Comparative evaluation of remineralizing potential of Galla chinensis with nanohydroxyapatite, chicken egg shell and fish scale derived nanohydroxyapatite on early enamel caries- A single-blinded in vitro study

Comparative evaluation of remineralizing potential of Galla chinensis with nanohydroxyapatite, chicken egg shell and fish scale derived nanohydroxyapatite on early enamel caries- A single-blinded in vitro study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Comparative evaluation of remineralizing potential of Galla chinensis with nanohydroxyapatite, chicken egg shell and fish scale derived nanohydroxyapatite on early enamel caries- A single-blinded in vitro study Sonali Taneja, Bazila Malik, Abhik Mukherjee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7031614/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 Introduction: The earliest clinical sign of enamel caries is subsurface demineralization beneath an intact surface. At this stage, remineralizing agents can potentially reverse or halt lesion progression. Aim: To evaluate and compare the remineralization potential of plant-based Galla chinensis extract (GCE) with nanohydroxyapatite (nHAp), chicken eggshell-derived nHAp (CES nHAp), and fish scale-derived nHAp (FS nHAp), against casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), on early artificial enamel lesions in human premolars. Design: In-vitro study performed under pH cycling stimulating oral conditions. Methodology: A total of 148 specimens were taken. Among these, 48 samples were used for microhardness testing by Vicker’s hardness test, 60 for lesion depth evaluation using Polarized Light Microscopy (PLM), 30 for Energy Dispersive X-ray (EDX) analysis, and 10 for Scanning Electron Microscopy (SEM). All samples were demineralized except for five intact controls designated for baseline EDX assessment. Microhardness was measured before demineralization (SMH1), after demineralization (SMH2), and following remineralization (SMH3). These values were used to calculate the Surface Microhardness Recovery percentage (SMHR%). The demineralized samples were categorized into the following experimental groups based on the remineralizing agent applied: Demineralized control (no treatment); Group 1: CPP-ACPF; Group 2: GCE with nHAp; Group 3: CES nHAp; and Group 4: FS nHAp. Each group included 12 samples for microhardness testing, 12 for lesion depth evaluation, 5 for EDX, and 2 for SEM. A seven-day pH cycling model simulated oral conditions before post-remineralization analysis. PLM was utilized for morphometric lesion depth assessment. EDX was used to assess changes in calcium-to-phosphorus (Ca/P) ratio, and SEM evaluated surface topography. Results: GCE nHAp showed the highest surface microhardness recovery and the shallowest lesion depth, indicating effective, uniform remineralization. CES nHAp and CPP-ACPF demonstrated moderate potential, while FS nHAp was the least effective. EDX analysis revealed a significantly higher calcium-to-phosphorus (Ca/P) ratio in the GCE nHAp group. No significant difference was found between CPP-ACPF and FS nHAp. SEM images confirmed smoother, more uniform surfaces in the GCE group. Conclusion: GCE combined with nHAp was the most effective remineralizing agent, followed by CES nHAp and CPP-ACPF. These results suggest biogenic materials from plant and animal sources may offer effective, biocompatible alternatives for non-invasive enamel caries treatment. Health sciences/Health care/Dentistry/Preventive dentistry/Minimal intervention dentistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Dental caries is a multifactorial, biofilm-mediated disease resulting in enamel demineralization due to acidic conditions caused by bacterial metabolites. ¹ Enamel, composed of hydroxyapatite crystals, maintains equilibrium with saliva in a neutral pH environment. 2 , 3 However, when pH falls below 5.5, hydrogen ions disrupt the crystal lattice, causing mineral loss and enamel breakdown. Remineralization occurs when pH is neutralized, and calcium and phosphate ions from external sources rebuild hydroxyapatite crystals.⁴ If demineralization exceeds remineralization over time, early enamel caries are formed, progressing to cavitations if untreated.⁵ Fluoride is a widely used remineralizing agent that strengthens enamel by forming fluorapatite, which is less soluble in acidic environments.⁶ However, fluoride alone cannot completely remineralize carious lesions, as calcium and phosphate ions are also required.⁷ Additionally, excessive fluoride exposure may lead to fluorosis and other health issues. These limitations have prompted research into alternative remineralizing agents, such as casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), which furnishes calcium and phosphate ions to enhance remineralization.⁸ Combining CPP-ACP with fluoride (CPP-ACPF) has demonstrated superior results and is considered a gold standard.⁹ Biomimetic materials like nano-hydroxyapatite (nHA) offer promising remineralization potential due to their structural and chemical similarity to enamel crystals.¹⁰ nHA particles (nHAp), being smaller, dissolve more readily, fill enamel defects efficiently, and promote compact surface formation. ¹¹ Plant- and animal-derived products have also emerged as sustainable remineralizing agents. Galla chinensis extract (GCE), a natural Chinese herbal medicine, has been shown to prevent demineralization and enhance mineral deposition. 12 , 13 Animal-based sources like chicken eggshell powder and fish scales, rich in hydroxyapatite and calcium, offer biocompatible and biodegradable options. ¹⁴ Eggshell-derived nHA can penetrate enamel pores, while fish scales, containing collagen and hydroxyapatite, are a valuable biomaterial for remineralization. 15 , 16 To the best of our knowledge, there are no studies that compare the remineralizing potential of plant-based remineralizing agents and animal-derived nanohydroxyapatite on the reversal of early enamel caries. Thus, this study aimed to evaluate the remineralizing potential of Galla chinensis with nanohydroxyapatite, chicken egg shell-derived nanohydroxyapatite, and fish scale-derived nanohydroxyapatite particles in comparison with CPP-ACPF on early enamel caries. In this study, the first null hypothesis tested was that there was no significant difference in the microhardness recovery of early enamel caries subjected to remineralization by Galla Chinensis and nanohydroxyapatite, Chicken egg shell derived nanohydroxyapatite, Fish scale derived nanohydroxyapatite, and CPP-ACPF. The second hypothesis tested was that there was no significant difference in the lesion depth of early enamel caries subjected to remineralization by Galla Chinensis and nanohydroxyapatite, Chicken egg shell derived nanohydroxyapatite, Fish scale derived nanohydroxyapatite and CPP-ACPF. The third hypothesis tested was that there was no significant difference in the Ca/P ratio of early enamel caries subjected to remineralization by Galla Chinensis and nanohydroxyapatite, Chicken egg shell derived nanohydroxyapatite, Fish scale derived nanohydroxyapatite and CPP-ACPF. Materials & Methodology Specimen collection This study was conducted in the Department of Conservative Dentistry & Endodontics in collaboration with Centre for Advanced Research at I.T.S Centre for Dental Studies & Research, Ghaziabad and KIET School of Pharmacy, Ghaziabad. The protocol was reviewed and approved by the institutional ethical clearance committee under protocol number ITSCDSR/IIEC/LD/CONS/2022-25/002. Specimen size determination The sample size was calculated using the sample size calculator software program G. Power (version 3.1.9.7). The sample size calculation was based on 95% confidence interval and a power of 90%. The minimum sample size estimated for this study was one hundred forty eight. Specimen selection With the patient’s consent, one hundred forty-eight human maxillary premolars extracted for orthodontic or periodontal reasons were collected for the study. The inclusion criteria included non-carious, intact crowns and teeth free from restorations. The samples were examined under an operating microscope (10X) to ensure there were no cracks, fractures, or anatomical developmental anomalies. The teeth were then disinfected by autoclaving at 121°C at 15 lbs psi and stored in freshly prepared artificial saliva Fusayama artificial saliva described by Olssom et al. 17 containing NaCl (6.8 mmol/L), KCl (5.4 mmol/L), CaCl 2 .2H 2 O (2.7 mmol/L), NaH 2 PO 4 .H 2 O (5.0 mmol/L), Na 2 S.9H 2 O (21 µmol/L), and Urea (16.7 mmol/L) 18 titrated to pH 7.0 with NaOHuntil further use. Specimen preparation The crowns were detached from the roots using a diamond disc on a slow-speed handpiece and then divided into two halves along the mesio-distal axis to obtain buccal enamel samples (N = 148). The samples were embedded in acrylic resin with the buccal surface facing upward, and the enamel was sequentially polished using silicon carbide abrasive papers with grit sizes 1000, 600, and 320 to create flat surfaces. A 3x3 wax sheet was applied to the buccal surfaces to form a window, while the rest of the enamel was coated with acid-resistant nail varnish. 