Effects of Four Different Desensitizing Agents on the Dentin Permeability of Teeth: An In Vitro Study

Effects of Four Different Desensitizing Agents on the Dentin Permeability of Teeth: An 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 Article Effects of Four Different Desensitizing Agents on the Dentin Permeability of Teeth: An In Vitro Study Lorenzo Bevilacqua, Gloria Driussi, Costanza Frattini, Gianluca Turco This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7772183/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 Objectives: Dentin hypersensitivity results from external stimuli altering dentinal fluid dynamics and activating nociceptors. Although multiple desensitizing agents are available, their comparative efficacy remains debated. This study aimed to evaluate four molecules—arginine-phosphate-zinc, calcium sodium phosphosilicate, nano-hydroxyapatite, and hydroxyapatite crystals—in reducing dentin permeability after 30 days in vitro. Materials and Methods: Fifty dentin sections from intact third molars were randomly assigned to experimental or control groups. Samples were brushed twice daily with toothpaste containing one of the test agents or fluoride alone (control). Dentin permeability was measured at baseline, 7, 15, and 30 days using a liquid mass flow meter. Remineralization was assessed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Surface changes and elemental composition were analyzed by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). Results: All desensitizing agents induced progressive reductions in dentin permeability. By day 15, permeability decreased by approximately 50% in all treated groups, with stabilization by day 30, except for a transient increase in one group. ATR-FTIR spectra revealed no significant changes in hydroxyapatite or organic peaks. SEM showed gradual dentinal tubule occlusion, while EDX confirmed the presence of calcium, phosphorus, oxygen, and carbon in treated samples. Conclusion: All tested agents effectively reduced dentin permeability after 30 days. These results support their clinical use and provide a basis for future randomized clinical trials to validate in vivo outcomes. Clinical Relevance: Use of toothpastes containing the tested desensitizing agents significantly reduced dentinal permeability and achieved occlusion of dentinal tubules as early as 14 days. dental hypersensitivity dentinal permeability desensitising agents hydrodynamic theory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 INTRODUCTION Dentinal hypersensitivity is defined as a brief, acute pain that originates from exposed dentin in response to a thermal, osmotic, chemical, or evaporative stimulus and that cannot be attributed to any other dental defect or pathology. (1–2) The pathogenesis of dentinal hypersensitivity is not fully clarified, and currently the most accepted theory is Brännströmm's 1963 hydrodynamic theory: an external stimulus causes a change in the flow of intratubular dentinal fluid that activates nerve fibers and mechanoreceptors leading to the nociceptive stimulus. (3) The prevalence of dentinal hypersensitivity reported in the literature is variable. Most review articles conclude that the prevalence of dentinal hypersensitivity ranges from 3 to 57% depending on the study sample. (4,5) In terms of gender, females tend to be affected more frequently than men. (6,7) Cuhna-Cruz et al. in 2013 report how the prevalence of hypersensitivity is higher in women with gum recessions who use toothpastes and/or whitening products. (8) Locally, it generally occurs in the buccal and cervical areas of the teeth and is very common in premolars and canines. (9) In addition, there may be local conditions that facilitate its occurrence: 49% of teeth with hypersensitivity are characterized by the presence of restorations, 54% by dentinal exposure, and 70% have gingival recessions. (10) General causes that may facilitate its occurrence may be non-carious enamel lesions, periodontal disease and occlusal trauma (11), surgical and non-surgical therapy (12) and bleaching treatments. (13) Pain caused by dentinal hypersensitivity has an impact on the level of perceived quality of life related to patients' oral health, leading to impairments in daily activities such as speaking, drinking, eating, and brushing teeth. (14) The stimuli that cause the onset of pain can be thermal (the most common being that caused by a cold stimulus), chemical (intake of acidic foods) and mechanical (brushing). (15,16) A proper differential diagnosis must be combined with a thorough history and clinical and instrumental examinations to rule out other pathologies that could affect the tooth: incipient caries, incongruous restorations, fractures, or inflammatory pulp disease. Diagnosis is performed using an exploratory probe run over the tooth surface (tactile test) or by applying a cold stimulus to the sensitive tooth (thermal test) or an air blast (air blast test) from the air-water syringe to evoke a response from the patient. The degree of pain severity can be quantifiable subjectively by the patient or by a VAS visual representation scale of pain intensity. The air blast test is the most accurate and easiest system for assessing dentin hypersensitivity: it involves a large area of dentin and is more widely used as a method of assessment than the tactile test, or the thermal test. (17) The main treatment strategies involve reducing the movement of dentinal fluid within the tubules or blocking the pulpal nerve response, but to significantly reduce hypersensitivity, there must also be a reduction in dentinal permeability and occlusion of the dentinal tubules. (18) The hydraulic conductance of a tissue expresses the rate at which a fluid can move through an area with a given pressure in a unit of time. Its value gives us an indication of dentinal permeability. (19) Permeability is determined by several variables: the pressure of the fluids moving in the dentin, the length of the tubules, the viscosity of the fluid, and the tubule radius. (20) Changes in the latter are used as a screening method on the effectiveness of desensitizing agents, including for domiciliary molecules. (21) Home treatment with toothpastes and mouthwashes is proven to be convenient, inexpensive, practical, and noninvasive. In most cases, the active ingredients used with desensitizing action work by blocking the exposure of the dentinal tubules by precipitation of salts, interrupting, thus, the pain caused by external stimuli. (22) Nowadays, there are different desensitizing molecules on the market, and some of the most effective ones in the literature are: Arginine, Novamin, HAF + bioactive complex and nano-hydroxyapatite. The technology that uses arginine as a desensitizing molecule for topical use is based on the mechanism proposed by Kleinberg et al. in 2002: the combination of arginine and calcium works by forming a plug that occludes the dentinal tubules. Being positively charged, arginine is attracted to the negative charge of the dentin surface; this promotes its adhesion together with calcium carbonate at both the dentinal and tubular levels. Studies also show that the association between arginine and calcium