Effects of Calcium-Based Solutions on Enamel Remineralization and Cariogenicity: Evidence from In Vitro and Clinical Studies

Effects of Calcium-Based Solutions on Enamel Remineralization and Cariogenicity: Evidence from In Vitro and Clinical Studies | 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 Effects of Calcium-Based Solutions on Enamel Remineralization and Cariogenicity: Evidence from In Vitro and Clinical Studies Zhi-Yun Lin, Earl Fu, Chung-Hsing Li, Ting-Han Chang, Da-Yo Yuh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8513583/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background. Calcium has been proposed as an alternative for promoting remineralization of dental caries. This study evaluated the effects of calcium-based solutions on tooth cariogenicity through an in vitro experiment and a randomized clinical trial. Methods. Teeth with carious lesions were rinsed in vitro with water, artificial saliva, or 0.3% calcium solution. Using scanning electron microscopy, micro-computed tomography, and quantitative light-induced fluorescence (QLF), the mineralization volumes (MVs) and the relative QLF-covered areas (QLF-areas) were assessed. In the clinical trial, fifteen children were randomly assigned to placebo, 0.3% calcium (formula 1), or 0.3% calcium plus 225 ppm fluoride (formula 2) oral sprays for two months. Plaque bacterial composition was analyzed using 16S rRNA gene sequencing, salivary calcium levels using enzyme-linked immunosorbent assay, and the cariogenicity area by QLF. Results. In vitro, calcium and artificial saliva rinses increased MV and reduced QLF-areas, with a significant negative correlation between their changes. Clinically, salivary calcium increased significantly with formulas 1 and 2 but not placebo. Both calcium sprays showed significant reductions in QLF-areas, while the placebo did not. Conclusions. Combined with in vitro (MVs increasing but QLF areas reducing after calcium rinsing) and clinical findings (salivary calcium level elevations but QLF-area reductions after using calcium sprays) indicate enhanced enamel mineralization but reduced biofilm cariogenicity by calcium supplementation. Clinical significance. Calcium-containing solutions may aid in the management of early enamel demineralization. Clinical Trials Identifier NCT07269730, retrospectively registered at 2025/11/25. URL: https://register.clinicaltrials.gov. Dental Caries tooth remineralization calcium compounds dental plaque randomized controlled trial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 One sentence summary Calcium supplementation may reduce tooth cariogenicity and enhance enamel remineralization based on our findings of in vitro and clinical cariogenicity area reductions, as well as increased mineralization volume in vitro and salivary calcium elevations in the clinical trial. Introduction Dental caries is the most prevalent dental disease worldwide, primarily caused by cariogenic bacteria such as Streptococcus mutans [ 1 ]. These bacteria metabolize dietary sugars into acids that demineralize tooth enamel. Thus, theoretically, caries lesions develop due to an imbalance between demineralization and remineralization processes [ 2 ]. Plaque accumulation initiates enamel demineralization [ 3 ], forming porous white spot lesions with an opaque appearance. In contrast, remineralization redeposits minerals into enamel, reversing early caries [ 4 , 5 ]. Fluoride, calcium, and phosphate ions enhance remineralization [ 6 , 7 ]; however, concerns regarding fluoride overexposure and its potential toxicity have led to conflicting reports in the literature [ 8 ]. Since calcium and phosphate ions are required by fluoride to promote the natural remineralization process of enamel, calcium phosphates has been applied as biomimetics in preventing dental caries [ 9 – 11 ]. Studies have shown enhanced enamel remineralization with calcium-containing chewing gum [ 12 ], casein phosphopeptide-amorphous calcium phosphate paste [ 13 ], and hydroxyapatite toothpaste [ 14 ]. Moreover, in a randomized clinical trial, combining functionalized tricalcium phosphate with fluoride improved caries arrest in preschool children compared to fluoride varnish alone [ 15 ]. As maintaining oral hygiene in young children remains difficult, chemical control methods such as mouthwashes or sprays may offer a more convenient alternative [ 16 ]. This study evaluated the effects of calcium solutions on mineralization volume and cariogenic areas in vitro , and of calcium sprays on cariogenic areas in a randomized clinical trial. Additionally, bacterial composition in plaques and salivary calcium levels in children were compared. Materials and methods In Vitro Experiment Sample Processing and Grouping Twenty human teeth with caries lesions were obtained with approval from the Tri-Service General Hospital Institutional Review Board (TSGHIRB No: A202105108). After cleaning with deionized water and drying, the teeth were randomly divided into four groups (n = 5/group): ultrapure water (control), artificial saliva (AS), calcium solution, and AS plus calcium solution. Artificial saliva was prepared following a previous study,[ 17 ] consisting of 0.1029 g CaCl₂·2H₂O, 0.04066 g MgCl₂, 0.544 g KH₂PO₄, 4.766 g HEPES buffer acid form, and 2.2365 g KCl in 1000 mL distilled water (pH 7). The calcium solution, commercially available (Kids Oral Spray, Toothfilm Inc., Taipei, Taiwan), contained 0.3% calcium lactate, 0.01% dicalcium phosphate, and 0.01% mesoporous bioactive glass. In Vitro Experimental Procedures Caries lesions on the tooth surfaces were identified using a scanning electron microscope (SEM) and then examined three-dimensionally via micro-computed tomography (µ-CT) in sagittal, coronal, and horizontal views. Additionally, bacterial plaque on the tooth surfaces was captured using quantitative light-induced fluorescence (QLF). These analyses allowed for the quantification of mineralization volumes and cariogenicity areas before and after treatment with the four solutions. Quantitative Light-Induced Fluorescence (QLF) Bacterial plaque on tooth surfaces was visualized using QLF (Metal Industries Research & Development Centre, Kaohsiung, Taiwan). Three types of images were captured: (1) an original photograph, (2) a binary bacterial plaque image, and (3) a fluorescent bacterial plaque image. Through the images, the relative cariogenicity area was presented as the red fluorescent plaque (%, percentage of QLF-observed red patch in total area, Fig. 2 C), while the difference in the areas before and after treatment was recorded as ΔQLF-area [ 18 ]. Scanning Electron Microscopy and Micro-Computerized Tomography Caries lesions were identified using SEM (Apreo2S, Thermo Fisher Scientific, Waltham, USA) at 130X magnification and 5.0 kV, as well as µ-CT (Quantum FX, Perkin Elmer, Waltham, USA). The acquired µ-CT images were processed using Materialize Mimics software to calculate the enamel mineralization volume (MV, mm 3 ). Boolean operations were applied to calculate the volume change before and after treatment, recorded as the Δ mineralization volume (ΔMV) [ 19 ]. Randomized Clinical Trials Experimental Design and Patient Selection Randomized clinical trial (RCT) was double-blindly conducted with 15 children presenting early carious lesions (Fig. 1 ). (CK 3, 8) Participants were recruited between January 2023 and December 2023. Inclusion criteria required participants to have an ICDAS code ≥ 1 and ≤ 3 as determined by a clinician (Dr. CHL). The mean age of the participants was 6.26 ± 3.0 years. The random allocation