19 Grouping of samples Out of a total of 148 samples, 48 were assigned for microhardness testing using Vicker’s Microhardness tester, 60 for mean lean depth using PLM, 10 for SEM analysis and 30 for EDX analysis .(Fig. 1) Microhardness testing Forty-eight samples were used for microhardness testing. A Vickers hardness tester (Banbros, India) with a diamond indenter was used to measure the baseline surface microhardness (SMH 1 ) of all 48 samples, applying a 500g load for 15 seconds. 20 The samples were then demineralized to mimic early enamel caries using a solution containing 2.2 mM calcium chloride, 2.2 mM monosodium phosphate, and 0.05 M lactic acid. The pH was adjusted to 4.5 with 50% sodium hydroxide and confirmed using a calibrated digital pH meter. 21 Samples were immersed in this demineralizing solution in glass containers and incubated at 37°C for 96 hours. The solution was regularly replaced to maintain pH and prevent mineral buildup. After 96 hours, samples were rinsed with deionized water for 30 seconds, air-dried for 5 seconds, and stored in sterile containers. Microhardness was then re-evaluated to obtain the post-demineralization value (SMH 2 ). Following this, the remineralization was done according to the assigned groups. Preparation of tested remineralizing agents : I) CPP-ACPF The commercially available GC Tooth Mousse Plus ® was used. II) GCE with nHAp 4000ppm GCE aqueous solution was made from GCE powder (BulkSupplemets.com) and mixed with equal proportion of 10% aqueous slurry of commercially available nanohydroxyapatite powder (Vedayukt India Private Limited). 22 III) CES nHAp Eggshells from a local hatchery were cleaned, dried, ground, and sieved. The powder was ball-milled for 5 days, and nanoparticles were formed through a precipitation reaction: CaCO 3 + 2HCl → CaCl 2 + CO 2 + H 2 O. The solution was mixed with water and surfactant, then centrifuged. The particles were dried and used to prepare a 10% aqueous slurry of CES nHAp. 23 IV) FS nHAp Fish scales from Labeo rohita were sourced from a local market, washed, soaked in hydrochloric acid (HCl) to remove impurities, then dried. The scales were ground into particles, heated in a furnace, and wet-ground using ball milling for 48 hours. The resulting slurry was dried using a spray dryer to obtain FS nHAp powder. 16 A 10% aqueous slurry of FS nHAp was then made with distilled water. The pH cycling model 0.1M tris buffer (hydroxymethyl aminomethane), 0.05 ppm fluoride in the form of NaF, 0.9 mM phosphorus in the form of KH 2 PO 4 , and 1.5 mM calcium in the form of Ca(NO 3 ) 2 with a pH of 7 was used to prepare the remineralizing solution. In the pH cycling model, the remineralizing agent from respective groups were applied to the samples for 5 minutes, followed by a 5-second rinse with deionized water. To mimic the daily acidic challenges in the oral environment, the teeth were then immersed in the demineralizing solution for 3 hours. Then the samples were placed in the prepared remineralizing solution for the remaining 21 hours of the 24-hour cycle. This process was repeated daily for 7 days, with the demineralizing and remineralizing solutions being replaced every 3 days. 24 After remineralization, microhardness was again evaluated to obtain post-remineralization surface microhardness (SMH 3 ) in a similar manner. The percentage surface microhardness recovery (%SMHR) was recorded. 25 The formula used was: %SMHR = (SMH 3 − SMH 2 )/( SMH 1 − SMH 2 ) × 100% Mean depth of lesion : Sixty samples were used to evaluate the mean depth of enamel lesions. Demineralization was carried out as previously described. Twelve of the demineralized samples (demineralized control) were assessed for mean lesion depth using morphometric analysis and examined under polarized light microscopy (PLM). For this, the buccal surfaces were sectioned longitudinally into mesial and distal halves and ground to a thickness of 100–150 µm. After rinsing, the sections were placed on slides and viewed under PLM at 40x magnification. Photomicrographs were taken, and the demineralized areas were measured three times using ImageJ software to determine the mean lesion depth for each sample. 26 , 27 The remaining 48 samples were divided into four remineralization groups of twelve and treated with different remineralizing agents using the pH cycling model described earlier. After remineralization, lesion depth was re-evaluated. SEM Analysis : Ten samples were used for SEM analysis. Demineralized was done as previously described. Two samples (demineralized control) were then prepared for SEM by undergoing ultrasonication for 10 minutes, rinsed with deionized water, air-dried, gold-sputtered, and examined under a scanning electron microscope (SEM) (Nova Nano SEM™, Thermo Fisher, USA) at 5000x magnification to observe enamel surface microstructure. 28 , 29 After remineralization using the pH cycling model, the remaining eight samples were similarly evaluated under SEM. EDX analysis : Thirty samples were used for EDX analysis out of which 5 served as intact controls. The remianing 25 samples underwent demineralization. After this, 5 samples (demineralized control) were first subjected to EDX analysis (Bruker, UK) in order to assess the Calcium:Phosphte (Ca/P) ratio of enamel. Following this, the remineralization of 20 samples with 5 in each reminerlization group wasd done by the procedure as described earlier. Finally, EDX anaylisis of these remineralized samples was done. Data Analysis: All the collected data was entered in MS Excel. Descriptive statistics was performed by calculating mean and standard deviation for the continuous variables. The data was analyzed using IBM SPSS (Statistical Package for Social Sciences) 25.0 Version (IBM Corp 2013, New York, USA). Paired t-test was conducted for comparison of the hardness after demineralization and after remineralization. The intergroup comparison of the % SMHR was conducted using One-way ANOVA test (level of significance < 0.05). Pairwise comparison of %SMHR was done by Post hoc Tukey test (level of significance < 0.05). Inter-group comparison of mean lesion depth was done by One-way ANOVA test (level of significance < 0.05). Pairwise comparison of mean lesion depth was done using Post hoc Tukey test (level of significance < 0.005). Paired t-test was conducted for comparison of baseline, remineralization and demineralization Ca/P ratio (level of significance < 0.05). Inter-group comparison of Ca/P was done by One-way ANOVA test (level of significance < 0.05). Results The percentage of Surface Microhardness Recovery (%SMHR) was greatest in the GCE nHAp group, followed by the CES nHAp group, while the FS nHAp group exhibited the lowest %SMHR.Furthermore, the %SMHR in the FS nHAp group was significantly lower compared to both the GCE nHAp and CES nHAp groups, with no significant difference observed between the FS nHAp and CPP ACPF groups. (Table 1) The demineralized control group displayed the maximum mean lesion depth (µm), succeeded by the CPP ACPF group, while the GCE nHAp group had the least mean lesion depth (in Table 2, Fig. 2) Intergroup analysis of Ca/P revealed that the most substantial increase was seen after remineralization in the GCE nHAp group, followed by the CES nHAp group, with the CPP ACPF and FS nHAp groups showing the lowest increase with no significant difference found between the CPP ACPF and FS nHAp groups.