water cooling. After preparation, the basal and lateral walls, as well as one-third of the surface of the discs were covered with acid-resistant cosmetic nail polish. The baseline surface hardness (SH) of each disc was evaluated through three indentations, each spaced 100 \u0026micro;m apart, made at the center of the dentin disc using a Knoop microhardness indenter with a 25g load applied for 5 seconds (Hardness Tester, HMV 2, Shimadzu, Tokyo, Japan). The values of the three indentations were averaged within each disc. Discs with overall average baseline SH of 45.65\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26 KHN were selected and randomly distributed into individual support units, totaling 27 samples (n\u0026thinsp;=\u0026thinsp;9/group). The supports units containing the specimens were sterilized with ethylene oxide (MIC Sterilization services LTDA).\u003c/p\u003e\n\u003ch3\u003eBiofilm model\u003c/h3\u003e\n\u003cp\u003eThe MOCS (Odeme Dental Research, Luzerna-SC, Brazil) consist of a system with three cylindrical chambers on a heating base (37\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C), featuring three individual inlets connected to silicone tubes to allow liquid`s flow through the system (artificial saliva, sucrose solution and treatments if needed). Support units are located under each group of inlets. The device is gas supplemented (10% CO\u003csub\u003e2\u003c/sub\u003e, 10% H\u003csub\u003e2\u003c/sub\u003e, and 80% N\u003csub\u003e2\u003c/sub\u003e). The silicone tubes are originated from artificial saliva and sucrose reservoirs and passed through an infusion system composed of two 10-channel computer-controlled peristaltic pumps (FC 100, Chao Chuing, China), and reached to the specific inlets. The over-flow is directed to a waste reservoir located at the bottom of the chambers (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The experimental setup and methodological details have been previously described in [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eInoculation and biofilm growth\u003c/h3\u003e\n\u003cp\u003eSaliva (2.5 mL) was collected from a caries-free donor (female, 36 yearsold) who exhibited normal salivary flow (stimulated\u0026thinsp;\u0026gt;\u0026thinsp;1 mL/min), was systemically healthy, and showed no clinical signs of periodontal disease, and who had not used antimicrobial agents in the past 3 months. Saliva collection was performed under mechanical stimuli (paraffin chewing for 10 minutes; Parafilm \"M\", American National Can TM, Chicago, Ill., USA). The collected saliva was vortexed and added to 50 ml of sterile DMM (Defined Medium Mucin) artificial saliva to form the microbial inoculum solution. Three mL of this mixture was perfused over the root dentin specimens and remained on the top of dentin discs for 1 hour to allow microbial adhesion. After this period, the DMM was perfused at a rate of 0.06 ml/min. The cariogenic challenge was simulated with an intermittent flow of 5% sucrose for 6 minutes, applied 3 times a day with 2-hour intervals, at a flow rate of 0.25 ml/min. The biofilms were formed under anaerobic conditions (80% N\u003csub\u003e2\u003c/sub\u003e, 10% CO\u003csub\u003e2\u003c/sub\u003e, and 10% H\u003csub\u003e2\u003c/sub\u003e), provided by a gas flow at 0.006 N/mm\u0026sup2;, applied twice a day for 1 minute during the 4 days of experiment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm treatment\u003c/h2\u003e \u003cp\u003eTwenty-four hours after the initial microbial adhesion, the biofilms were treated twice daily (every 12 hours) for 1 minute with 5 mL of NaF solutions at concentrations of 0, 450, and 1,350 ppmF. These solutions were dripped over the biofilms by a syringe inserted into an extra-inlet located above each support units of the cylindrical chambers.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collections and analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial analysis\u003c/h2\u003e \u003cp\u003eAfter 4 days of growth, the biofilms formed over the dentin discs were collected. This procedure was done using a sterile loop positioned at the center of each disc and inserted into the biofilm until it reached to the dentin disc surface. The biofilm attached to the loop and to its surrounding was collected and added to pre-weighted microtube for microbial analysis. The remained biofilm was also collected and added to another pre-weighted microtubes for biochemical analysis. The wet weight of biofilms was determined. For microbial analysis, the biofilms were resuspended in one ml of sterile saline solution and the suspension was vortexed, sonicated (20W, 30 s), serially diluted (10\u003csup\u003e0\u003c/sup\u003e-10⁶), and plated in duplicate (20 \u0026micro;L) in Brain Heart Infusion Agar (BHI), in BHI with pH adjusted to 4.8 and in Mitis Salivarius Agar (MSB) (supplemented with 0.2 U of bacitracin per mL) for the cultivation of total microorganisms, total aciduric microorganisms