sequence was generated by an independent researcher using a computer-generated random number table. Participants were randomly assigned to one of three groups: placebo (control) and two formulations of calcium spray. Eligibility was determined by the enrolling clinician, whereas group assignment was conducted by an independent researcher using sequentially numbered, opaque, sealed envelopes to ensure allocation concealment. Participants, caregivers, and outcome assessors were blinded to group allocation. All sprays were packaged in identical bottles with standardized appearance and labeling, and were coded by an independent researcher (Ms. ZYL) to maintain blinding until data analysis was completed. Formulation 1 contained 0.3% calcium lactate, while Formulation 2 contained 0.3% calcium lactate plus 225 ppm fluoride (0.05% NaF). Differences before and after treatment were analyzed. Plaque and saliva samples were collected from all participants. All procedures were conducted following ethical guidelines and regulations, with informed consent obtained from all participants (TSGHIRB No: A202305084 and NCT07269730). No important changes to the trial design, eligibility criteria, interventions, outcomes, or statistical analysis plan were made after the trial commenced. Experimental Procedures At the initial clinic visit, plaque and saliva samples were collected. Participants were instructed on product use, and follow-up visits ensured compliance. Participants used the assigned spray twice daily under caregiver supervision. Adherence was verbally confirmed at each follow-up visit, with no deviations from the intended intervention reported. Harms, defined as any unexpected oral symptoms such as tooth sensitivity, gingival irritation, or mucosal discomfort, were systematically assessed at each visit through clinician questioning and oral examination. After two months, dental conditions were assessed using QLF, and additional plaque and saliva samples were collected. In the clinical trial, QLF imaging produced three outputs: (1) an original photograph, (2) a binary image highlighting carious lesions, and (3) a fluorescent image. The QLF-area (%) and its change (ΔQLF-area) were measured and compared. 16S rRNA Gene Sequence Analyses To examine bacterial changes in plaque before and after calcium spray use, three out of 15 participants were selected for bacterial sampling (pre- and post-treatment). Plaque samples were collected using a periodontal probe and stored in 1.5 mL tubes containing 500 µL of preservation solution at -80°C. Bacterial DNA was extracted following the manufacturer's instructions using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Cat No. EE161). DNA purity and concentration were verified using a Nanodrop spectrophotometer. The V3 and V4 regions of the 16S rRNA gene were amplified with primers (CCTACGGRRBGCASCAGKVRVGAAT and GGACTACNVGGGTWTCTAATCC) and the MetaVX Library Preparation Kit. PCR products (~ 600 bp) were confirmed via 1% agarose gel electrophoresis. Sequencing reads were processed using QIIME2, with quality filtering, denoising, and operational taxonomic unit (OTU) clustering at a 97% similarity threshold. Taxonomic classification was performed using the Silva 138 reference database and the Ribosomal Database Project (RDP). Salivary Calcium Content Analyses Saliva samples collected during the clinical trial were centrifuged at 3000 rpm for 10 minutes to obtain the clarified supernatant, which was then diluted tenfold for calcium measurement. Calcium concentration was determined colorimetrically using arsenazo III in an ELISA reader. The calcium-arsenazo III complex was measured spectrophotometrically at 652 nm, with absorbance proportional to calcium concentration in the sample [ 20 ]. Statistical Analysis Data aggregation and calculations were performed using Excel 2013. Statistical analyses of MV, QLF-area, and salivary calcium content were conducted using Prism® 9 (v9.5). Paired t-test was used to compare differences before and after treatment, an independent t-test to compare differences between treatment groups, and a linear regression model to assess the correlation between ΔMV and ΔQLF-area. The level of p < 0.05 was selected as statistically significant. All randomized participants were included in the analyses, and no missing data occurred; therefore, no imputation was required. The full trial protocol and statistical analysis plan are available from the corresponding author upon reasonable request, and de-identified participant data and statistical code may also be provided upon request. Results In vitro Experiment Tooth surfaces were examined using SEM, and the changes before and after treatment were clearly observed (Fig. 2 A). The µ-CT was used for quantitative measurement of MV. MV remained unchanged in the water group but significantly increased after treatment with artificial saliva, calcium solution, or their combination (Fig. 2 B). QLF analysis showed reduced cariogenic areas in all groups, including water (Fig. 2 C). Furthermore, △QLF-area was negatively correlated with △MV in the artificial saliva, calcium, and calcium + saliva groups, but not in the water group (Fig. 2 D). Randomized Clinical Trials Bacterial Shifting in Dental Plaque after Calcium-Spray Using 16S rRNA sequencing identified 30 bacterial genera in plaque samples (Fig. 3 ). After two months of calcium spray use, bacterial compositions shifted across all formulations. Streptococcus proportions remained stable in the placebo group (19.41% before vs 21.91% after) but markedly decreased in the calcium spray groups—Formulation 1 (29.99 vs 17.03%) and Formulation 2 (43.52 vs 29.55%). Changes in Saliva Calcium Levels after Calcium-Spray Use Salivary calcium levels significantly increased after treatment in all groups (Fig. 4 A). However, the increase (△calcium) was significantly greater in both calcium spray groups than in the placebo, with no difference between formulations (Fig. 4 B). Reduction in QLF-Identified Plaque after Calcium-Spray Use The clinical photographs in Fig. 5 show QLF results, where red fluorescence on the tooth surfaces in the left column of QLF combinations indicates cariogenic areas. Two-month calcium sprays significantly reduced cariogenic areas (Formulation 1: 5.13 ± 0.6% vs 2.38 ± 0.68%; Formulation 2: 6.81 ± 3.05% vs 3.57 ± 2.15%), while no change was seen in the placebo group (2.82 ± 1.68% vs 4.03 ± 2.47%) (Fig. 5 A–B). The △QLF-areas in both calcium formulations were significantly lower than those in the placebo group (Fig. 5 C). Compared with placebo, formulation 1 reduced ΔQLF-area by − 3.26% (95% CI − 5.20 to − 1.32), and formulation 2 reduced ΔQLF-area by − 3.75% (95% CI − 6.71 to − 0.79). No adverse events were observed in any group throughout the study. Discussion This study assessed the effects of calcium-based oral supplementations on enamel remineralization and biofilm cariogenicity. Early caries detection is essential for prevention. Although radiography remains the diagnostic standard [ 21 ], it is less suitable for children [ 22 ]. Noninvasive tools such as QLF permit early detection by visualizing red fluorescence from bacterial porphyrins, which reflects biofilm acidogenicity [ 23 – 25 ]. µ-CT provides quantitative mineralization data but is limited to in vitro use [ 26 ]. By µ-CT and QLF, our in vitro results showed that a 6-hour rinse with artificial saliva or calcium solutions significantly increased mineralization volume (by µ-CT) but reduced cariogenic area (by QLF), indicating lesion reversal (Fig. 2 ). The negative correlation between △MV and △QLF-area suggests that increased mineralization and reduced biofilm-associated fluorescence occurred in parallel. By contrast, water rinsing reduced QLF-areas (Fig. 2 C) without a detectable increase in mineralization (Fig. 2 B), indicating a limited effect on enamel mineral gain under the present experimental conditions. In the clinical trial, calcium sprays (Formulations 1 and 2) were associated with a reduction in the relative abundance of Streptococcus in plaque samples, whereas minimal changes were observed in the placebo group (Fig. 3 ), suggesting a potential trend toward altered plaque composition. This finding is consistent with studies linking caries progression to oral microbial dysbiosis [ 27 , 28 ]. During caries development, frequent sugar exposure increases acid production, favoring acidogenic and aciduric bacteria such as Streptococcus mutans while suppressing acid-sensitive species [ 29 ]. Although this study did not investigate the mechanisms by which calcium influences plaque composition, prior studies suggest that calcium may contribute to maintaining microbial balance in the oral environment [ 30 ]. In the present trial, salivary calcium levels also increased significantly after using calcium sprays (Fig. 4 ), indicating enhanced buffering and remineralization potential. The elevated salivary calcium observed here may therefore support conditions favorable for enamel remineralization [ 31 , 32 ]. It has been shown that calcium and phosphate combined with fluoride enhance in situ remineralization more effectively than fluoride alone [ 33 ]; the elevated salivary calcium observed here may therefore support enamel repair and slow caries progression [ 34 ]. The QLF images used in this study provided visual and quantitative information related to plaque-associated red fluorescence, which has been associated with biofilm maturity and acidogenic potential [ 35 ]. QLF imaging demonstrated a reduction in red fluorescence–covered areas after two months of calcium spray use, whereas no significant change was observed in the placebo group (Fig. 5 ). However, the details in calcium’s action on reducing the cariogenicity of dental biofilms are likely multifaceted [ 5 , 36 ]. Calcium ions are known to contribute to the formation and stabilization of hydroxyapatite crystals and have been reported to support enamel remineralization in previous studies [ 37 , 38 ]. Additionally, calcium strengthens the salivary pellicle, creating a protective layer against acid attacks [ 39 ]. Taken together, the observed microbial trends, increased salivary calcium levels, and reductions in QLF-identified plaque fluorescence suggest that calcium sprays may contribute to a less cariogenic plaque environment and support enamel remineralization. These findings should be interpreted in light of the pilot nature of the clinical trial, the limited sample size, and the exploratory microbiome analysis, which preclude definitive conclusions regarding long-term caries prevention or microbial causality. Conclusions Our in vitro findings showed that rinsing with calcium solutions increased mineralization volume and decreased QLF-areas, whereas clinical results demonstrated a favorable shift in plaque microbiota, a significant rise in salivary calcium levels, and reduced QLF-covered tooth surfaces. These observations indicate enhanced remineralization and reduced cariogenicity after using of calcium supplementation. Further research is needed to elucidate the underlying microbial mechanisms. Abbreviations QLF Quantitative Light-induced Fluorescence MV Mineralization Volume QLF-areas QLF-covered areas ELISA Enzyme-Linked Immunosorbent Assay SEM Scanning electron microscope µ-CT Micro-computed tomography RCT Randomized clinical trial Declarations Ethics approval and consent to participate All research procedures were approved by the Tri-Service General Hospital Institutional Review Board (TSGHIRB No. A202305084; approval period: June 11, 2023 to June 10, 2026). Written informed consent was obtained from all study participants prior to enrollment. Protocol in the approval IRB, TSGHIRB No. A202305084, was retrospctively registered at November 25,2025 and approved at ClinicalTrials.gov (Identifier: NCT07269730). Consent for publication Not applicable. Availability of data and materials The 16S rRNA sequencing data generated in this study have been deposited in the NCBI BioProject database under accession number PRJNA1404812 . (The dataset is currently not publicly available but is accessible to editors and reviewers during the peer review process under following link : (https://dataview.ncbi.nlm.nih.gov/object/PRJNA1404812?reviewer=2d8ock4ig62poujantj544ev4q) and will be made publicly available upon acceptance and publication.) Competing interests The authors have no conflicts of interest relevant to this article. Acknowledgments The authors acknowledge the technical services provided by the Instrument Center of National Defense Medical Center and the Tri-Service General Hospital, as well as the support from the Metal Industries Research & Development Center for providing QLF technology. Funding This study was supported by research grants from the Ministry of National Defense (MND-MAB-110-095, MND-MAB-110-123, and MND-MAB-C08-112032), and Tri-Service General Hospital Research Grant (TSGH-D-110060, TSGH-E-111198, TSGH-E-112202, TSGH-A-113003, 801GB112200, and TSGH-E-112202). Author Contributions Lin, ZY and Yuh, DY contributed to the conception and design of the study, acquisition, analysis, and interpretation of data, drafted the manuscript, and critically revised it; Fu, E contributed to the conception and design, interpretation of data, drafted the manuscript, and critically revised it; Chang, TH contributed to the conception and design, acquisition, analysis, and interpretation of data, drafted the manuscript, and critically revised it; Li, CH contributed to the conception and design, interpretation of data, drafted the manuscript, and critically revised it. All authors gave their final approval and agree to be accountable for all aspects of the work. 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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-8513583","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":628295507,"identity":"13264bb3-e2ad-4e11-8fea-5ec2522949a2","order_by":0,"name":"Zhi-Yun Lin","email":"","orcid":"","institution":"National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Yun","middleName":"","lastName":"Lin","suffix":""},{"id":628295508,"identity":"75bdd99a-57a0-4b48-940a-811e3762163c","order_by":1,"name":"Earl Fu","email":"","orcid":"","institution":"Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation","correspondingAuthor":false,"prefix":"","firstName":"Earl","middleName":"","lastName":"Fu","suffix":""},{"id":628295509,"identity":"4a5ef7d7-89f9-489f-973b-3dda4496861c","order_by":2,"name":"Chung-Hsing Li","email":"","orcid":"","institution":"Tri-Service General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chung-Hsing","middleName":"","lastName":"Li","suffix":""},{"id":628295510,"identity":"5cc6487b-0f1f-4e07-be76-05819ecca510","order_by":3,"name":"Ting-Han Chang","email":"","orcid":"","institution":"National Yang Ming Chiao Tung University","correspondingAuthor":false,"prefix":"","firstName":"Ting-Han","middleName":"","lastName":"Chang","suffix":""},{"id":628295511,"identity":"1fd14965-81b4-4c6f-b300-21621394bfea","order_by":4,"name":"Da-Yo Yuh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYFACHgYJICkHYh54QIoWY7CWBFK0JDaA2ERpMbiRe/DGzzab9Plhhx8CbbGT020gqCUv2bK3LS134+00A6CWZGOzAwS15JhJ8LYdzt04OwGk5UDiNmK0SP5tO5xuODv9A/FapIG2JMhL5xBpi+SZN8bWMufSDDdI5xQcSDAgwi98x3MMb74ps5GXn52++cOHCjs5gloUQAoY2YAuBKs0IKAcBOQbQOQfGGMUjIJRMApGARYAANUySOtgsUbfAAAAAElFTkSuQmCC","orcid":"","institution":"National Defense Medical University","correspondingAuthor":true,"prefix":"","firstName":"Da-Yo","middleName":"","lastName":"Yuh","suffix":""}],"badges":[],"createdAt":"2026-01-04 14:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8513583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8513583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107871260,"identity":"8ad48097-4a6f-412b-8ec6-453d33e54226","added_by":"auto","created_at":"2026-04-27 07:47:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10071510,"visible":true,"origin":"","legend":"\u003cp\u003eCONSORT flow diagram showing enrolment, allocation, follow-up, and analysis of participants (n = 15). All were randomized to the group of placebo (n = 5), formulation 1 (n = 5), or formulation 2 (n = 5), with no losses or exclusions.