( Table 3, Fig. 3) The SEM pictures revealed that after demineralization, the treated enamel surface exhibited a loss of the enamel prism core while retaining the periphery. In contrast, the remineralized groups displayed a mineral layer formed on the enamel surface, effectively restoring the enamel prism defect. (Fig. 4) Discussion Early enamel caries is the first sign of demineralization and can be reversed or arrested with remineralization techniques. In recent years, natural plant- and animal-based products have garnered attention for their potential as remineralizing agents. Galla chinensis extract (GCE) is a plant-based traditional Chinese medicine that has demonstrated potential in remineralizing enamel. In addition to plant-based agents, animal-derived nHAp from sources such as chicken eggshells, fish scales, and other biowaste materials has shown promise for remineralization. The novelty of this study emerged from the use of natural agents for remineralization. In the present study, GCE with nHAp (Group 1) exhibited the highest remineralizing potential, as seen in the highest % surface microhardness recovery (%SMHR) among all groups. The possible reason for this might be the chemical components of GCE, such as tannins and gallic acid which act as calcium ion carriers, transferring calcium ions to enamel and promoting mineral crystal reproduction, thus aiding remineralization (Chu et al. 2007), 30 Zhang et al. 2009). 31 GCE combined with nano-HA showed a higher mineral density compared to nHAp alone, as it enhanced mineral deposition, creating a synergistic effect. Huang et al. 2010 22 also reported that GCE + nHAp formed more uniform and regular surface crystals compared to nHAp alone. CES nHAp showed better remineralization than CPP-ACPF and FS nHAp. It could be attributed to the higher crystallinity and buffering properties of CES nHAp. Its irregular rod-like structure and smaller particle size provide a larger surface area for acid-neutralizing reactions, thus better protecting enamel from demineralization. This observation is supported by the study conducted by Mkhize et al. in 2023, 16 which determined that CES nHAp possesses a superior remineralizing capability in comparison to FS nHAp. CES nHAp demonstrated higher microhardness than CPP-ACPF, possibly due to its greater surface area and hydrophilic properties, which facilitated the precipitation of calcium and phosphate ions on the enamel surface (Kunam et al. 2019, 32 Hassan et al. 2023). 33 Meanwhile, CPP-ACPF also showed increased %SMHR, as CPP-ACP’s ability to release calcium and phosphate ions, in combination with fluoride, forms acid-resistant fluorapatite (Reynolds et al. 1998)⁷. Also, FS nHAp demonstrated significant microhardness recovery, comparable to CPP-ACPF, likely due to its HA content (Mkhize et al. 2023). 16 Thus, the first null hypothesis was rejected. There was a significant reduction in mean lesion depth in Groups 1, 2, 3, and 4 compared to the demineralized control group, with the GCE nHAp group showing the greatest reduction. This may be due to mineral ions being deposited deeper in the lesion body rather than just the outer layer when GCE is applied. Studies by Cheng et al. (2008) 13 and Huang et al. (2010) 22 confirm that GCE facilitates ion transfer into the lesion body, enhancing remineralization. When combined with nHAp, GCE showed improved results compared to nHAp alone, which was effective only at depths of 20–40 µm, with minimal remineralization beyond that. The mean lesion depth in the CPP-ACPF, CES nHAp, and FS nHAp groups was also significantly reduced. nHAp aids remineralization by filling enamel defects and attracting calcium and phosphate ions to the surface, promoting crystal growth (Kunam et al. 2019). 32 However, the reduction in lesion depth of CES nHAp was less than that of GCE nHAp, possibly due to CES nHAp's mineral deposition being more concentrated in the outer layer, inhibiting deeper ion diffusion. CPP-ACPF, which supersaturates the micro-environment with calcium, phosphate, and fluoride ions, also showed reduced lesion depth which was less effective than GCE nHAp. The reason could be attributed to the shorter application period (7 days) which might have limited its efficacy, which is also supported by Thierens et al. (2019), 34 who found better results with longer application periods. Polarized microscopy at 40x magnification confirmed these findings, with GCE nHAp showing the most extensive remineralization. In all treatment groups, the lesions showed a change from positive to negative birefringence, reflecting a notable reduction in lesion depth, which aligns with the results of the morphometric analysis. Thus, the second null hypothesis was rejected. In the present study, SEM images demonstrated the presence of a newly developed apatite layer on the surface in all the groups, which envelops the prismatic and interprismatic enamel structures. Moreover, the EDX analysis results also corroborated with that of microhardness and mean lesion depth, with the highest ratio seen in GCE nHAp group followed by CES nHAp group and lowest in CPP ACPF and FS nHAp. Thus, the third null hypothesis was rejected. The strength of this study lies in its comprehensive evaluation of various remineralizing agents which addresses a gap in remineralization research, particularly regarding sustainable and cost-effective alternatives to fluoride-based therapies. Furthermore, the use of multiple analysis methods, such as Vickers microhardness testing, PLM, and EDX allow for a detailed understanding of how each treatment interacts with enamel at both the structural and elemental levels. The incorporation of real-world conditions, such as using human maxillary premolars and simulation of the oral environment, enhances the study’s relevance and applicability. Inevitably, certain in-vitro limitations exist, such as the absence of human saliva, plaque, and varying susceptibility of teeth. Future in-vivo studies are needed to validate these results over longer periods. Conclusions Within the limitations of this study, all the tested agents demonstrated a remineralizing potential on early enamel caries. Galla chinensis extract combined with nanohydroxyapatite showed the highest remineralization potential across microhardness recovery, lesion depth reduction, and Ca/P ratio enhancement, outperforming other alternatives as well as the conventional CPP-ACPF. Chicken eggshell-derived nanohydroxyapatite showed superior outcomes compared to CPP-ACPF, whereas fish scale-derived nanohydroxyapatite exhibited a remineralization effect comparable to CPP-ACPF. These findings suggest that natural biomimetic materials, particularly plant-based combinations, represent promising adjuncts for enamel caries management. Declarations Conflict of Interest Statement The authors have no conflicts of interest to declare. Author Contributions Credit roles –Malik B: data curation, formal analysis, investigation, methodology, writing;Taneja S: conceptualization, validation, review and editing ;Mukherjee A: validation, review and editing, methodology;Pathak A: data curation, methodology, validation. 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Contemporary Clinical Dentistry [Internet]. 2019];10(3):489–93 Hassan NM, Zainab Juma Jafar, Abdul MH. Nano-hydroxyapatite preparation for the remineralization of primary tooth enamel surface subjected to liquid medication: An observational study. Health science reports. 2023;6(4). Thierens LAM, Moerman S, Elst C van, Vercruysse C, Maes P, Temmerman L, et al. The in vitro remineralizing effect of CPP-ACP and CPP-ACPF after 6 and 12 weeks on initial caries lesion. Journal of Applied Oral Science. 