and mutans streptococci, respectively. The agar plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC in anaerobiosis jars for 4 days. Subsequently, the agar plates were observed under a stereoscope and the colony-forming units (CFU) were counted. Results were expressed as CFU (log)/mg of wet biofilm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFluoride bound to biofilm analysis\u003c/h2\u003e \u003cp\u003eBiofilm samples were dried for 24 hours in a vacuum desiccator with P₂O₅ and the dry weight was determined. The dried biofilms were resuspended in 100 \u0026micro;L of HCl (per mg/dry-weight). After 3 hours under agitation, an equal volume of TISAB II NaOH was added. The samples were then centrifuged for 10 minutes at 14,000 rpm, and the supernatant was carefully removed and frozen for further analysis. All samples were analyzed using a fluoride ion-specific electrode (Orion Research Inc., Beverly, USA), previously calibrated with standard solutions of known fluoride concentration (from 0.1 ppmF to 16 ppmF) (Orion Research Inc., Beverly, USA), connected to an ion analyzer (Procyon, S\u0026atilde;o Paulo, Brazil). The sample readings were recorded in millivolts (mV) and converted using linear regression from the calibration curve into fluoride concentrations expressed as \u0026micro;mol/L and \u0026micro;g/mg [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDentin carious lesion assessment\u003c/h2\u003e \u003cp\u003eDentin discs were cleaned with distilled water and brushed with a soft-bristle toothbrush. The SH was again assessed by placing three indentations at the center of each disc, spaced 100 \u0026micro;m apart from each other and from the baseline indentations. The values of the three indentations were averaged within each disc. Root dentin demineralization was expressed as the percentage of surface hardness change (%SHC) which was calculated using following formula: (SH baseline \u0026ndash; SH post treatment) / (SH baseline) x 100 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe assumptions of equality of variances and normal distribution of errors were checked for each response variable. If the variable did not fulfill these assumptions, data transformations were applied [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Fluoride concentration data were rank transformed, and One-way ANOVA followed by Tukey's post-hoc test was used for all the outcomes. Additionally, a linear regression was employed to evaluate the relationship between fluoride concentrations and %SHC values. The significance level was set at 5%. All statistical analyses were carried out using Jamovi software (THE JAMOMI PROJECT, 2021; version 1.6).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn respect to the microbial composition, no effect of any treatment was observed for CFU counts of total microorganisms, mutans streptococci and total aciduric microorganisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Both fluoridated solutions (450 and 1,350 ppmF) significantly reduced dentin demineralization compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.03)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Biofilms exposed to 1,350 ppmF presented higher fluoride concentration than those exposed to 450 ppmF or to control group (p\u0026thinsp;\u0026le;\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLinear regression analysis showed that the exposure to 450 ppmF and to 1,350 ppmF significantly reduced the %SHC by 25.1% (p\u0026thinsp;=\u0026thinsp;0.013) and by 27.1% (p\u0026thinsp;=\u0026thinsp;0.006), respectively, compared to the control solution (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLinear regression analysis for the effect of fluoride concentrations in %SHC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePredictor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEffect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e95% Confidence Interval\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLower\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUpper\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntercept \u003csup\u003eᵃ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e77.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e450 ppm F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-25.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-44.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1350 ppm F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-27.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-45.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-8.