carbonate results in an alkaline environment, which encourages endogenous calcium and phosphate to be deposed by occluding dentinal tubules. (23) Bae JH et al. in 2015 showed reduced hypersensitivity support use of desensitizing toothpastes containing potassium, stannous fluoride, potassium and stannous fluoride, sodium and calcium phosphosilicate, and arginine for dentinal hypersensitivity, but not use of desensitizing toothpastes containing strontium. (24) Finally, Saba Arshad et al; 2021 showed that the Pro-Argin ™ molecule had significant effect in reducing hypersensitivity by 45.7%, 37.6% with the addition of strontium acetate 8%, 18.1% with fluoro-calcium phospho-silicate, and 15.2% with NaF-based toothpastes after one-minute application on sensitive teeth. In contrast, there was a reduction of 61.1% with fluoro-calcium phospho-silicate, 60.1% with Pro-ArginTM, 53.4% with strontium acetate 8% and 25% with NaF-based toothpastes after six consecutive weeks of application. (25) Up to now in the literature there is a lot of interest on the efficacy of bioactive molecules. Amorphous calcium and sodium phosphosilicate are a bioactive material composed of molecules also present in the human body. Calcium and phosphate ions interact with demineralized dentin and collagen present, allowing, by high affinity, to create new hydroxyapatite with protective function. (26) SEM analysis for evaluation of the remineralization and reduction potential of dentinal tubules. Calcium silicate and sodium phosphate completely occluded the dentinal tubules and formed the mineral HAP. The deposition of dentin on and within the dentinal tubules allows them to better resist acidic insults. (27) Zhu M et al. in 2015 demonstrated how sodium calcium phosphosilicate was more effective than negative group control in relieving dentin hypersensitivity at 6 weeks in both thermal and evaporative stimulus. (28) Nano-hydroxyapatite is considered one of the most compatible bioactive materials and is widely used in medicine and dentistry. Evidence has shown that nano-sized particles have very similar morphology, structure and crystallinity when compared to hydroxyapatite. The mechanism of action of nano-hydroxyapatite consists of the deposition of a layer of nanometer-sized particles of zinc carbonate and nano-hydroxyapatite on the surface of enamel and dentin: this fills enamel defects and seals exposed dentinal tubules, thus promoting regeneration and desensitizing effect. (29) Several studies have demonstrated the efficacy of nano-hydroxyapatite in reducing dentin hypersensitivity at 2 and 4 weeks. (30) Nowadays, hydroxyapatite partially substituted with fluoride and conjugated to a complex including carbonate, magnesium, strontium, chitosan, and finally fluoride that strengthens the newly formed mineral phase is also available. Specifically, chitosan, a natural polymer obtained by de-acetylation of chitin, improves attachment and residence time on tooth surfaces due to the adhesive properties of the biopolymer. (31) Degli Espositi L et al. in 2020 showed how treatment with this molecule can have a remineralising and desensitizing effect. (32) Up to now, several systematic reviews and meta-analyses have questioned the efficacy of toothpastes containing the above agents, but these studies were based on the desensitizing effect of a specific toothpaste component often using few samples. (33) Although there are studies in the literature demonstrating the efficacy of the above-mentioned desensitizing molecules, there are no studies comparing their objective efficacy and testing their actual duration of desensitizing effect. The objective of the present in vitro study is to understand which of the modern desensitized molecule arginine, calcium and sodium phosphosilicate, nano-hydroxyapatite, and hydroxyapatite crystals is most effective in reducing dentinal permeability in dental elements extracted at different time points. Dentinal permeability will be used as the primary objective variable of the effectiveness of different desensitizing principles in reducing intratubular fluid flow and consequently subjective dentinal hypersensitivity. Samples will also be analyzed by ATR/FTIR spectroscopy to understand the level of remineralization and by SEM for surface microanalysis of the sample through detailed dentinal microstructural analysis. The results will allow the identification of which desensitizing molecule is most effective and which clinical protocol can be suggested in the different clinical situations faced by the dental hygienist daily. MATERIALS AND METHODS Teeth and Dentin Preparation Twenty-five third molars extracted due to impaction or for orthodontic reasons were selected from healthy adult donors, following a protocol previously approved by the local Ethics Committee (protocol code 194/2019). The teeth were preserved by freezing and subsequently thawed on the day of testing. Two sections, each measuring 4.0 ± 0.1 mm in thickness, were obtained from each tooth using a low-speed water-cooled diamond blade microtome (Isomet 1000, Buehler Ltd, Lake Bluff, IL, USA) [Figure 1 ]. The sections were obtained by first sectioning each tooth along its longitudinal axis, followed by additional cuts made 4 mm to the right and 4 mm to the left of the central section [Figure 2 ]. Following meticulous removal of the pulp tissues, taking care not to contact the dentin walls, the pulp chamber was thoroughly rinsed with deionized water. External dentin side of the dental element was etched with 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4, for 2 min to remove the smear layer. Using a dedicated adhesive (Zapit, Dental Ventures of America Inc., Lewis Ct., Corona, CA) each section was bonded to a custom-designed support, which was 3D-printed using a polylactic acid (PLA) filament (Ultimaker 3 Extended, Utrecht, The Netherlands). The support featured a central channel with a diameter of 2 mm to allow for flow measurements [Figure 3 ]. The section was positioned with the residual pulp chamber facing the channel of the support [Figure 4 ]. The samples were then randomly divided into five groups according to four desensitizing agents contained in commercially available toothpastes used for at-home treatment of dentin hypersensitivity: Group A – Arginine (Elmex Sensitive Professional, Colgate-Palmolive, NYC) Group B – Nano-Hydroxyapatite (Biorepair Plus Sensitive Teeth, Coswell, Bologna, Italy) Group C – Sodium Calcium Phosphosilicate (Sensodyne Repair and Protect, Haleon, UK) Group D – Fluoride-substituted Hydroxyapatite conjugated with Chitosan and Potassium Salts (H.A.F.) + Bio-active complex (Carbonate, Magnesium, Strontium) (Curasept Biosmalto Sensitive Teeth, Curasept S.p.A., Varese, Italy) Group E – Control After being divided and labelled using a combination of letters and sequential numbers, the samples were placed into five separate containers and immersed in laboratory-prepared artificial saliva (AS), formulated as follows: KCl 12.92 mM, KSCN 1.95 mM, Na₂SO₄·10H₂O 2.37 mM, NH₄Cl 3.33 mM, CaCl₂·2H₂O 1.55 mM, NaHCO₃ 7.51 mM, ZnCl₂ 0.02 mM, MES 5 mM, at pH 5.0. The artificial saliva was replaced every 24 hours. The containers were kept in an incubator