2019;27 Tables Table 1 to 3 are available in the Supplementary Files section. Additional Declarations There is no duality of interest Supplementary Files table1.docx Table 1 table2.docx Table 2 table3.docx Table 3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Taneja","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYLCCBwZgKoHhA4RhAEZ4QYIBWEUC4wwIgxgtUBXMPAwwLXiAfHvvwQ8JBX/kzdkbHn62Kfhjz8DevE2CoeAOTi0GZ84lSwAdZriz50CydI6BQWIDz7EyCQaDZ7i1SOQYgLQwbriRkADSksAgkWMG1HIYt8Nm5Bj/AGqx33D/QfJvCyCDQf4Nfi0MN4BmArUkbrjBkCbNALSuQYIHvxaDM2fMLBIMjJM3nElIs+wxME5s40krBorgcVh7j/GND3/kbDccP5N848cfOXt+9sMbgSJ4HIYAPAlgig1EJBCjgYGB/QBx6kbBKBgFo2DEAQCDC0+bX3RAtwAAAABJRU5ErkJggg==","orcid":"","institution":"I.T.S-CDSR","correspondingAuthor":true,"prefix":"","firstName":"Sonali","middleName":"","lastName":"Taneja","suffix":""},{"id":489934991,"identity":"1ac14872-3d77-46e5-badb-dc16c3a2ef18","order_by":1,"name":"Bazila Malik","email":"","orcid":"","institution":"I.T.S-CDSR","correspondingAuthor":false,"prefix":"","firstName":"Bazila","middleName":"","lastName":"Malik","suffix":""},{"id":489934992,"identity":"2db49dcf-6825-455e-b2dd-6f837f494484","order_by":2,"name":"Abhik Mukherjee","email":"","orcid":"","institution":"I.T.S-CDSR","correspondingAuthor":false,"prefix":"","firstName":"Abhik","middleName":"","lastName":"Mukherjee","suffix":""}],"badges":[],"createdAt":"2025-07-02 17:15:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7031614/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7031614/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87696583,"identity":"4452acf3-ca30-4f06-a98f-59211f3e2166","added_by":"auto","created_at":"2025-07-28 06:14:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":491486,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig121.png","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/e2ee6b6e65e7b88185ee17fb.png"},{"id":87696584,"identity":"e2df93de-2e24-4fe2-95bd-be47b39d5f4a","added_by":"auto","created_at":"2025-07-28 06:14:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1203081,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig122.png","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/e3465d9e664c2382dd1c1e0b.png"},{"id":87696582,"identity":"c699de52-ab89-49bd-afee-4d142d8eaacc","added_by":"auto","created_at":"2025-07-28 06:14:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1503630,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig123.png","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/4474238321ae8bbe0c5d0123.png"},{"id":87696586,"identity":"c246ca91-68ff-40d0-a55e-9a46985aba23","added_by":"auto","created_at":"2025-07-28 06:14:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98327,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig124.png","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/f0a8142800a89befa0b3c32c.png"},{"id":92406354,"identity":"2e7f503e-3f5d-46ba-aec9-6f200abed420","added_by":"auto","created_at":"2025-09-29 11:19:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4341048,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/d004ebe6-4b5c-4f42-bba2-72362b7218af.pdf"},{"id":87696577,"identity":"d7b85624-eecd-44bb-8b7d-1d9257f16412","added_by":"auto","created_at":"2025-07-28 06:14:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14821,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/fd939cd61dbf7f14fd19db39.docx"},{"id":87696579,"identity":"fd097088-6396-42a6-b601-d8fe91d8689d","added_by":"auto","created_at":"2025-07-28 06:14:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14950,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/215d66793d78be023c86752b.docx"},{"id":87696578,"identity":"9bafa17f-3db3-497d-8599-84ea50f9db4d","added_by":"auto","created_at":"2025-07-28 06:14:05","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14929,"visible":true,"origin":"","legend":"Table 3","description":"","filename":"table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7031614/v1/edde30f6887255a1bcc20a44.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Comparative evaluation of remineralizing potential of Galla chinensis with nanohydroxyapatite, chicken egg shell and fish scale derived nanohydroxyapatite on early enamel caries- A single-blinded in vitro study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDental caries is a multifactorial, biofilm-mediated disease resulting in enamel demineralization due to acidic conditions caused by bacterial metabolites. ¹ Enamel, composed of hydroxyapatite crystals, maintains equilibrium with saliva in a neutral pH environment.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e However, when pH falls below 5.5, hydrogen ions disrupt the crystal lattice, causing mineral loss and enamel breakdown. Remineralization occurs when pH is neutralized, and calcium and phosphate ions from external sources rebuild hydroxyapatite crystals.⁴ If demineralization exceeds remineralization over time, early enamel caries are formed, progressing to cavitations if untreated.⁵\u003c/p\u003e \u003cp\u003eFluoride is a widely used remineralizing agent that strengthens enamel by forming fluorapatite, which is less soluble in acidic environments.⁶ However, fluoride alone cannot completely remineralize carious lesions, as calcium and phosphate ions are also required.⁷ Additionally, excessive fluoride exposure may lead to fluorosis and other health issues. These limitations have prompted research into alternative remineralizing agents, such as casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), which furnishes calcium and phosphate ions to enhance remineralization.⁸ Combining CPP-ACP with fluoride (CPP-ACPF) has demonstrated superior results and is considered a gold standard.⁹\u003c/p\u003e \u003cp\u003eBiomimetic materials like nano-hydroxyapatite (nHA) offer promising remineralization potential due to their structural and chemical similarity to enamel crystals.¹⁰ nHA particles (nHAp), being smaller, dissolve more readily, fill enamel defects efficiently, and promote compact surface formation.\u003csup\u003e¹¹\u003c/sup\u003e Plant- and animal-derived products have also emerged as sustainable remineralizing agents. Galla chinensis extract (GCE), a natural Chinese herbal medicine, has been shown to prevent demineralization and enhance mineral deposition.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Animal-based sources like chicken eggshell powder and fish scales, rich in hydroxyapatite and calcium, offer biocompatible and biodegradable options. ¹⁴ Eggshell-derived nHA can penetrate enamel pores, while fish scales, containing collagen and hydroxyapatite, are a valuable biomaterial for remineralization.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, there are no studies that compare the remineralizing potential of plant-based remineralizing agents and animal-derived nanohydroxyapatite on the reversal of early enamel caries. Thus, this study aimed to evaluate the remineralizing potential of Galla chinensis with nanohydroxyapatite, chicken egg shell-derived nanohydroxyapatite, and fish scale-derived nanohydroxyapatite particles in comparison with CPP-ACPF on early enamel caries. In this study, the first null hypothesis tested was that there was no significant difference in the microhardness recovery of early enamel caries subjected to remineralization by Galla Chinensis and nanohydroxyapatite, Chicken egg shell derived nanohydroxyapatite, Fish scale derived nanohydroxyapatite, and CPP-ACPF. The second hypothesis tested was that there was no significant difference in the lesion depth of early enamel caries subjected to remineralization by Galla Chinensis and nanohydroxyapatite, Chicken egg shell derived nanohydroxyapatite, Fish scale derived nanohydroxyapatite and CPP-ACPF. The third hypothesis tested was that there was no significant difference in the Ca/P ratio of early enamel caries subjected to remineralization by Galla Chinensis and nanohydroxyapatite, Chicken egg shell derived nanohydroxyapatite, Fish scale derived nanohydroxyapatite and CPP-ACPF.\u003c/p\u003e "},{"header":"Materials \u0026 Methodology","content":"\u003cp\u003e \u003cem\u003eSpecimen collection\u003c/em\u003e \u003c/p\u003e\u003cp\u003eThis study was conducted in the Department of Conservative Dentistry \u0026amp; Endodontics in collaboration with Centre for Advanced Research at I.T.S Centre for Dental Studies \u0026amp; Research, Ghaziabad and KIET School of Pharmacy, Ghaziabad. The protocol was reviewed and approved by the institutional ethical clearance committee under protocol number ITSCDSR/IIEC/LD/CONS/2022-25/002.