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003e reference: 0 ppmF. R\u0026thinsp;=\u0026thinsp;0.57\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn vitro biofilm models have been used to investigate pathogenic mechanisms involved in the carious lesion development and progression, as well as to assess the effects of anticariogenic strategies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Despite their widespread application, relatively few models have been validated regarding their response to fluoride in carious lesion development [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, the MOCS, a dynamic microcosm biofilm model, presented an appropriate fluoride remineralization response. Therefore, the hypotheses proposed in this study were accepted, and the results support the suitability of the MOCS as a validated preclinical model for investigating root caries lesions and anticariogenic properties.\u003c/p\u003e \u003cp\u003eThe %SHC data showed that both 450 ppmF and 1,350 ppmF treatments significantly reduced demineralization compared with the control group, with a greater reduction observed at 1,350 ppmF. Linear regression analysis confirmed that fluoride solutions decreased SHC by 25.1% and 27.1% in the 450 ppmF and 1,350 ppmF groups, respectively. These findings demonstrate the fluoride effect in reducing demineralization, and thus validate the response ofthe lesions induced in the MOCS to fluoride concentrations.\u003c/p\u003e \u003cp\u003eThe microbiological analysis revealed no significant differences in the counts of total microorganisms, mutans streptococci, or total acidurics among the treatment groups. While fluoride is widely recognized for its effects on de- and remineralization, its antimicrobial properties remain less consistent and not fully understood. Most studies examining fluoride\u0026rsquo;s antimicrobial effects have focused on individual species rather than complex microbial communities, with limited data available on fluoride\u0026rsquo;s broader impact on the oral microbiome [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Koopman et al. (2015) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], for example, found that fluoride mouthwash (100ppm - AmF and 150ppm - SnF2) used in adolescents induced only minor and transient changes in the microbiota. Similarly, Rosin-Grget et al. (2013) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] observed minimal effects of fluoride mouthwashes on oral microbiota composition. These findings are consistent with the present study, which also found no significant microbiological shifts in a microcosm biofilm model following fluoride exposure. These findings reinforce that fluoride's primary anticaries mechanism is physicochemical\u0026mdash;reducing demineralization and enhancing remineralization\u0026mdash;rather than antimicrobial effect [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding biofil fluoride retention, statistically significant differences were observed among the groups. Biofilms formed under 1,350 ppmF treatment exhibited higher fluoride concentrations than those exposed to 450 ppmF or no fluoride (0 ppmF), demonstrating greater fluoride uptake with increased treatment concentration. These findings align with previous results where higher fluoride exposure enhances biofilm fluoride content and contributes to reduced demineralization [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, no significant difference in fluoride concentration was found between the control and 450 ppmF groups, which may be attributed to the use of a saliva-derived inoculum from a donor with prior fluoride exposure through toothbrushing with fluoridated dentifrice and drinking of fluoridated water (0.7 ppmF), potentially influencing baseline fluoride levels.\u003c/p\u003e \u003cp\u003eIt is also important to consider the behavior of fluoride in contact with dentin substrate. It is known that dentin is more reactive to fluoride than enamel [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. At lower concentrations, fluoride may be quickly incorporated into the dentin surface, reducing the amount available to accumulate in the biofilm. In contrast, at higher concentrations, there may be sufficient fluoride to both react with the dentin and be retained in the biofilm, which could explain the observed differences in biofilm fluoride concentrations and similar mineral protection.\u003c/p\u003e \u003cp\u003eAdditionally, it is important to note that no control for biofilm thickness was performed in this study, which limits the interpretation of fluoride penetration and its action in deeper biofilm layers. Future research should address this limitation and further refine the model to better evaluate subtle differences in fluoride effect, including potential dose-response behaviors.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the MOCS, as a dynamic biofilm model, was able to produce dentin root carious lesions whose demineralization was reduced by fluoride, indicating its potential as validated pre-clinical model for testing anticariogenic agents.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStatement of Ethics\u003c/p\u003e \u003cp\u003eThis study was approved by the Research Committee of the School of Dentistry and the Ethics Committee on Human Research at the Federal University of Rio Grande do Sul (UFRGS) under CAAE number 69918823.6.0000.5347. Written informed consent was obtained from the volunteer who provided the saliva sample.