at a constant temperature of 37°C for the entire duration of the experiment (30 days). Dentin Permeability Dentin permeability was measured using an ASL1600-10 Media Isolated Liquid Mass Flow Meter (Sensirion AG, Staefa ZH, Switzerland) within a sealed chamber unit under a simulated pulpal pressure of 70 cm H₂O (equivalent to 6.86 kPa) (34). The upper reservoir was filled with room temperature deionized water and positioned 70 cm above the sample, which was connected to the flow meter located below [Figure 5 ]. After allowing the flow to stabilize, the measurement was conducted over a period of two minutes. Permeability flow rate was calculated as the average of the recorded flow values within this time interval. Attenuated Total Reflectance/Fourier Transform Infrared (ATR/FTIR) spectroscopy Attenuated Total Reflectance/Fourier Transform Infrared (ATR/FTIR) spectroscopy was subsequently used to assess the degree of sample remineralisation. ATR/FTIR spectra were acquired before and after treatment using a Nicolet iS50 FTIR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a diamond ATR accessory. Following the permeability testing, the samples were placed on the ATR diamond crystal with the pulpal surface facing upwards. Spectra were collected in the range of 800–1800 cm⁻¹ at a resolution of 4 cm⁻¹, using 64 scans. ATR/FTIR spectra, corrected for environmental background contributions, were analysed using OMNIC 8 software (Nicolet, Madison, WI, USA), and atmospheric interference was subtracted from each original spectrum. Scanning Electron Microscope Energy Dispersive X-ray Spectroscopy (SEM-EDS) Subsequently to ATR-FTIR spectroscopy, the same two samples from each group were dried and degassed in a glass desiccator connected to a vacuum pump for 48 hours, in order to remove as much air as possible and enable scanning electron microscopy analysis. The samples were then coated with a thin carbon layer using a vacuum sputter coater (Sputter Coater K550X, Emitech, Quorum Technologies Ltd, U.K.). A Scanning Electron Microscope (SEM) (FEI QUANTA-250, FEI, Oregon, USA) was then used to perform elemental surface analysis of the dentin following the different treatments. The SEM provided high-resolution, high-magnification images to examine dentinal tubule morphology and their degree of occlusion. In combination with SEM, Energy Dispersive X-ray Spectroscopy (EDS) was used to perform elemental analysis. This technique detects X-rays emitted by the sample when irradiated with electrons, with each element producing a characteristic X-ray wavelength. The technique allows both qualitative and quantitative identification of the chemical elements present in the sample. Desensitizing agents Subsequently, the application of the desensitizing agents to the dentin sections was carried out using an Oral-B Smart 4 oscillating-rotating toothbrush equipped with a pressure sensor and Oral-B Ultrathin bristle head. Brushing was performed directly on the dentin surface for 5 seconds per section, twice a day (morning and evening) for all five groups. Assessments of intratubular fluid flow permeability, ATR/FTIR spectroscopy, and SEM analysis were repeated for the two samples in each group at every time point (T1 = 7 days, T2 = 14 days, T3 = 21 days, and T4 = 28 days). RESULTS Table 1 reports the mean percentage values of dentin permeability measured using the flowmeter after flow stabilisation. At T0, all test groups exhibited 100% dentin permeability, followed by a progressive decrease at subsequent experimental time points. At T2, permeability was reduced by half in all groups, except for the control group, where it remained unchanged. At T3, dentin permeability was further reduced and stabilised across all groups, except for Group A, which showed an anomalous increase likely due to a laboratory error. At T4, permeability remained stable across all groups. Table 1 Dentin permeability expressed as percentage values, reported as mean ± standard deviation, Summary table showing dentin permeability measurements over time for all experimental groups. GROUP A GROUP B GROUP C GROUP D GROUP E T0 100 100 100 100 100 T1 80 ± 3 91 ± 5 72 ± 9 81 ± 9 93 ± 9 T2 46 ± 5 46 ± 5 51 ± 6 51 ± 8 91 ± 1 T3 63 ± 8 48 ± 7 44 ± 3 40 ± 5 90 ± 6 T4 39 ± 4 46 ± 6 51 ± 7 38 ± 11 92 ± 7 The infrared spectroscopy graphs (Fig. 6 and Fig. 7 ), obtained from the FT-IR analyses of the samples at different time points, show absorbance peaks corresponding to the functional chemical groups of the dentin components. Focusing on the spectral region between 800 and 1800 cm⁻¹, peaks are observed for inorganic phosphate groups PO₄³⁻ (1400–1000 cm⁻¹), characteristic of hydroxyapatite, as well as for carboxylic and secondary amine groups (1600–1400 cm⁻¹), which are attributable to the organic components of the dentin surface. Comparison of the spectra across different time points revealed no significant changes or shifts in the peak positions. In the SEM analysis (Fig. 8 and Fig. 9 ), whose images are shown below, a qualitative observation reveals the progressive occlusion of dentinal tubules: at baseline (T0), the tubules appear open, whereas at T4, they appear partially occluded. Figure 10 shows the graph resulting from the EDS (Energy Dispersive X-ray Spectroscopy) analysis, performed in combination with Scanning Electron Microscopy (SEM). This technique detects X-rays emitted by the sample when it is struck by an incident electron beam. On each sample at T4, the most relevant elements identified were carbon (C), oxygen (O), phosphorus (P), and calcium (Ca), which are all key constituents of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). DISCUSSION The results of the present study demonstrate significant efficacy of the tested molecules in occluding dentinal tubules, evidenced by the decrease of dentinal permeability as measured by flowmetry. Permeability halves in all treated groups after 14 days of treatment, in contrast to the control group, which maintains stable permeability between 90–93% over time. This finding clearly highlights the action of the tested molecules in reducing dentin permeability, a key mechanism in reducing hypersensitivity. The further reduction and stabilization of permeability observed at 21 and 28 days confirms the lasting efficacy of the treatment, suggesting stable tubule occlusion over time. The abnormal increase observed in group A at T3 could be attributable to a laboratory error, as hypothesized, and does not compromise the overall interpretation of the results. FT-IR analyses confirm the preservation of the chemical structure of dentin. The lack of significant changes or shifts in absorbance peaks related to inorganic phosphate PO 4 -3 functional groups and organic groups indicates that the molecules tested do not alter the intrinsic chemical composition of dentin. This finding supports the hypothesis that tubule occlusion occurs by a mechanism of "filling" and not altering the mineral structure. SEM-EDS analysis provides further visual support for the effectiveness of occlusion. SEM images show progressive occlusion of the dentinal tubules, confirming the quantitative data obtained by flowmetry. EDS analysis confirms the predominantly