\u003c/p\u003e\u003cp\u003e \u003cem\u003eSpecimen size determination\u003c/em\u003e \u003c/p\u003e\u003cp\u003eThe sample size was calculated using the sample size calculator software program G. Power (version 3.1.9.7). The sample size calculation was based on 95% confidence interval and a power of 90%. The minimum sample size estimated for this study was one hundred forty eight.\u003c/p\u003e\u003cp\u003e \u003cem\u003eSpecimen selection\u003c/em\u003e \u003c/p\u003e\u003cp\u003eWith the patient’s consent, one hundred forty-eight human maxillary premolars extracted for orthodontic or periodontal reasons were collected for the study. The inclusion criteria included non-carious, intact crowns and teeth free from restorations. The samples were examined under an operating microscope (10X) to ensure there were no cracks, fractures, or anatomical developmental anomalies. The teeth were then disinfected by autoclaving at 121°C at 15 lbs psi and stored in freshly prepared artificial saliva Fusayama artificial saliva described by Olssom et al.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e containing NaCl (6.8 mmol/L), KCl (5.4 mmol/L), CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO (2.7 mmol/L), NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO (5.0 mmol/L), Na\u003csub\u003e2\u003c/sub\u003eS.9H\u003csub\u003e2\u003c/sub\u003eO (21 µmol/L), and Urea (16.7 mmol/L) \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e titrated to pH 7.0 with NaOHuntil further use.\u003c/p\u003e\u003cp\u003e \u003cem\u003eSpecimen preparation\u003c/em\u003e \u003c/p\u003e\u003cp\u003eThe crowns were detached from the roots using a diamond disc on a slow-speed handpiece and then divided into two halves along the mesio-distal axis to obtain buccal enamel samples (N = 148). The samples were embedded in acrylic resin with the buccal surface facing upward, and the enamel was sequentially polished using silicon carbide abrasive papers with grit sizes 1000, 600, and 320 to create flat surfaces. A 3x3 wax sheet was applied to the buccal surfaces to form a window, while the rest of the enamel was coated with acid-resistant nail varnish.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e \u003cem\u003eGrouping of samples\u003c/em\u003e \u003c/p\u003e\u003cp\u003eOut of a total of 148 samples, 48 were assigned for microhardness testing using Vicker’s Microhardness tester, 60 for mean lean depth using PLM, 10 for SEM analysis and 30 for EDX analysis .(Fig.\u0026nbsp;1)\u003c/p\u003e\u003cp\u003e \u003cem\u003eMicrohardness testing\u003c/em\u003e \u003c/p\u003e\u003cp\u003eForty-eight samples were used for microhardness testing. A Vickers hardness tester (Banbros, India) with a diamond indenter was used to measure the baseline surface microhardness (SMH\u003csub\u003e1\u003c/sub\u003e) of all 48 samples, applying a 500g load for 15 seconds.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e The samples were then demineralized to mimic early enamel caries using a solution containing 2.2 mM calcium chloride, 2.2 mM monosodium phosphate, and 0.05 M lactic acid. The pH was adjusted to 4.5 with 50% sodium hydroxide and confirmed using a calibrated digital pH meter.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Samples were immersed in this demineralizing solution in glass containers and incubated at 37°C for 96 hours. The solution was regularly replaced to maintain pH and prevent mineral buildup. After 96 hours, samples were rinsed with deionized water for 30 seconds, air-dried for 5 seconds, and stored in sterile containers. Microhardness was then re-evaluated to obtain the post-demineralization value (SMH\u003csub\u003e2\u003c/sub\u003e). Following this, the remineralization was done according to the assigned groups.\u003c/p\u003e\u003cp\u003e \u003cem\u003ePreparation of tested remineralizing agents\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eI) CPP-ACPF\u003c/p\u003e\u003cp\u003eThe commercially available GC Tooth Mousse Plus \u003csup\u003e®\u003c/sup\u003e was used.\u003c/p\u003e\u003cp\u003eII) GCE with nHAp\u003c/p\u003e\u003cp\u003e4000ppm GCE aqueous solution was made from GCE powder (BulkSupplemets.com) and mixed with equal proportion of 10% aqueous slurry of commercially available nanohydroxyapatite powder (Vedayukt India Private Limited). \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIII) CES nHAp\u003c/p\u003e\u003cp\u003eEggshells from a local hatchery were cleaned, dried, ground, and sieved. The powder was ball-milled for 5 days, and nanoparticles were formed through a precipitation reaction:\u003c/p\u003e\u003cp\u003eCaCO\u003csub\u003e3\u003c/sub\u003e + 2HCl → CaCl\u003csub\u003e2\u003c/sub\u003e + CO\u003csub\u003e2\u003c/sub\u003e + H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\u003cp\u003eThe solution was mixed with water and surfactant, then centrifuged. The particles were dried and used to prepare a 10% aqueous slurry of CES nHAp.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIV) FS nHAp\u003c/p\u003e\u003cp\u003eFish scales from Labeo rohita were sourced from a local market, washed, soaked in hydrochloric acid (HCl) to remove impurities, then dried. The scales were ground into particles, heated in a furnace, and wet-ground using ball milling for 48 hours. The resulting slurry was dried using a spray dryer to obtain FS nHAp powder.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e A 10% aqueous slurry of FS nHAp was then made with distilled water.\u003c/p\u003e\u003cp\u003e \u003cem\u003eThe pH cycling model\u003c/em\u003e \u003c/p\u003e\u003cp\u003e0.1M tris buffer (hydroxymethyl aminomethane), 0.05 ppm fluoride in the form of NaF, 0.9 mM phosphorus in the form of KH\u003csub\u003e2\u003c/sub\u003e PO\u003csub\u003e4\u003c/sub\u003e, and 1.5 mM calcium in the form of Ca(NO\u003csub\u003e3\u003c/sub\u003e )\u003csub\u003e2\u003c/sub\u003e with a pH of 7 was used to prepare the remineralizing solution. In the pH cycling model, the remineralizing agent from respective groups were applied to the samples for 5 minutes, followed by a 5-second rinse with deionized water. To mimic the daily acidic challenges in the oral environment, the teeth were then immersed in the demineralizing solution for 3 hours. Then the samples were placed in the prepared remineralizing solution for the remaining 21 hours of the 24-hour cycle. This process was repeated daily for 7 days, with the demineralizing and remineralizing solutions being replaced every 3 days.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAfter remineralization, microhardness was again evaluated to obtain post-remineralization surface microhardness (SMH\u003csub\u003e3\u003c/sub\u003e) in a similar manner.\u003c/p\u003e\u003cp\u003eThe percentage surface microhardness recovery (%SMHR) was recorded. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe formula used was:\u003c/p\u003e\u003cp\u003e%SMHR = (SMH\u003csub\u003e3\u003c/sub\u003e − SMH\u003csub\u003e2\u003c/sub\u003e)/( SMH\u003csub\u003e1\u003c/sub\u003e − SMH\u003csub\u003e2\u003c/sub\u003e) × 100%\u003c/p\u003e\u003cp\u003e \u003cem\u003eMean depth of lesion\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eSixty samples were used to evaluate the mean depth of enamel lesions. Demineralization was carried out as previously described. Twelve of the demineralized samples (demineralized control) were assessed for mean lesion depth using morphometric analysis and examined under polarized light microscopy (PLM). For this, the buccal surfaces were sectioned longitudinally into mesial and distal halves and ground to a thickness of 100–150 µm. After rinsing, the sections were placed on slides and viewed under PLM at 40x magnification. Photomicrographs were taken, and the demineralized areas were measured three times using ImageJ software to determine the mean lesion depth for each sample.