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8513583/v1/1527109ede4ddfc81d47ce18.png"},{"id":107872225,"identity":"f535c18c-ee7b-44f1-becc-3ef6e1eb46a3","added_by":"auto","created_at":"2026-04-27 07:56:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45396285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e effects of calcium solutions on QLF-area and MV changes. (\u003cstrong\u003eA\u003c/strong\u003e) SEM images of tooth lesions before and after calcium solution treatment (Red box and blue arrow indicate the area where the 130X magnification pictures are shown; Red arrows indicate the lesion with the demineralization). (\u003cstrong\u003eB\u003c/strong\u003e) Micro-CT images of teeth in four solution groups, while the mineralization volumes (MVs) were analyzed and compared quantitatively before and after rinsing (Red arrows indicate the demineralization lesion; AS + Calcium: the group of combination of artificial saliva and calcium solution). (\u003cstrong\u003eC\u003c/strong\u003e) QLF images of teeth in four groups, with quantitative analysis of QLF-areas (Red fluorescent plaque indicates the QLF-area). (Box plots indicate the interquartile range, mean, and median, with individual data points shown as dots; n = 5 per group; *: significant difference at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, by paired \u003cem\u003et \u003c/em\u003etest in B and C). (\u003cstrong\u003eD\u003c/strong\u003e) Correlation between ΔQLF-areas and ΔMVs across groups (n = 5 per group; *: significant difference at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, by linear regression; AS + Calcium: the group of combination of artificial saliva and calcium solution).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8513583/v1/78163859f6c8732f011ec7e8.png"},{"id":107872089,"identity":"637f36ac-d71f-4786-9828-97e260fd718f","added_by":"auto","created_at":"2026-04-27 07:55:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2083665,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of calcium sprays on shifting of \u003cem\u003estreptococcus\u003c/em\u003e in dental plaque microbiota\u003cstrong\u003e.\u003c/strong\u003e The 16S rRNA sequencing of dental plaque samples before and after 2-month treatments of three oral spray formulations (6 samples, paired before/after, red arrowhead indicates \u003cem\u003estreptococcus\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8513583/v1/bad9d5bdd43b08701dddaa15.png"},{"id":107874091,"identity":"34c81f21-4b02-49fc-a35a-307410cacd18","added_by":"auto","created_at":"2026-04-27 08:05:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4650390,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of calcium sprays on salivary calcium levels in the clinical trials. \u003cstrong\u003e(A)\u003c/strong\u003e Salivary calcium levels before and after using the placebo, calcium spray of formulation 1, and that of 2 (Box plots indicate the interquartile range, mean, and median, with individual data points shown as dots; n = 5 per group; *: significant difference at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, by paired \u003cem\u003et \u003c/em\u003etest). \u003cstrong\u003e(B)\u003c/strong\u003e Changes in the salivary calcium levels before and after using sprays among the patients with placebo and two calcium groups (formulae 1 and 2) for 2 months (n = 5 per group; *: significant difference at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 by independent \u003cem\u003et\u003c/em\u003e-test, data shown as mean ± SEM).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8513583/v1/d423bfee2fdd70e8a9bdbe9b.png"},{"id":107872220,"identity":"f5ded3ff-0ffd-43d8-93b9-f1a8ccedcdd9","added_by":"auto","created_at":"2026-04-27 07:56:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49524074,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of calcium-sprays on QLF-identified plaque cariogenicity in the clinical trial. (\u003cstrong\u003eA\u003c/strong\u003e) Representative clinical and QLF images before and after the 2-month intervention. Images were processed using a combination of clinical photography (pictures in the left column) and binarized QLF images, where the red fluorescent plaque indicated the QLF-area (pictures in the right column). Formulations included the placebo, calcium spray (Formula 1), and calcium spray with fluoride (Formula 2). (\u003cstrong\u003eB\u003c/strong\u003e) Comparison of QLF-detected plaque areas before and after using the sprays within each of the test groups (Box plots indicate the interquartile range, mean, and median, with individual data points shown as dots; n = 5 per group; *: significant difference at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, by paired \u003cem\u003et \u003c/em\u003etest). (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative changes in ΔQLF-area across groups (n = 5 per group; *: significant difference at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 by independent \u003cem\u003et\u003c/em\u003e-test, data shown as mean ± SEM).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8513583/v1/20be64ec65e28823d048b63b.png"},{"id":108006535,"identity":"efa31c73-91b1-4ce1-888c-d64594661442","added_by":"auto","created_at":"2026-04-28 12:55:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":76793206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8513583/v1/cc65b9bf-84f8-4312-91c3-627a0e840de8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Calcium-Based Solutions on Enamel Remineralization and Cariogenicity: Evidence from In Vitro and Clinical Studies","fulltext":[{"header":"One sentence summary","content":"\u003cp\u003eCalcium supplementation may reduce tooth cariogenicity and enhance enamel remineralization based on our findings of \u003cem\u003ein vitro\u003c/em\u003e and clinical cariogenicity area reductions, as well as increased mineralization volume \u003cem\u003ein vitro\u003c/em\u003e and salivary calcium elevations in the clinical trial.