hydroxyapatite-based chemical composition (Ca, P, O) in the treated samples, excluding the presence of foreign elements or significant alterations in mineral composition. These results are consistent with the scientific literature demonstrating the efficacy of in reducing dentinal hypersensitivity (28, 35, 36, 37). The early reduction in dentinal permeability observed at T2, is consistent with studies that report a rapid desensitizing action of these compounds (30). The stability observed at successive time points (T3 and T4) suggests a lasting action, although further long-term studies would be needed to confirm this hypothesis. It is important to consider the limitations of the “in vitro” study. Experimental conditions, while attempting to mimic the oral environment, may differ from those “in vivo.” Factors such as saliva pH variation, presence of bacterial biofilms and occlusal forces were not considered in the present study. Further “in vivo” studies are therefore needed to validate the results obtained and evaluate the real clinical efficacy of the molecules tested. CONCLUSION The study demonstrates the efficacy of Arginine, Nano-Hydroxyapatite, Sodium Calcium Phosphosilicate, Fluoride-substituted Hydroxyapatite conjugated with Chitosan and Potassium Salts (H.A.F.) + Bio-active complex in occluding dentinal tubules and reducing dentinal permeability “in vitro.” Data obtained by flowmetry, FT-IR spectroscopy and SEM-EDS microscopy provide strong evidence to support this conclusion. The data obtained suggest promising potential for the use of these molecules in dentifrices for the treatment of dentin hypersensitivity. Declarations Conflict of interest and source of fundings The study was independently designed. The authors declare that they have no conflict of interest in relation of this paper. The study won the research prize Colgate-Palmolive Company and was supported by the fund of this prize. and source of fundings Author Contribution Lorenzo Bevilacqua contributed to conceptualization and design, methodology and interpretation, and critically revised the manuscript.Gloria Driussi participated as investigator and data curation.Costanza Frattini participated as critically revised the manuscriptGianluca Turco contributed to conceptualization and design, methodology, analysis and interpretation. Acknowledgement The authors gratefully acknowledge the linguistic expertise provided by Dr. Silvia Filippini. Data Availability The data that support the finding of this study are available from the corresponding author upon reasonable request. References Liu XX, Tenenbaum HC, Wilder RS, Quock R, Hewlett ER, Ren YF (2020) Pathogenesis, diagnosis and management of dentin hypersensitivity: an evidence-based overview for dental practitioners. 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PMID: 29787782 de Melo Alencar C, de Paula BLF, Guanipa Ortiz MI, Baraúna Magno M, Martins Silva C, Cople Maia L (2019) Clinical efficacy of nano-hydroxyapatite in dentin hypersensitivity: A systematic review and meta-analysis. J Dent. ;82:11–21. doi: 10.1016/j.jdent.2018.12.014. Epub 2019 Jan 3. PMID: 30611773 Lin PY, Cheng YW, Chu CY, Chien KL, Lin CP, Tu YK (2013) In-office treatment for dentin hypersensitivity: a systematic review and network meta-analysis. J Clin Periodontol. ;40(1):53–64. 10.1111/jcpe.12011 . Epub 2012 Oct 11. PMID: 23057701 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7772183","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":531697890,"identity":"6a15ec54-0027-462b-bb34-7818e318a50b","order_by":0,"name":"Lorenzo 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1","display":"","copyAsset":false,"role":"figure","size":505354,"visible":true,"origin":"","legend":"\u003cp\u003eSample’s sectioning.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/1d0026c57545b24a0c95313b.png"},{"id":93935561,"identity":"b0aeba1a-86c2-4ca7-a9ac-362b262a4c55","added_by":"auto","created_at":"2025-10-20 12:45:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":183094,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of sample’s orientation.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/c653f24ab02024cb33f32398.png"},{"id":93934642,"identity":"aff3c64f-4ccd-4000-9e79-63cb7a21001f","added_by":"auto","created_at":"2025-10-20 12:37:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126131,"visible":true,"origin":"","legend":"\u003cp\u003eCustom 3D-printed support (PLA) used to hold and bond dentin samples during permeability testing.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/d3dcb27897ce38a6e66e3194.png"},{"id":93934057,"identity":"09505425-98d4-4ecc-953a-3baa3ff3af4d","added_by":"auto","created_at":"2025-10-20 12:29:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":705995,"visible":true,"origin":"","legend":"\u003cp\u003eImage of a bonded sample positioned on the support during flowmeter measurement at baseline (T0), showing the outflow of deionized water from the open dentinal 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graph showing ATR/FT-IR spectrophotometry measurements at baseline (T0) for all experimental groups.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/e8c761cbaa52ce639ce1eed6.png"},{"id":93934650,"identity":"2da0c74f-3766-47de-9c15-bccc7c0358d2","added_by":"auto","created_at":"2025-10-20 12:37:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":242406,"visible":true,"origin":"","legend":"\u003cp\u003eSummary graph showing ATR/FT-IR spectrophotometry measurements at T4 (28 days) for all experimental groups.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/b81a8d998b6c849938c80345.png"},{"id":93934072,"identity":"c4072998-2485-4350-85e7-2ec994e1f48a","added_by":"auto","created_at":"2025-10-20 12:29:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1418234,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative scanning electron microscopy (SEM) images of dentin samples captured at different time points, illustrating the progression of dentinal tubule occlusion following treatment.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/984f4b806dfa4b960d67f865.png"},{"id":93934651,"identity":"10874315-205b-4bca-9225-e1aebfaaaa75","added_by":"auto","created_at":"2025-10-20 12:37:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":499959,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of images at 5000x and 50,000x.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/60e0be7289de885466224bc8.png"},{"id":93934647,"identity":"4f88b866-002e-4c69-8ef1-1a89f1e01cfb","added_by":"auto","created_at":"2025-10-20 12:37:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":77160,"visible":true,"origin":"","legend":"\u003cp\u003eGraph generated from SEM-EDS (Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy) analysis, showing the elemental composition of the dentin surface.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/c2eb7bbc42e32f0a384a2e3c.png"},{"id":96605547,"identity":"2b8872b5-cbe0-4300-8734-ee0f2db45731","added_by":"auto","created_at":"2025-11-24 09:23:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5042133,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7772183/v1/d807452e-d8df-4516-b723-e431c28ab163.