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The remaining 48 samples were divided into four remineralization groups of twelve and treated with different remineralizing agents using the pH cycling model described earlier. After remineralization, lesion depth was re-evaluated.\u003c/p\u003e\u003cp\u003e \u003cem\u003eSEM Analysis\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eTen samples were used for SEM analysis. Demineralized was done as previously described. Two samples (demineralized control) were then prepared for SEM by undergoing ultrasonication for 10 minutes, rinsed with deionized water, air-dried, gold-sputtered, and examined under a scanning electron microscope (SEM) (Nova Nano SEM™, Thermo Fisher, USA) at 5000x magnification to observe enamel surface microstructure. \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e After remineralization using the pH cycling model, the remaining eight samples were similarly evaluated under SEM.\u003c/p\u003e\u003cp\u003e \u003cem\u003eEDX analysis\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eThirty samples were used for EDX analysis out of which 5 served as intact controls. The remianing 25 samples underwent demineralization. After this, 5 samples (demineralized control) were first subjected to EDX analysis (Bruker, UK) in order to assess the Calcium:Phosphte (Ca/P) ratio of enamel. Following this, the remineralization of 20 samples with 5 in each reminerlization group wasd done by the procedure as described earlier. Finally, EDX anaylisis of these remineralized samples was done.\u003c/p\u003e\u003ch2\u003eData Analysis:\u003c/h2\u003e\u003cp\u003eAll the collected data was entered in MS Excel. Descriptive statistics was performed by calculating mean and standard deviation for the continuous variables. The data was analyzed using IBM SPSS (Statistical Package for Social Sciences) 25.0 Version (IBM Corp 2013, New York, USA). Paired t-test was conducted for comparison of the hardness after demineralization and after remineralization. The intergroup comparison of the % SMHR was conducted using One-way ANOVA test (level of significance \u0026lt; 0.05). Pairwise comparison of %SMHR was done by Post hoc Tukey test (level of significance \u0026lt; 0.05). Inter-group comparison of mean lesion depth was done by One-way ANOVA test (level of significance \u0026lt; 0.05). Pairwise comparison of mean lesion depth was done using Post hoc Tukey test (level of significance \u0026lt; 0.005). Paired t-test was conducted for comparison of baseline, remineralization and demineralization Ca/P ratio (level of significance \u0026lt; 0.05). Inter-group comparison of Ca/P was done by One-way ANOVA test (level of significance \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe percentage of Surface Microhardness Recovery (%SMHR) was greatest in the GCE nHAp group, followed by the CES nHAp group, while the FS nHAp group exhibited the lowest %SMHR.Furthermore, the %SMHR in the FS nHAp group was significantly lower compared to both the GCE nHAp and CES nHAp groups, with no significant difference observed between the FS nHAp and CPP ACPF groups. (Table\u0026nbsp;1)\u003c/p\u003e \u003cp\u003eThe demineralized control group displayed the maximum mean lesion depth (\u0026micro;m), succeeded by the CPP ACPF group, while the GCE nHAp group had the least mean lesion depth (in Table\u0026nbsp;2, Fig.\u0026nbsp;2)\u003c/p\u003e \u003cp\u003eIntergroup analysis of Ca/P revealed that the most substantial increase was seen after remineralization in the GCE nHAp group, followed by the CES nHAp group, with the CPP ACPF and FS nHAp groups showing the lowest increase with no significant difference found between the CPP ACPF and FS nHAp groups.( Table\u0026nbsp;3, Fig.\u0026nbsp;3)\u003c/p\u003e \u003cp\u003eThe SEM pictures revealed that after demineralization, the treated enamel surface exhibited a loss of the enamel prism core while retaining the periphery. In contrast, the remineralized groups displayed a mineral layer formed on the enamel surface, effectively restoring the enamel prism defect. (Fig.\u0026nbsp;4)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEarly enamel caries is the first sign of demineralization and can be reversed or arrested with remineralization techniques. In recent years, natural plant- and animal-based products have garnered attention for their potential as remineralizing agents. Galla chinensis extract (GCE) is a plant-based traditional Chinese medicine that has demonstrated potential in remineralizing enamel. In addition to plant-based agents, animal-derived nHAp from sources such as chicken eggshells, fish scales, and other biowaste materials has shown promise for remineralization. The novelty of this study emerged from the use of natural agents for remineralization.\u003c/p\u003e \u003cp\u003eIn the present study, GCE with nHAp (Group 1) exhibited the highest remineralizing potential, as seen in the highest % surface microhardness recovery (%SMHR) among all groups. The possible reason for this might be the chemical components of GCE, such as tannins and gallic acid which act as calcium ion carriers, transferring calcium ions to enamel and promoting mineral crystal reproduction, thus aiding remineralization (Chu et al. 2007), \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Zhang et al. 2009).\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e GCE combined with nano-HA showed a higher mineral density compared to nHAp alone, as it enhanced mineral deposition, creating a synergistic effect. Huang et al. 2010\u003csup\u003e22\u003c/sup\u003e also reported that GCE\u0026thinsp;+\u0026thinsp;nHAp formed more uniform and regular surface crystals compared to nHAp alone. CES nHAp showed better remineralization than CPP-ACPF and FS nHAp. It could be attributed to the higher crystallinity and buffering properties of CES nHAp. Its irregular rod-like structure and smaller particle size provide a larger surface area for acid-neutralizing reactions, thus better protecting enamel from demineralization. This observation is supported by the study conducted by Mkhize et al. in 2023,\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e which determined that CES nHAp possesses a superior remineralizing capability in comparison to FS nHAp. CES nHAp demonstrated higher microhardness than CPP-ACPF, possibly due to its greater surface area and hydrophilic properties, which facilitated the precipitation of calcium and phosphate ions on the enamel surface (Kunam et al. 2019,\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Hassan et al. 2023).\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Meanwhile, CPP-ACPF also showed increased %SMHR, as CPP-ACP\u0026rsquo;s ability to release calcium and phosphate ions, in combination with fluoride, forms acid-resistant fluorapatite (Reynolds et al. 1998)⁷. Also, FS nHAp demonstrated significant microhardness recovery, comparable to CPP-ACPF, likely due to its HA content (Mkhize et al. 2023).\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Thus, the first null hypothesis was rejected.\u003c/p\u003e \u003cp\u003eThere was a significant reduction in mean lesion depth in Groups 1, 2, 3, and 4 compared to the demineralized control group, with the GCE nHAp group showing the greatest reduction. This may be due to mineral ions being deposited deeper in the lesion body rather than just the outer layer when GCE is applied. Studies by Cheng et al. (2008)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and Huang et al. (2010)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e confirm that GCE facilitates ion transfer into the lesion body, enhancing remineralization. When combined with nHAp, GCE showed improved results compared to nHAp alone, which was effective only at depths of 20\u0026ndash;40 \u0026micro;m, with minimal remineralization beyond that. The mean lesion depth in the CPP-ACPF, CES nHAp, and FS nHAp groups was also significantly reduced. nHAp aids remineralization by filling enamel defects and attracting calcium and phosphate ions to the surface, promoting crystal growth (Kunam et al. 2019).