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eDental caries is the most prevalent dental disease worldwide, primarily caused by cariogenic bacteria such as \u003cem\u003eStreptococcus mutans\u003c/em\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These bacteria metabolize dietary sugars into acids that demineralize tooth enamel. Thus, theoretically, caries lesions develop due to an imbalance between demineralization and remineralization processes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Plaque accumulation initiates enamel demineralization [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], forming porous white spot lesions with an opaque appearance. In contrast, remineralization redeposits minerals into enamel, reversing early caries [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFluoride, calcium, and phosphate ions enhance remineralization [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; however, concerns regarding fluoride overexposure and its potential toxicity have led to conflicting reports in the literature [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Since calcium and phosphate ions are required by fluoride to promote the natural remineralization process of enamel, calcium phosphates has been applied as biomimetics in preventing dental caries [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies have shown enhanced enamel remineralization with calcium-containing chewing gum [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], casein phosphopeptide-amorphous calcium phosphate paste [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and hydroxyapatite toothpaste [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, in a randomized clinical trial, combining functionalized tricalcium phosphate with fluoride improved caries arrest in preschool children compared to fluoride varnish alone [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As maintaining oral hygiene in young children remains difficult, chemical control methods such as mouthwashes or sprays may offer a more convenient alternative [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study evaluated the effects of calcium solutions on mineralization volume and cariogenic areas \u003cem\u003ein vitro\u003c/em\u003e, and of calcium sprays on cariogenic areas in a randomized clinical trial. Additionally, bacterial composition in plaques and salivary calcium levels in children were compared.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro Experiment\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eSample Processing and Grouping\u003c/h2\u003e \u003cp\u003eTwenty human teeth with caries lesions were obtained with approval from the Tri-Service General Hospital Institutional Review Board (TSGHIRB No: A202105108). After cleaning with deionized water and drying, the teeth were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;5/group): ultrapure water (control), artificial saliva (AS), calcium solution, and AS plus calcium solution.\u003c/p\u003e \u003cp\u003eArtificial saliva was prepared following a previous study,[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] consisting of 0.1029 g CaCl₂\u0026middot;2H₂O, 0.04066 g MgCl₂, 0.544 g KH₂PO₄, 4.766 g HEPES buffer acid form, and 2.2365 g KCl in 1000 mL distilled water (pH 7). The calcium solution, commercially available (Kids Oral Spray, Toothfilm Inc., Taipei, Taiwan), contained 0.3% calcium lactate, 0.01% dicalcium phosphate, and 0.01% mesoporous bioactive glass.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eIn Vitro Experimental Procedures\u003c/h3\u003e\n\u003cp\u003eCaries lesions on the tooth surfaces were identified using a scanning electron microscope (SEM) and then examined three-dimensionally \u003cem\u003evia\u003c/em\u003e micro-computed tomography (\u0026micro;-CT) in sagittal, coronal, and horizontal views. Additionally, bacterial plaque on the tooth surfaces was captured using quantitative light-induced fluorescence (QLF). These analyses allowed for the quantification of mineralization volumes and cariogenicity areas before and after treatment with the four solutions.\u003c/p\u003e\n\u003ch3\u003eQuantitative Light-Induced Fluorescence (QLF)\u003c/h3\u003e\n\u003cp\u003eBacterial plaque on tooth surfaces was visualized using QLF (Metal Industries Research \u0026amp; Development Centre, Kaohsiung, Taiwan). Three types of images were captured: (1) an original photograph, (2) a binary bacterial plaque image, and (3) a fluorescent bacterial plaque image. Through the images, the relative \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ecariogenicity\u003c/span\u003e area was presented as the red fluorescent plaque (%, percentage of QLF-observed red patch in total area, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), while the difference in the areas before and after treatment was recorded as ΔQLF-area [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy and Micro-Computerized Tomography\u003c/h3\u003e\n\u003cp\u003eCaries lesions were identified using SEM (Apreo2S, Thermo Fisher Scientific, Waltham, USA) at 130X magnification and 5.0 kV, as well as \u0026micro;-CT (Quantum FX, Perkin Elmer, Waltham, USA). The acquired \u0026micro;-CT images were processed using Materialize Mimics software to calculate the enamel mineralization volume (MV, mm\u003csup\u003e3\u003c/sup\u003e). Boolean operations were applied to calculate the volume change before and after treatment, recorded as the Δ mineralization volume (ΔMV) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRandomized Clinical Trials\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eExperimental Design and Patient Selection\u003c/h2\u003e \u003cp\u003eRandomized clinical trial (RCT) was double-blindly conducted with 15 children presenting early carious lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). (CK 3, 8) Participants were recruited between January 2023 and December 2023. Inclusion criteria required participants to have an ICDAS code\u0026thinsp;\u0026ge;\u0026thinsp;1 and \u0026le;\u0026thinsp;3 as determined by a clinician (Dr. CHL). The mean age of the participants was 6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 years. The random allocation sequence was generated by an independent researcher using a computer-generated random number table. Participants were randomly assigned to one of three groups: placebo (control) and two formulations of calcium spray. Eligibility was determined by the enrolling clinician, whereas group assignment was conducted by an independent researcher using sequentially numbered, opaque, sealed envelopes to ensure allocation concealment. Participants, caregivers, and outcome assessors were blinded to group allocation. All sprays were packaged in identical bottles with standardized appearance and labeling, and were coded by an independent researcher (Ms. ZYL) to maintain blinding until data analysis was completed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFormulation 1 contained 0.3% calcium lactate, while Formulation 2 contained 0.3% calcium lactate plus 225 ppm fluoride (0.05% NaF). Differences before and after treatment were analyzed. Plaque and saliva samples were collected from all participants. All procedures were conducted following ethical guidelines and regulations, with informed consent obtained from all participants (TSGHIRB No: A202305084 and NCT07269730). No important changes to the trial design, eligibility criteria, interventions, outcomes, or statistical analysis plan were made after the trial commenced.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental Procedures\u003c/h3\u003e\n\u003cp\u003eAt the initial clinic visit, plaque and saliva samples were collected. Participants were instructed on product use, and follow-up visits ensured compliance. Participants used the assigned spray twice daily under caregiver supervision. Adherence was verbally confirmed at each follow-up visit, with no deviations from the intended intervention reported. Harms, defined as any unexpected oral symptoms such as tooth sensitivity, gingival irritation, or mucosal discomfort, were systematically assessed at each visit through clinician questioning and oral examination. After two months, dental conditions were assessed using QLF, and additional plaque and saliva samples were collected.