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Four Different Desensitizing Agents on the Dentin Permeability of Teeth: An In Vitro Study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDentinal hypersensitivity is defined as a brief, acute pain that originates from exposed dentin in response to a thermal, osmotic, chemical, or evaporative stimulus and that cannot be attributed to any other dental defect or pathology. (1\u0026ndash;2)\u003c/p\u003e\u003cp\u003eThe pathogenesis of dentinal hypersensitivity is not fully clarified, and currently the most accepted theory is Br\u0026auml;nnstr\u0026ouml;mm's 1963 hydrodynamic theory: an external stimulus causes a change in the flow of intratubular dentinal fluid that activates nerve fibers and mechanoreceptors leading to the nociceptive stimulus. (3)\u003c/p\u003e\u003cp\u003eThe prevalence of dentinal hypersensitivity reported in the literature is variable. Most review articles conclude that the prevalence of dentinal hypersensitivity ranges from 3 to 57% depending on the study sample. (4,5)\u003c/p\u003e\u003cp\u003eIn terms of gender, females tend to be affected more frequently than men. (6,7) Cuhna-Cruz et al. in 2013 report how the prevalence of hypersensitivity is higher in women with gum recessions who use toothpastes and/or whitening products. (8)\u003c/p\u003e\u003cp\u003eLocally, it generally occurs in the buccal and cervical areas of the teeth and is very common in premolars and canines. (9)\u003c/p\u003e\u003cp\u003eIn addition, there may be local conditions that facilitate its occurrence: 49% of teeth with hypersensitivity are characterized by the presence of restorations, 54% by dentinal exposure, and 70% have gingival recessions. (10)\u003c/p\u003e\u003cp\u003eGeneral causes that may facilitate its occurrence may be non-carious enamel lesions, periodontal disease and occlusal trauma (11), surgical and non-surgical therapy (12) and bleaching treatments. (13)\u003c/p\u003e\u003cp\u003ePain caused by dentinal hypersensitivity has an impact on the level of perceived quality of life related to patients' oral health, leading to impairments in daily activities such as speaking, drinking, eating, and brushing teeth. (14)\u003c/p\u003e\u003cp\u003eThe stimuli that cause the onset of pain can be thermal (the most common being that caused by a cold stimulus), chemical (intake of acidic foods) and mechanical (brushing). (15,16)\u003c/p\u003e\u003cp\u003eA proper differential diagnosis must be combined with a thorough history and clinical and instrumental examinations to rule out other pathologies that could affect the tooth: incipient caries, incongruous restorations, fractures, or inflammatory pulp disease.\u003c/p\u003e\u003cp\u003eDiagnosis is performed using an exploratory probe run over the tooth surface (tactile test) or by applying a cold stimulus to the sensitive tooth (thermal test) or an air blast (air blast test) from the air-water syringe to evoke a response from the patient. The degree of pain severity can be quantifiable subjectively by the patient or by a VAS visual representation scale of pain intensity.\u003c/p\u003e\u003cp\u003eThe air blast test is the most accurate and easiest system for assessing dentin hypersensitivity: it involves a large area of dentin and is more widely used as a method of assessment than the tactile test, or the thermal test. (17)\u003c/p\u003e\u003cp\u003eThe main treatment strategies involve reducing the movement of dentinal fluid within the tubules or blocking the pulpal nerve response, but to significantly reduce hypersensitivity, there must also be a reduction in dentinal permeability and occlusion of the dentinal tubules. (18)\u003c/p\u003e\u003cp\u003eThe hydraulic conductance of a tissue expresses the rate at which a fluid can move through an area with a given pressure in a unit of time. Its value gives us an indication of dentinal permeability. (19)\u003c/p\u003e\u003cp\u003ePermeability is determined by several variables: the pressure of the fluids moving in the dentin, the length of the tubules, the viscosity of the fluid, and the tubule radius. (20)\u003c/p\u003e\u003cp\u003eChanges in the latter are used as a screening method on the effectiveness of desensitizing agents, including for domiciliary molecules. (21)\u003c/p\u003e\u003cp\u003eHome treatment with toothpastes and mouthwashes is proven to be convenient, inexpensive, practical, and noninvasive. In most cases, the active ingredients used with desensitizing action work by blocking the exposure of the dentinal tubules by precipitation of salts, interrupting, thus, the pain caused by external stimuli. (22)\u003c/p\u003e\u003cp\u003eNowadays, there are different desensitizing molecules on the market, and some of the most effective ones in the literature are: Arginine, Novamin, HAF\u0026thinsp;+\u0026thinsp;bioactive complex and nano-hydroxyapatite.\u003c/p\u003e\u003cp\u003eThe technology that uses arginine as a desensitizing molecule for topical use is based on the mechanism proposed by Kleinberg et al. in 2002: the combination of arginine and calcium works by forming a plug that occludes the dentinal tubules. Being positively charged, arginine is attracted to the negative charge of the dentin surface; this promotes its adhesion together with calcium carbonate at both the dentinal and tubular levels.\u003c/p\u003e\u003cp\u003eStudies also show that the association between arginine and calcium carbonate results in an alkaline environment, which encourages endogenous calcium and phosphate to be deposed by occluding dentinal tubules. (23)\u003c/p\u003e\u003cp\u003eBae JH et al. in 2015 showed reduced hypersensitivity support use of desensitizing toothpastes containing potassium, stannous fluoride, potassium and stannous fluoride, sodium and calcium phosphosilicate, and arginine for dentinal hypersensitivity, but not use of desensitizing toothpastes containing strontium. (24)\u003c/p\u003e\u003cp\u003eFinally, Saba Arshad et al; \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e showed that the Pro-Argin\u003csup\u003e\u0026trade;\u003c/sup\u003e molecule had significant effect in reducing hypersensitivity by 45.7%, 37.6% with the addition of strontium acetate 8%, 18.1% with fluoro-calcium phospho-silicate, and 15.2% with NaF-based toothpastes after one-minute application on sensitive teeth. In contrast, there was a reduction of 61.1% with fluoro-calcium phospho-silicate, 60.1% with Pro-ArginTM, 53.4% with strontium acetate 8% and 25% with NaF-based toothpastes after six consecutive weeks of application. (25)\u003c/p\u003e\u003cp\u003eUp to now in the literature there is a lot of interest on the efficacy of bioactive molecules.