\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e However, the reduction in lesion depth of CES nHAp was less than that of GCE nHAp, possibly due to CES nHAp's mineral deposition being more concentrated in the outer layer, inhibiting deeper ion diffusion. CPP-ACPF, which supersaturates the micro-environment with calcium, phosphate, and fluoride ions, also showed reduced lesion depth which was less effective than GCE nHAp. The reason could be attributed to the shorter application period (7 days) which might have limited its efficacy, which is also supported by Thierens et al. (2019), \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e who found better results with longer application periods. Polarized microscopy at 40x magnification confirmed these findings, with GCE nHAp showing the most extensive remineralization. In all treatment groups, the lesions showed a change from positive to negative birefringence, reflecting a notable reduction in lesion depth, which aligns with the results of the morphometric analysis. Thus, the second null hypothesis was rejected.\u003c/p\u003e \u003cp\u003eIn the present study, SEM images demonstrated the presence of a newly developed apatite layer on the surface in all the groups, which envelops the prismatic and interprismatic enamel structures. Moreover, the EDX analysis results also corroborated with that of microhardness and mean lesion depth, with the highest ratio seen in GCE nHAp group followed by CES nHAp group and lowest in CPP ACPF and FS nHAp. Thus, the third null hypothesis was rejected.\u003c/p\u003e \u003cp\u003eThe strength of this study lies in its comprehensive evaluation of various remineralizing agents which addresses a gap in remineralization research, particularly regarding sustainable and cost-effective alternatives to fluoride-based therapies. Furthermore, the use of multiple analysis methods, such as Vickers microhardness testing, PLM, and EDX allow for a detailed understanding of how each treatment interacts with enamel at both the structural and elemental levels. The incorporation of real-world conditions, such as using human maxillary premolars and simulation of the oral environment, enhances the study\u0026rsquo;s relevance and applicability. Inevitably, certain in-vitro limitations exist, such as the absence of human saliva, plaque, and varying susceptibility of teeth. Future in-vivo studies are needed to validate these results over longer periods.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWithin the limitations of this study, all the tested agents demonstrated a remineralizing potential on early enamel caries. Galla chinensis extract combined with nanohydroxyapatite showed the highest remineralization potential across microhardness recovery, lesion depth reduction, and Ca/P ratio enhancement, outperforming other alternatives as well as the conventional CPP-ACPF. Chicken eggshell-derived nanohydroxyapatite showed superior outcomes compared to CPP-ACPF, whereas fish scale-derived nanohydroxyapatite exhibited a remineralization effect comparable to CPP-ACPF. These findings suggest that natural biomimetic materials, particularly plant-based combinations, represent promising adjuncts for enamel caries management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eCredit roles \u0026ndash;Malik B: data curation, formal analysis, investigation, methodology, writing;Taneja S: conceptualization, validation, review and editing ;Mukherjee A: validation, review and editing, methodology;Pathak A: data curation, methodology, validation.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors would like to express their sincere gratitude to ITS-CDSR and KIET School of Pharmacy for providing the facilities and support necessary for the successful completion of this study. We are grateful to our colleagues and mentors for their constructive feedback and encouragement throughout the course of this work.\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. Terminology of Dental Caries and Dental Caries Management: Consensus Report of a Workshop Organized by ORCA and Cariology Research Group of IADR. Caries Res. 2020;54(1):7\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarsh PD, Nyvad B. The oral microflora and biofilms on teeth. In Fejerskov O, Kidd EAM, editors, Dental Caries. The Disease and its Clinical Management. Oxford: Blackwell Publishing. 2003. p. 29\u0026ndash;48\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLARSEN MJ. Dissolution of enamel. European Journal of Oral Sciences. 1973;81(7):518\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato Y, Sato T, Niwa M, Aoki H. Precipitation of octacalcium phosphates on artificial enamel in artificial saliva. Journal of Materials Science: Materials in Medicine. 2006;17(11):1173\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGon\u0026ccedil;alves FMC, Delbem ACB, Gomes LF, Emerenciano NG, Pessan JP, Romero GDA, et al. Effect of fluoride, casein phosphopeptide-amorphous calcium phosphate and sodium trimetaphosphate combination treatment on the remineralization of caries lesions: An in vitro study. Archives of Oral Biology [Internet]. 2021 Feb 1 [cited 2021 Nov 14];122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeatherstone JDB. Prevention and reversal of dental caries: role of low level fluoride. Community Dentistry and Oral Epidemiology. 1999;27(1):31\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReynolds E. Calcium phosphate-based remineralization systems: scientific evidence? Australian Dental Journal. 2008;53(3):268\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbahrawy E, El -Dosoky A. Remineralization Potential of Chicken Eggshell Powder in The Treatment of Artificially Induced Enamel Carious lesion in Permanent Teeth. Egyptian Dental Journal. 2019;65(4):3581\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar V, Itthagarun A, King N. The effect of casein phosphopeptide-amorphous calcium phosphate on remineralization of artificial caries-like lesions: an in vitro study. Australian Dental Journal. 2008;53(1):34\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuntavee A, Juntavee N, Hirunmoon P. Remineralization Potential of Nanohydroxyapatite Toothpaste Compared with Tricalcium Phosphate and Fluoride Toothpaste on Artificial Carious Lesions. Pagano S, editor. International Journal of Dentistry. 2021;2021:1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. Journal of Dentistry. 2011;39(6):430\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou L, Zhang L, Li J, Hao Y, Cheng L, Li W, et al. Effect of Galla chinensis extract and chemical fractions on demineralization of bovine enamel in vitro. Journal of Dentistry. 2008;36(12):999\u0026ndash;1004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng L, Li J, Hao Y, Zhou X. Effect of compounds of Galla chinensis on remineralization of enamel surface in vitro. Archives of Oral Biology. 2010;55(6):435\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiva Rama Krishna D, Siddharthan A, Seshadri SK, Sampath Kumar TS. A novel route for synthesis of nanocrystalline hydroxyapatite from eggshell waste. Journal of Materials Science: Materials in Medicine. 2007;18(9):1735\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSampath V, Kunam D, Manimaran S, Sekar M. Evaluation of dentinal tubule occlusion and depth of penetration of nano-hydroxyapatite derived from chicken eggshell powder with and without addition of sodium fluoride: An in vitro study. Journal of Conservative Dentistry. 2016;19(3):239.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandile Cromwell Mkhize, Stanley Chibuzor Onwubu, Thabang Hendrica Mokhothu, Phumlane Selby Mdluli, Ajay Kumar Mishra. Comparative assessment of the remineralization characteristics of nano-hydroxyapatite extracted from fish scales and eggshells. Journal of Applied Biomaterials \u0026amp; Functional Materials. 2023;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlssom S., Berglund A., Bergman M. Release of elements due to electrochemical corrosion of dental amalgam. J. Dent. Res. 1994; 73:33\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMueller H.J. In vitro tarnish and corrosion of a consolidated silver material for direct filling applications. Dent. Mater. 