\u003c/p\u003e \u003cp\u003eIn the clinical trial, QLF imaging produced three outputs: (1) an original photograph, (2) a binary image highlighting carious lesions, and (3) a fluorescent image. The QLF-area (%) and its change (ΔQLF-area) were measured and compared.\u003c/p\u003e \u003cp\u003e \u003cb\u003e16S rRNA Gene Sequence Analyses\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo examine bacterial changes in plaque before and after calcium spray use, three out of 15 participants were selected for bacterial sampling (pre- and post-treatment). Plaque samples were collected using a periodontal probe and stored in 1.5 mL tubes containing 500 \u0026micro;L of preservation solution at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eBacterial DNA was extracted following the manufacturer's instructions using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Cat No. EE161). DNA purity and concentration were verified using a Nanodrop spectrophotometer. The V3 and V4 regions of the 16S rRNA gene were amplified with primers (CCTACGGRRBGCASCAGKVRVGAAT and GGACTACNVGGGTWTCTAATCC) and the MetaVX Library Preparation Kit. PCR products (~\u0026thinsp;600 bp) were confirmed via 1% agarose gel electrophoresis. Sequencing reads were processed using QIIME2, with quality filtering, denoising, and operational taxonomic unit (OTU) clustering at a 97% similarity threshold. Taxonomic classification was performed using the Silva 138 reference database and the Ribosomal Database Project (RDP).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSalivary Calcium Content Analyses\u003c/h2\u003e \u003cp\u003eSaliva samples collected during the clinical trial were centrifuged at 3000 rpm for 10 minutes to obtain the clarified supernatant, which was then diluted tenfold for calcium measurement. Calcium concentration was determined colorimetrically using arsenazo III in an ELISA reader. The calcium-arsenazo III complex was measured spectrophotometrically at 652 nm, with absorbance proportional to calcium concentration in the sample [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData aggregation and calculations were performed using Excel 2013. Statistical analyses of MV, QLF-area, and salivary calcium content were conducted using Prism\u0026reg; 9 (v9.5). Paired t-test was used to compare differences before and after treatment, an independent t-test to compare differences between treatment groups, and a linear regression model to assess the correlation between ΔMV and ΔQLF-area. The level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was selected as statistically significant. All randomized participants were included in the analyses, and no missing data occurred; therefore, no imputation was required. The full trial protocol and statistical analysis plan are available from the corresponding author upon reasonable request, and de-identified participant data and statistical code may also be provided upon request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eExperiment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTooth surfaces were examined using SEM, and the changes before and after treatment were clearly observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The \u0026micro;-CT was used for quantitative measurement of MV. MV remained unchanged in the water group but significantly increased after treatment with artificial saliva, calcium solution, or their combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eQLF analysis showed reduced cariogenic areas in all groups, including water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, △QLF-area was negatively correlated with △MV in the artificial saliva, calcium, and calcium\u0026thinsp;+\u0026thinsp;saliva groups, but not in the water group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRandomized Clinical Trials\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003eBacterial Shifting in Dental Plaque after Calcium-Spray Using\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e16S rRNA sequencing identified 30 bacterial genera in plaque samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After two months of calcium spray use, bacterial compositions shifted across all formulations. Streptococcus proportions remained stable in the placebo group (19.41% before \u003cem\u003evs\u003c/em\u003e 21.91% after) but markedly decreased in the calcium spray groups\u0026mdash;Formulation 1 (29.99 \u003cem\u003evs\u003c/em\u003e 17.03%) and Formulation 2 (43.52 \u003cem\u003evs\u003c/em\u003e 29.55%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChanges in Saliva Calcium Levels after Calcium-Spray Use\u003c/h2\u003e \u003cp\u003eSalivary calcium levels significantly increased after treatment in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, the increase (△calcium) was significantly greater in both calcium spray groups than in the placebo, with no difference between formulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eReduction in QLF-Identified Plaque after Calcium-Spray Use\u003c/h2\u003e \u003cp\u003eThe clinical photographs in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e show QLF results, where red fluorescence on the tooth surfaces in the left column of QLF combinations indicates cariogenic areas. Two-month calcium sprays significantly reduced cariogenic areas (Formulation 1: 5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% \u003cem\u003evs\u003c/em\u003e 2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68%; Formulation 2: 6.81\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05% \u003cem\u003evs\u003c/em\u003e 3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15%), while no change was seen in the placebo group (2.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68% \u003cem\u003evs\u003c/em\u003e 4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B). The △QLF-areas in both calcium formulations were significantly lower than those in the placebo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Compared with placebo, formulation 1 reduced ΔQLF-area by \u0026minus;\u0026thinsp;3.26% (95% CI \u0026minus;\u0026thinsp;5.20 to \u0026minus;\u0026thinsp;1.32), and formulation 2 reduced ΔQLF-area by \u0026minus;\u0026thinsp;3.75% (95% CI \u0026minus;\u0026thinsp;6.71 to \u0026minus;\u0026thinsp;0.79). No adverse events were observed in any group throughout the study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study assessed the effects of calcium-based oral supplementations on enamel remineralization and biofilm cariogenicity. Early caries detection is essential for prevention. Although radiography remains the diagnostic standard [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], it is less suitable for children [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Noninvasive tools such as QLF permit early detection by visualizing red fluorescence from bacterial porphyrins, which reflects biofilm acidogenicity [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u0026micro;-CT provides quantitative mineralization data but is limited to \u003cem\u003ein vitro\u003c/em\u003e use [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy \u0026micro;-CT and QLF, our \u003cem\u003ein vitro\u003c/em\u003e results showed that a 6-hour rinse with artificial saliva or calcium solutions significantly increased mineralization volume (by \u0026micro;-CT) but reduced cariogenic area (by QLF), indicating lesion reversal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The negative correlation between △MV and △QLF-area suggests that increased mineralization and reduced biofilm-associated fluorescence occurred in parallel. By contrast, water rinsing reduced QLF-areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) without a detectable increase in mineralization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating a limited effect on enamel mineral gain under the present experimental conditions.