\u003c/p\u003e\u003cp\u003eAmorphous calcium and sodium phosphosilicate are a bioactive material composed of molecules also present in the human body. Calcium and phosphate ions interact with demineralized dentin and collagen present, allowing, by high affinity, to create new hydroxyapatite with protective function. (26)\u003c/p\u003e\u003cp\u003eSEM analysis for evaluation of the remineralization and reduction potential of dentinal tubules. Calcium silicate and sodium phosphate completely occluded the dentinal tubules and formed the mineral HAP. The deposition of dentin on and within the dentinal tubules allows them to better resist acidic insults. (27)\u003c/p\u003e\u003cp\u003eZhu M et al. in 2015 demonstrated how sodium calcium phosphosilicate was more effective than negative group control in relieving dentin hypersensitivity at 6 weeks in both thermal and evaporative stimulus. (28)\u003c/p\u003e\u003cp\u003eNano-hydroxyapatite is considered one of the most compatible bioactive materials and is widely used in medicine and dentistry. Evidence has shown that nano-sized particles have very similar morphology, structure and crystallinity when compared to hydroxyapatite.\u003c/p\u003e\u003cp\u003eThe mechanism of action of nano-hydroxyapatite consists of the deposition of a layer of nanometer-sized particles of zinc carbonate and nano-hydroxyapatite on the surface of enamel and dentin: this fills enamel defects and seals exposed dentinal tubules, thus promoting regeneration and desensitizing effect. (29)\u003c/p\u003e\u003cp\u003eSeveral studies have demonstrated the efficacy of nano-hydroxyapatite in reducing dentin hypersensitivity at 2 and 4 weeks. (30)\u003c/p\u003e\u003cp\u003eNowadays, hydroxyapatite partially substituted with fluoride and conjugated to a complex including carbonate, magnesium, strontium, chitosan, and finally fluoride that strengthens the newly formed mineral phase is also available. Specifically, chitosan, a natural polymer obtained by de-acetylation of chitin, improves attachment and residence time on tooth surfaces due to the adhesive properties of the biopolymer. (31)\u003c/p\u003e\u003cp\u003eDegli Espositi L et al. in 2020 showed how treatment with this molecule can have a remineralising and desensitizing effect. (32)\u003c/p\u003e\u003cp\u003eUp to now, several systematic reviews and meta-analyses have questioned the efficacy of toothpastes containing the above agents, but these studies were based on the desensitizing effect of a specific toothpaste component often using few samples. (33)\u003c/p\u003e\u003cp\u003eAlthough there are studies in the literature demonstrating the efficacy of the above-mentioned desensitizing molecules, there are no studies comparing their objective efficacy and testing their actual duration of desensitizing effect.\u003c/p\u003e\u003cp\u003eThe objective of the present in vitro study is to understand which of the modern desensitized molecule arginine, calcium and sodium phosphosilicate, nano-hydroxyapatite, and hydroxyapatite crystals is most effective in reducing dentinal permeability in dental elements extracted at different time points. Dentinal permeability will be used as the primary objective variable of the effectiveness of different desensitizing principles in reducing intratubular fluid flow and consequently subjective dentinal hypersensitivity. Samples will also be analyzed by ATR/FTIR spectroscopy to understand the level of remineralization and by SEM for surface microanalysis of the sample through detailed dentinal microstructural analysis.\u003c/p\u003e\u003cp\u003eThe results will allow the identification of which desensitizing molecule is most effective and which clinical protocol can be suggested in the different clinical situations faced by the dental hygienist daily.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTeeth and Dentin Preparation\u003c/h2\u003e\u003cp\u003eTwenty-five third molars extracted due to impaction or for orthodontic reasons were selected from healthy adult donors, following a protocol previously approved by the local Ethics Committee (protocol code 194/2019). The teeth were preserved by freezing and subsequently thawed on the day of testing. Two sections, each measuring 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mm in thickness, were obtained from each tooth using a low-speed water-cooled diamond blade microtome (Isomet 1000, Buehler Ltd, Lake Bluff, IL, USA) [Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]. The sections were obtained by first sectioning each tooth along its longitudinal axis, followed by additional cuts made 4 mm to the right and 4 mm to the left of the central section [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]. Following meticulous removal of the pulp tissues, taking care not to contact the dentin walls, the pulp chamber was thoroughly rinsed with deionized water. External dentin side of the dental element was etched with 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4, for 2 min to remove the smear layer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing a dedicated adhesive (Zapit, Dental Ventures of America Inc., Lewis Ct., Corona, CA) each section was bonded to a custom-designed support, which was 3D-printed using a polylactic acid (PLA) filament (Ultimaker 3 Extended, Utrecht, The Netherlands). The support featured a central channel with a diameter of 2 mm to allow for flow measurements [Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e]. The section was positioned with the residual pulp chamber facing the channel of the support [Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe samples were then randomly divided into five groups according to four desensitizing agents contained in commercially available toothpastes used for at-home treatment of dentin hypersensitivity:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eGroup A \u0026ndash; Arginine (Elmex Sensitive Professional, Colgate-Palmolive, NYC)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup B \u0026ndash; Nano-Hydroxyapatite (Biorepair Plus Sensitive Teeth, Coswell, Bologna, Italy)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup C \u0026ndash; Sodium Calcium Phosphosilicate (Sensodyne Repair and Protect, Haleon, UK)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup D \u0026ndash; Fluoride-substituted Hydroxyapatite conjugated with Chitosan and Potassium Salts (H.A.F.)\u0026thinsp;+\u0026thinsp;Bio-active complex (Carbonate, Magnesium, Strontium) (Curasept Biosmalto Sensitive Teeth, Curasept S.p.A., Varese, Italy)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup E \u0026ndash; Control\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAfter being divided and labelled using a combination of letters and sequential numbers, the samples were placed into five separate containers and immersed in laboratory-prepared artificial saliva (AS), formulated as follows:\u003c/p\u003e\u003cp\u003eKCl 12.92 mM, KSCN 1.95 mM, Na₂SO₄\u0026middot;10H₂O 2.37 mM, NH₄Cl 3.33 mM, CaCl₂\u0026middot;2H₂O 1.55 mM, NaHCO₃ 7.51 mM, ZnCl₂ 0.02 mM, MES 5 mM, at pH 5.0.\u003c/p\u003e\u003cp\u003eThe artificial saliva was replaced every 24 hours.\u003c/p\u003e\u003cp\u003eThe containers were kept in an incubator at a constant temperature of 37\u0026deg;C for the entire duration of the experiment (30 days).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDentin Permeability\u003c/h3\u003e\n\u003cp\u003eDentin permeability was measured using an ASL1600-10 Media Isolated Liquid Mass Flow Meter (Sensirion AG, Staefa ZH, Switzerland) within a sealed chamber unit under a simulated pulpal pressure of 70 cm H₂O (equivalent to 6.86 kPa) (34).