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors declare a conflict of interest with Odeme Dental Research. The system was received by UFRGS (T.T.M., R.A.A., and L.N.H.) to be assembled and tested as a laboratory cavity simulator.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eGenerative Artificial Intelligence (AI)\u003c/h2\u003e \u003cp\u003eGPT-4.0 mini was used to improve grammar and fluency in the text originally generated by the authors. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the publication.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThe authors declare a conflict of interest with Odeme Dental Research. The system was received by UFRGS (T.T.M., R.A.A., and L.N.H.) to be assembled and tested as a laboratory cavity simulator.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding Sources\u003c/h2\u003e \u003cp\u003eThis study was supported by Brazilian research agency FAPERGS (Foundation for the Support of Research in the State of Rio Grande do Sul; application ARD 10/2020).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.T.M., and R.A.A. conceived and designed the study. G.S, G.A.M, B.MK, L.M.C carried out the study and and G.S and L.NH collected the data. T.T.M and G.S analysed the data and written the draft of the paper. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the technicians Luiza Weber Mercado and Douglas Fernando Paix\u0026atilde;o for their support during the laboratory procedures at LABIM (Laboratory of Biochemical and Microbiology of Dentistry School - UFRGS). Their assistance was important for the successful execution of this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMachiulskiene V, Campus G, Carvalho JC, Dige I, Ekstrand KR, Jablonski-Momeni A et al (2020) Terminology of dental caries and dental caries management: consensus report of a workshop organized by ORCA and Cariology Research Group of IADR. Caries Res 54(1):7\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelwitz RH, Ismail AI, Pitts NB (2007) Dental caries. \u003cem\u003eLancet.\u003c/em\u003e ;369(9555):51\u0026ndash;9. 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Eur J Oral Sci 103(6):362\u0026ndash;367\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Biofilm. Fluorides. Demineralization. Microcosm. Root caries","lastPublishedDoi":"10.21203/rs.3.rs-8484242/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8484242/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effect of distinct fluoride concentrations ([F]) on root dentin carious lesions produced by the MOCS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrocosm biofilms were grown for 4 days on bovine root dentin discs from saliva of caries-free donor, under intermittent sucrose flow (5%, 0.25 ml/min, 6 min, 3×/day), and treated with NaF solutions at 0 (control), 450, or 1,350 ppmF, twice daily. Response variables included microbial composition (CFU/mg biofilm), biofilm fluoride concentration (µgF/mg biofilm), and dentin demineralization (% surface hardness change; %SHC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluoride treatments at 450 ppmF and 1,350 ppmF significantly reduced %SHC compared to 0 ppmF (p ≤ 0.035, Tukey). The %SHC was 64.1 in the control group, and 25.1 and 27.1 in biofilms exposed to 450 ppmF and 1,350 ppmF, respectively (linear regression, R = 0.57, p \u0026lt; 0.013). No significant differences were found in microbial composition in respect to total microorganisms, total aciduric microorganisms and mutans streptococci counts among the groups (p \u0026gt; 0.05). Biofilms exposed to 1,350 ppmF present [F] statistically higher than control group (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MOCS demonstrated an appropriate fluoride remineralization response, supporting its validity as a pre-clinical model for testing anticariogenic agents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Relevance\u003c/strong\u003e: This study contributes to scientific advancement in cariology by validating the MOCS as a suitable model for studies aiming at preventing and controlling root caries.\u003c/p\u003e","manuscriptTitle":"Fluoride-Based Validation of a Dynamic Microcosm Biofilm Model for Root Caries-Like Lesions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-07 12:28:57","doi":"10.21203/rs.3.rs-8484242/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":"b16db8ee-4334-4b00-9370-26938d255555","owner":[],"postedDate":"January 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-30T15:13:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-07 12:28:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8484242","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8484242","identity":"rs-8484242","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}