\u003c/p\u003e \u003cp\u003eIn the clinical trial, calcium sprays (Formulations 1 and 2) were associated with a reduction in the relative abundance of Streptococcus in plaque samples, whereas minimal changes were observed in the placebo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting a potential trend toward altered plaque composition. This finding is consistent with studies linking caries progression to oral microbial dysbiosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. During caries development, frequent sugar exposure increases acid production, favoring acidogenic and aciduric bacteria such as \u003cem\u003eStreptococcus mutans\u003c/em\u003e while suppressing acid-sensitive species [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Although this study did not investigate the mechanisms by which calcium influences plaque composition, prior studies suggest that calcium may contribute to maintaining microbial balance in the oral environment [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the present trial, salivary calcium levels also increased significantly after using calcium sprays (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating enhanced buffering and remineralization potential. The elevated salivary calcium observed here may therefore support conditions favorable for enamel remineralization [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It has been shown that calcium and phosphate combined with fluoride enhance in situ remineralization more effectively than fluoride alone [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; the elevated salivary calcium observed here may therefore support enamel repair and slow caries progression [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe QLF images used in this study provided visual and quantitative information related to plaque-associated red fluorescence, which has been associated with biofilm maturity and acidogenic potential [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. QLF imaging demonstrated a reduction in red fluorescence\u0026ndash;covered areas after two months of calcium spray use, whereas no significant change was observed in the placebo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the details in calcium\u0026rsquo;s action on reducing the cariogenicity of dental biofilms are likely multifaceted [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Calcium ions are known to contribute to the formation and stabilization of hydroxyapatite crystals and have been reported to support enamel remineralization in previous studies [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additionally, calcium strengthens the salivary pellicle, creating a protective layer against acid attacks [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Taken together, the observed microbial trends, increased salivary calcium levels, and reductions in QLF-identified plaque fluorescence suggest that calcium sprays may contribute to a less cariogenic plaque environment and support enamel remineralization. These findings should be interpreted in light of the pilot nature of the clinical trial, the limited sample size, and the exploratory microbiome analysis, which preclude definitive conclusions regarding long-term caries prevention or microbial causality.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e findings showed that rinsing with calcium solutions increased mineralization volume and decreased QLF-areas, whereas clinical results demonstrated a favorable shift in plaque microbiota, a significant rise in salivary calcium levels, and reduced QLF-covered tooth surfaces. These observations indicate enhanced remineralization and reduced cariogenicity after using of calcium supplementation. Further research is needed to elucidate the underlying microbial mechanisms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQLF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative Light-induced Fluorescence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMineralization Volume\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQLF-areas\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQLF-covered areas\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-Linked Immunosorbent Assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eScanning electron microscope\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;-CT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicro-computed tomography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRandomized clinical trial\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll research procedures were approved by the Tri-Service General Hospital Institutional Review Board (TSGHIRB No. A202305084; approval period: June 11, 2023 to June 10, 2026). Written informed consent was obtained from all study participants prior to enrollment. Protocol in the approval IRB, TSGHIRB No. A202305084, was retrospctively registered at November 25,2025 and approved at ClinicalTrials.gov (Identifier: NCT07269730).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA sequencing data generated in this study have been deposited in the NCBI BioProject database under accession number \u003cstrong\u003ePRJNA1404812\u003c/strong\u003e. (The dataset is currently not publicly available but is accessible to editors and reviewers during the peer review process under following link : (https://dataview.ncbi.nlm.nih.gov/object/PRJNA1404812?reviewer=2d8ock4ig62poujantj544ev4q) and will be made publicly available upon acceptance and publication.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest relevant to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the technical services provided by the Instrument Center of National Defense Medical Center and the Tri-Service General Hospital, as well as the support from the Metal Industries Research \u0026amp; Development Center for providing QLF technology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by research grants from the Ministry of National Defense (MND-MAB-110-095, MND-MAB-110-123, and MND-MAB-C08-112032), and Tri-Service General Hospital Research Grant (TSGH-D-110060, TSGH-E-111198, TSGH-E-112202, TSGH-A-113003, 801GB112200, and TSGH-E-112202).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLin, ZY and Yuh, DY contributed to the conception and design of the study, acquisition, analysis, and interpretation of data, drafted the manuscript, and critically revised it; Fu, E contributed to the conception and design, interpretation of data, drafted the manuscript, and critically revised it; Chang, TH contributed to the conception and design, acquisition, analysis, and interpretation of data, drafted the manuscript, and critically revised it; Li, CH contributed to the conception and design, interpretation of data, drafted the manuscript, and critically revised it. All authors gave their final approval and agree to be accountable for all aspects of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eOkada M, Soda Y, Hayashi F, Doi T, Suzuki J, Miura K, Kozai K: \u003cstrong\u003eLongitudinal study of dental caries incidence associated with Streptococcus mutans and Streptococcus sobrinus in pre-school children\u003c/strong\u003e. \u003cem\u003eJournal of Medical Microbiology\u0026nbsp;\u003c/em\u003e2005, \u003cstrong\u003e54\u003c/strong\u003e(7):661-5.