\u003c/p\u003e\u003cp\u003eThe upper reservoir was filled with room temperature deionized water and positioned 70 cm above the sample, which was connected to the flow meter located below [Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter allowing the flow to stabilize, the measurement was conducted over a period of two minutes.\u003c/p\u003e\u003cp\u003ePermeability flow rate was calculated as the average of the recorded flow values within this time interval.\u003c/p\u003e\n\u003ch3\u003eAttenuated Total Reflectance/Fourier Transform Infrared (ATR/FTIR) spectroscopy\u003c/h3\u003e\n\u003cp\u003eAttenuated Total Reflectance/Fourier Transform Infrared (ATR/FTIR) spectroscopy was subsequently used to assess the degree of sample remineralisation.\u003c/p\u003e\u003cp\u003eATR/FTIR spectra were acquired before and after treatment using a Nicolet iS50 FTIR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a diamond ATR accessory.\u003c/p\u003e\u003cp\u003eFollowing the permeability testing, the samples were placed on the ATR diamond crystal with the pulpal surface facing upwards.\u003c/p\u003e\u003cp\u003eSpectra were collected in the range of 800\u0026ndash;1800 cm⁻\u0026sup1; at a resolution of 4 cm⁻\u0026sup1;, using 64 scans.\u003c/p\u003e\u003cp\u003eATR/FTIR spectra, corrected for environmental background contributions, were analysed using OMNIC 8 software (Nicolet, Madison, WI, USA), and atmospheric interference was subtracted from each original spectrum.\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscope Energy Dispersive X-ray Spectroscopy (SEM-EDS)\u003c/h3\u003e\n\u003cp\u003eSubsequently to ATR-FTIR spectroscopy, the same two samples from each group were dried and degassed in a glass desiccator connected to a vacuum pump for 48 hours, in order to remove as much air as possible and enable scanning electron microscopy analysis.\u003c/p\u003e\u003cp\u003eThe samples were then coated with a thin carbon layer using a vacuum sputter coater (Sputter Coater K550X, Emitech, Quorum Technologies Ltd, U.K.).\u003c/p\u003e\u003cp\u003eA Scanning Electron Microscope (SEM) (FEI QUANTA-250, FEI, Oregon, USA) was then used to perform elemental surface analysis of the dentin following the different treatments. The SEM provided high-resolution, high-magnification images to examine dentinal tubule morphology and their degree of occlusion.\u003c/p\u003e\u003cp\u003eIn combination with SEM, Energy Dispersive X-ray Spectroscopy (EDS) was used to perform elemental analysis. This technique detects X-rays emitted by the sample when irradiated with electrons, with each element producing a characteristic X-ray wavelength.\u003c/p\u003e\u003cp\u003eThe technique allows both qualitative and quantitative identification of the chemical elements present in the sample.\u003c/p\u003e\n\u003ch3\u003eDesensitizing agents\u003c/h3\u003e\n\u003cp\u003eSubsequently, the application of the desensitizing agents to the dentin sections was carried out using an Oral-B Smart 4 oscillating-rotating toothbrush equipped with a pressure sensor and Oral-B Ultrathin bristle head. Brushing was performed directly on the dentin surface for 5 seconds per section, twice a day (morning and evening) for all five groups. Assessments of intratubular fluid flow permeability, ATR/FTIR spectroscopy, and SEM analysis were repeated for the two samples in each group at every time point (T1\u0026thinsp;=\u0026thinsp;7 days, T2\u0026thinsp;=\u0026thinsp;14 days, T3\u0026thinsp;=\u0026thinsp;21 days, and T4\u0026thinsp;=\u0026thinsp;28 days).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e reports the mean percentage values of dentin permeability measured using the flowmeter after flow stabilisation. At T0, all test groups exhibited 100% dentin permeability, followed by a progressive decrease at subsequent experimental time points.\u003c/p\u003e\u003cp\u003eAt T2, permeability was reduced by half in all groups, except for the control group, where it remained unchanged.\u003c/p\u003e\u003cp\u003eAt T3, dentin permeability was further reduced and stabilised across all groups, except for Group A, which showed an anomalous increase likely due to a laboratory error.\u003c/p\u003e\u003cp\u003eAt T4, permeability remained stable across all groups.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDentin permeability expressed as percentage values, reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, Summary table showing dentin permeability measurements over time for all experimental groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGROUP A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGROUP B\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGROUP C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGROUP D\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGROUP E\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e91\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e46\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e63\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e92\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe infrared spectroscopy graphs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), obtained from the FT-IR analyses of the samples at different time points, show absorbance peaks corresponding to the functional chemical groups of the dentin components.\u003c/p\u003e\u003cp\u003eFocusing on the spectral region between 800 and 1800 cm⁻\u0026sup1;, peaks are observed for inorganic phosphate groups PO₄\u0026sup3;⁻ (1400\u0026ndash;1000 cm⁻\u0026sup1;), characteristic of hydroxyapatite, as well as for carboxylic and secondary amine groups (1600\u0026ndash;1400 cm⁻\u0026sup1;), which are attributable to the organic components of the dentin surface.\u003c/p\u003e\u003cp\u003eComparison of the spectra across different time points revealed no significant changes or shifts in the peak positions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the SEM analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), whose images are shown below, a qualitative observation reveals the progressive occlusion of dentinal tubules: at baseline (T0), the tubules appear open, whereas at T4, they appear partially occluded.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e shows the graph resulting from the EDS (Energy Dispersive X-ray Spectroscopy) analysis, performed in combination with Scanning Electron Microscopy (SEM).\u003c/p\u003e\u003cp\u003eThis technique detects X-rays emitted by the sample when it is struck by an incident electron beam.\u003c/p\u003e\u003cp\u003eOn each sample at T4, the most relevant elements identified were carbon (C), oxygen (O), phosphorus (P), and calcium (Ca), which are all key constituents of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe results of the present study demonstrate significant efficacy of the tested molecules in occluding dentinal tubules, evidenced by the decrease of dentinal permeability as measured by flowmetry.