2001; 17:60\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMukherjee, K., Ruan, Q., Moradian-Oldak, J. (2019). Peptide-Mediated Biomimetic Regrowth of Human Enamel In Situ. In: Papagerakis, P. (eds) Odontogenesis. Methods in Molecular Biology, vol 1922. Humana Press, New York, NY.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahiti JS, Krishna NV, Prasad SD, Kumar CS, Kumar SS, Babu KSC. Comparative evaluation of enamel microhardness after using two different remineralizing agents on artificially demineralized human enamel: An \u003cem\u003ein vitro\u003c/em\u003e study. J Clin Transl Res. 2020;6(3):87\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVicente, A., Ortiz-Ruiz, A.J., Gonz\u0026aacute;lez-Paz, B.M. \u003cem\u003eet al.\u003c/em\u003e Effectiveness of a toothpaste and a serum containing calcium silicate on protecting the enamel after interproximal reduction against demineralization. \u003cem\u003eSci Rep\u003c/em\u003e 11, 834 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang S, Gao S, Cheng L, Yu H. Combined effects of nano-hydroxyapatite and Galla chinensis on remineralisation of initial enamel lesion in vitro. Journal of Dentistry. 2010;38(10):811\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuamsrinuan N, Kaewwiset W, Limsuwan P, Naemchanthara K. Hydroxyapatite Synthesized from Waste Eggshell via Ball Milling. AMM. 2017;866:12\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuzalaf MAR, Hannas AR, Magalh\u0026atilde;es AC, Rios D, Hon\u0026oacute;rio HM, Delbem ACB. pH-cycling models for in vitro evaluation of the efficacy of fluoridated dentifrices for caries control: strengths and limitations. J Appl Oral Sci. 2010;18(4):316\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodrigues E, Delbem ACB, Pedrini D, Cavassan L. Enamel remineralization by fluoride-releasing materials: proposal of a pH-cycling model. Braz Dent J. 2010;21(5):446\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrundmann J, Golde J, Steuer S, Tetschke F, Kirsten L, Walther J, et al. Visualization of carious lesions with polarized and depolarized light microscopy. Biomed Opt Express. 2024;15(5):3018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajan R, Krishnan R, Bhaskaran B, Kumar SV. A Polarized Light Microscopic Study to Comparatively evaluate Four Remineralizing Agents on Enamel viz CPP-ACPF, ReminPro, SHY-NM and Colgate Strong Teeth. International Journal of Clinical Pediatric Dentistry. 2015;8(1):42\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng L, Shu M, Mei P, Liang Y, Xia L. Size-controllable synthesis of hydroxyapatite nanorods via fluorine modulation: applications in dental adhesives for enhanced enamel remineralization. BMC Oral Health. 2025;25(1):204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoggio C, Lombardini M, Vigorelli P, Ceci M. Analysis of dentin/enamel remineralization by a CPP-ACP paste: AFM and SEM study. Scanning. 2013;35(6):366\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu JP, Li JY, Hao YQ, Zhou XD. Effect of compounds of Galla chinensis on remineralisation of initial enamel carious lesions in vitro. Journal of Dentistry. 2007;35(5):383\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Deng X, Wu Y. Remineralizing Nanomaterials for Minimally Invasive Dentistry. Springer eBooks. 2015;173\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKunam D, Sampath V, Manimaran S, Sekar M. Effect of Indigenously Developed Nano-Hydroxyapatite Crystals from Chicken Egg Shell on the Surface Hardness of Bleached Human Enamel: An In Vitro Study. Contemporary Clinical Dentistry [Internet]. 2019];10(3):489\u0026ndash;93\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassan NM, Zainab Juma Jafar, Abdul MH. Nano-hydroxyapatite preparation for the remineralization of primary tooth enamel surface subjected to liquid medication: An observational study. Health science reports. 2023;6(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThierens LAM, Moerman S, Elst C van, Vercruysse C, Maes P, Temmerman L, et al. The in vitro remineralizing effect of CPP-ACP and CPP-ACPF after 6 and 12 weeks on initial caries lesion. Journal of Applied Oral Science. 2019;27\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 3 are available in the Supplementary Files section.\u003c/p\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":"","lastPublishedDoi":"10.21203/rs.3.rs-7031614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7031614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction: The earliest clinical sign of enamel caries is subsurface demineralization beneath an intact surface. At this stage, remineralizing agents can potentially reverse or halt lesion progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAim: To evaluate and compare the remineralization potential of plant-based Galla chinensis extract (GCE) with nanohydroxyapatite (nHAp), chicken eggshell-derived nHAp (CES nHAp), and fish scale-derived nHAp (FS nHAp), against casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), on early artificial enamel lesions in human premolars. Design: In-vitro study performed under pH cycling stimulating oral conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: A total of 148 specimens were taken. Among these, 48 samples were used for microhardness testing by Vicker’s hardness test, 60 for lesion depth evaluation using Polarized Light Microscopy (PLM), 30 for Energy Dispersive X-ray (EDX) analysis, and 10 for Scanning Electron Microscopy (SEM). All samples were demineralized except for five intact controls designated for baseline EDX assessment. Microhardness was measured before demineralization (SMH1), after demineralization (SMH2), and following remineralization (SMH3). These values were used to calculate the Surface Microhardness Recovery percentage (SMHR%). The demineralized samples were categorized into the following experimental groups based on the remineralizing agent applied: Demineralized control (no treatment); Group 1: CPP-ACPF; Group 2: GCE with nHAp; Group 3: CES nHAp; and Group 4: FS nHAp. Each group included 12 samples for microhardness testing, 12 for lesion depth evaluation, 5 for EDX, and 2 for SEM. A seven-day pH cycling model simulated oral conditions before post-remineralization analysis. PLM was utilized for morphometric lesion depth assessment. EDX was used to assess changes in calcium-to-phosphorus (Ca/P) ratio, and SEM evaluated surface topography.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults: GCE nHAp showed the highest surface microhardness recovery and the shallowest lesion depth, indicating effective, uniform remineralization. CES nHAp and CPP-ACPF demonstrated moderate potential, while FS nHAp was the least effective. EDX analysis revealed a significantly higher calcium-to-phosphorus (Ca/P) ratio in the GCE nHAp group. No significant difference was found between CPP-ACPF and FS nHAp. SEM images confirmed smoother, more uniform surfaces in the GCE group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusion: GCE combined with nHAp was the most effective remineralizing agent, followed by CES nHAp and CPP-ACPF. These results suggest biogenic materials from plant and animal sources may offer effective, biocompatible alternatives for non-invasive enamel caries treatment.\u003c/p\u003e","manuscriptTitle":"Comparative evaluation of remineralizing potential of Galla chinensis with nanohydroxyapatite, chicken egg shell and fish scale derived nanohydroxyapatite on early enamel caries- A single-blinded in vitro study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 06:14:00","doi":"10.21203/rs.3.rs-7031614/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":"9a4b4633-3617-473d-9229-02a7c9666df7","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52017254,"name":"Health sciences/Health care/Dentistry/Preventive dentistry/Minimal intervention dentistry"}],"tags":[],"updatedAt":"2025-09-29T11:10:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 06:14:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7031614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7031614","identity":"rs-7031614","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}