\u003c/li\u003e\n \u003cli\u003eZero DT: \u003cstrong\u003eDental caries process\u003c/strong\u003e. \u003cem\u003eDent Clin North Am\u0026nbsp;\u003c/em\u003e1999, \u003cstrong\u003e43\u003c/strong\u003e(4):635-64.\u003c/li\u003e\n \u003cli\u003eKidd EAM, Fejerskov O: \u003cstrong\u003eWhat Constitutes Dental Caries? 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results\u003c/strong\u003e. \u003cem\u003eBiomaterials Research\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e22\u003c/strong\u003e(1):26.\u003c/li\u003e\n \u003cli\u003eJiang W, Ling Z, Lin X, Chen Y, Zhang J, Yu J, Xiang C, Chen H: \u003cstrong\u003ePyrosequencing Analysis of Oral Microbiota Shifting in Various Caries States in Childhood\u003c/strong\u003e. \u003cem\u003eMicrobial Ecology\u0026nbsp;\u003c/em\u003e2014, \u003cstrong\u003e67\u003c/strong\u003e(4):962-9.\u003c/li\u003e\n \u003cli\u003eTeng F, Yang F, Huang S, Bo C, Xu ZZ, Amir A, Knight R, Ling J, Xu J: \u003cstrong\u003ePrediction of Early Childhood Caries via Spatial-Temporal Variations of Oral Microbiota\u003c/strong\u003e. \u003cem\u003eCell Host Microbe\u0026nbsp;\u003c/em\u003e2015, \u003cstrong\u003e18\u003c/strong\u003e(3):296-306.\u003c/li\u003e\n \u003cli\u003eTakahashi N, Nyvad B: \u003cstrong\u003eThe role of bacteria in the caries process: ecological perspectives\u003c/strong\u003e. \u003cem\u003eJ Dent Res\u0026nbsp;\u003c/em\u003e2011, \u003cstrong\u003e90\u003c/strong\u003e(3):294-303.\u003c/li\u003e\n \u003cli\u003eDipalma G, Inchingolo AD, Inchingolo F, Charitos IA, Di Cosola M, Cazzolla AP: \u003cstrong\u003eFocus on the cariogenic process: microbial and biochemical interactions with teeth and oral environment\u003c/strong\u003e. \u003cem\u003eJ Biol Regul Homeost Agents\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e35\u003c/strong\u003e(2).\u003c/li\u003e\n \u003cli\u003eShen P, Bagheri R, Walker G, Yuan Y, Stanton D, Reynolds C, Reynolds E: \u003cstrong\u003eEffect of calcium phosphate addition to fluoride containing dental varnishes on enamel demineralization\u003c/strong\u003e. \u003cem\u003eAustralian Dental Journal\u0026nbsp;\u003c/em\u003e2016, \u003cstrong\u003e61\u003c/strong\u003e(3):357-65.\u003c/li\u003e\n \u003cli\u003eVicente A, Ortiz-Ruiz AJ, Gonz\u0026aacute;lez-Paz BM, Mart\u0026iacute;nez-Beneyto Y, Bravo-Gonz\u0026aacute;lez LA: \u003cstrong\u003eEffectiveness of a toothpaste and a serum containing calcium silicate on protecting the enamel after interproximal reduction against demineralization\u003c/strong\u003e. \u003cem\u003eScientific Reports\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e11\u003c/strong\u003e(1):834.\u003c/li\u003e\n \u003cli\u003eShen P, Walker GD, Yuan Y, Reynolds C, Stanton DP, Fernando JR, Reynolds EC: \u003cstrong\u003eImportance of bioavailable calcium in fluoride dentifrices for enamel remineralization\u003c/strong\u003e. \u003cem\u003eJournal of Dentistry\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e78\u003c/strong\u003e:59-64.\u003c/li\u003e\n \u003cli\u003eMenon LU, Varma RB, Kumaran P, Xavier AM, Govinda BS, Kumar JS: \u003cstrong\u003eEfficacy of a Calcium Sucrose Phosphate Based Toothpaste in Elevating the Level of Calcium, Phosphate Ions in Saliva and Reducing Plaque: A Clinical Trial\u003c/strong\u003e. \u003cem\u003eContemp Clin Dent\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e9\u003c/strong\u003e(2):151-7.\u003c/li\u003e\n \u003cli\u003eAkifusa S, Isobe A, Kibata K, Oyama A, Oyama H, Ariyoshi W, Nishihara T: \u003cstrong\u003eComparison of dental plaque reduction after use of electric toothbrushes with and without QLF-D-applied plaque visualization: a 1-week randomized controlled trial\u003c/strong\u003e. \u003cem\u003eBMC Oral Health\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e20\u003c/strong\u003e(1):4.\u003c/li\u003e\n \u003cli\u003eCochrane NJ, Reynolds EC: \u003cstrong\u003eCalcium phosphopeptides -- mechanisms of action and evidence for clinical efficacy\u003c/strong\u003e. \u003cem\u003eAdv Dent Res\u0026nbsp;\u003c/em\u003e2012, \u003cstrong\u003e24\u003c/strong\u003e(2):41-7.\u003c/li\u003e\n \u003cli\u003eDong H, Wang D, Deng H, Yin L, Wang X, Yang W, Cai K: \u003cstrong\u003eApplication of a calcium and phosphorus biomineralization strategy in tooth repair: a systematic review\u003c/strong\u003e. \u003cem\u003eJournal of materials chemistry B\u0026nbsp;\u003c/em\u003e2024.\u003c/li\u003e\n \u003cli\u003eCochrane NJ, Cai F, Huq NL, Burrow MF, Reynolds EC: \u003cstrong\u003eNew Approaches to Enhanced Remineralization of Tooth Enamel\u003c/strong\u003e. \u003cem\u003eJournal of Dental Research\u0026nbsp;\u003c/em\u003e2010, \u003cstrong\u003e89\u003c/strong\u003e(11):1187-97.\u003c/li\u003e\n \u003cli\u003eChawhuaveang DD, Yu OY, Yin IX, Lam WY-H, Mei ML, Chu C-H: \u003cstrong\u003eAcquired salivary pellicle and oral diseases: A literature review\u003c/strong\u003e. \u003cem\u003eJournal of Dental Sciences\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e16\u003c/strong\u003e(1):523-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dental Caries, tooth remineralization, calcium compounds, dental plaque, randomized controlled trial","lastPublishedDoi":"10.21203/rs.3.rs-8513583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8513583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground. Calcium has been proposed as an alternative for promoting remineralization of dental caries. This study evaluated the effects of calcium-based solutions on tooth cariogenicity through an in vitro experiment and a randomized clinical trial.\u003c/p\u003e\n\u003cp\u003eMethods. Teeth with carious lesions were rinsed in vitro with water, artificial saliva, or 0.3% calcium solution. Using scanning electron microscopy, micro-computed tomography, and quantitative light-induced fluorescence (QLF), the mineralization volumes (MVs) and the relative QLF-covered areas (QLF-areas) were assessed. In the clinical trial, fifteen children were randomly assigned to placebo, 0.3% calcium (formula 1), or 0.3% calcium plus 225 ppm fluoride (formula 2) oral sprays for two months. Plaque bacterial composition was analyzed using 16S rRNA gene sequencing, salivary calcium levels using enzyme-linked immunosorbent assay, and the cariogenicity area by QLF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults. In vitro, calcium and artificial saliva rinses increased MV and reduced QLF-areas, with a significant negative correlation between their changes. Clinically, salivary calcium increased significantly with formulas 1 and 2 but not placebo. Both calcium sprays showed significant reductions in QLF-areas, while the placebo did not.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusions. Combined with in vitro (MVs increasing but QLF areas reducing after calcium rinsing) and clinical findings (salivary calcium level elevations but QLF-area reductions after using calcium sprays) indicate enhanced enamel mineralization but reduced biofilm cariogenicity by calcium supplementation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinical significance. Calcium-containing solutions may aid in the management of early enamel demineralization.\u003c/p\u003e\n\u003cp\u003eClinical Trials Identifier NCT07269730, retrospectively registered at 2025/11/25. 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