\u003c/p\u003e\u003cp\u003ePermeability halves in all treated groups after 14 days of treatment, in contrast to the control group, which maintains stable permeability between 90\u0026ndash;93% over time. This finding clearly highlights the action of the tested molecules in reducing dentin permeability, a key mechanism in reducing hypersensitivity. The further reduction and stabilization of permeability observed at 21 and 28 days confirms the lasting efficacy of the treatment, suggesting stable tubule occlusion over time. The abnormal increase observed in group A at T3 could be attributable to a laboratory error, as hypothesized, and does not compromise the overall interpretation of the results.\u003c/p\u003e\u003cp\u003eFT-IR analyses confirm the preservation of the chemical structure of dentin. The lack of significant changes or shifts in absorbance peaks related to inorganic phosphate PO\u003csub\u003e4\u003c/sub\u003e \u003csup\u003e-3\u003c/sup\u003e functional groups and organic groups indicates that the molecules tested do not alter the intrinsic chemical composition of dentin. This finding supports the hypothesis that tubule occlusion occurs by a mechanism of \"filling\" and not altering the mineral structure.\u003c/p\u003e\u003cp\u003eSEM-EDS analysis provides further visual support for the effectiveness of occlusion. SEM images show progressive occlusion of the dentinal tubules, confirming the quantitative data obtained by flowmetry. EDS analysis confirms the predominantly hydroxyapatite-based chemical composition (Ca, P, O) in the treated samples, excluding the presence of foreign elements or significant alterations in mineral composition.\u003c/p\u003e\u003cp\u003eThese results are consistent with the scientific literature demonstrating the efficacy of in reducing dentinal hypersensitivity (28, 35, 36, 37). The early reduction in dentinal permeability observed at T2, is consistent with studies that report a rapid desensitizing action of these compounds (30). The stability observed at successive time points (T3 and T4) suggests a lasting action, although further long-term studies would be needed to confirm this hypothesis.\u003c/p\u003e\u003cp\u003eIt is important to consider the limitations of the \u0026ldquo;in vitro\u0026rdquo; study. Experimental conditions, while attempting to mimic the oral environment, may differ from those \u0026ldquo;in vivo.\u0026rdquo; Factors such as saliva pH variation, presence of bacterial biofilms and occlusal forces were not considered in the present study. Further \u0026ldquo;in vivo\u0026rdquo; studies are therefore needed to validate the results obtained and evaluate the real clinical efficacy of the molecules tested.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe study demonstrates the efficacy of Arginine, Nano-Hydroxyapatite, Sodium Calcium Phosphosilicate, Fluoride-substituted Hydroxyapatite conjugated with Chitosan and Potassium Salts (H.A.F.)\u0026thinsp;+\u0026thinsp;Bio-active complex in occluding dentinal tubules and reducing dentinal permeability \u0026ldquo;in vitro.\u0026rdquo; Data obtained by flowmetry, FT-IR spectroscopy and SEM-EDS microscopy provide strong evidence to support this conclusion. The data obtained suggest promising potential for the use of these molecules in dentifrices for the treatment of dentin hypersensitivity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest and source of fundings\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was independently designed. The authors declare that they have no conflict of interest in relation of this paper. The study won the research prize Colgate-Palmolive Company and was supported by the fund of this prize.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eand source of fundings\u003c/span\u003e\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLorenzo Bevilacqua contributed to conceptualization and design, methodology and interpretation, and critically revised the manuscript.Gloria Driussi participated as investigator and data curation.Costanza Frattini participated as critically revised the manuscriptGianluca Turco contributed to conceptualization and design, methodology, analysis and interpretation.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the linguistic expertise provided by Dr. Silvia Filippini.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the finding of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu XX, Tenenbaum HC, Wilder RS, Quock R, Hewlett ER, Ren YF (2020) Pathogenesis, diagnosis and management of dentin hypersensitivity: an evidence-based overview for dental practitioners. 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PMID: 23057701\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"dental hypersensitivity, dentinal permeability, desensitising agents, hydrodynamic theory","lastPublishedDoi":"10.21203/rs.3.rs-7772183/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7772183/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eDentin hypersensitivity results from external stimuli altering dentinal fluid dynamics and activating nociceptors. Although multiple desensitizing agents are available, their comparative efficacy remains debated. This study aimed to evaluate four molecules—arginine-phosphate-zinc, calcium sodium phosphosilicate, nano-hydroxyapatite, and hydroxyapatite crystals—in reducing dentin permeability after 30 days in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods: \u003c/strong\u003eFifty dentin sections from intact third molars were randomly assigned to experimental or control groups. Samples were brushed twice daily with toothpaste containing one of the test agents or fluoride alone (control). Dentin permeability was measured at baseline, 7, 15, and 30 days using a liquid mass flow meter. Remineralization was assessed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Surface changes and elemental composition were analyzed by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAll desensitizing agents induced progressive reductions in dentin permeability. By day 15, permeability decreased by approximately 50% in all treated groups, with stabilization by day 30, except for a transient increase in one group. ATR-FTIR spectra revealed no significant changes in hydroxyapatite or organic peaks. SEM showed gradual dentinal tubule occlusion, while EDX confirmed the presence of calcium, phosphorus, oxygen, and carbon in treated samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eAll tested agents effectively reduced dentin permeability after 30 days. These results support their clinical use and provide a basis for future randomized clinical trials to validate in vivo outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Relevance: \u003c/strong\u003eUse of toothpastes containing the tested desensitizing agents significantly reduced dentinal permeability and achieved occlusion of dentinal tubules as early as 14 days.\u003c/p\u003e","manuscriptTitle":"Effects of Four Different Desensitizing Agents on the Dentin Permeability of Teeth: An In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 12:29:21","doi":"10.21203/rs.3.rs-7772183/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":"a21dbdc5-e438-49a6-93df-84dc82972062","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-22T17:23:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-20 12:29:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7772183","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7772183","identity":"rs-7772183","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}