Intratubular crystal formation in the exposed dentin from nano-sized calcium silicate for dentin hypersensitivity treatment

Intratubular crystal formation in the exposed dentin from nano-sized calcium silicate for dentin hypersensitivity treatment | 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 Article Intratubular crystal formation in the exposed dentin from nano-sized calcium silicate for dentin hypersensitivity treatment Mi-Jeong Jeon, Jeong-Won Park, Deog-Gyu Seo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2093584/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Aug, 2023 Read the published version in Scientific Reports → Version 2 posted 8 You are reading this latest preprint version Show more versions Abstract The aim of this study is to evaluate intratubular crystal formation from the experimental material consisting of dicalcium silicate (C 2 S) and tricalcium silicate (C 3 S) with nano-scaled particle size. A total of twenty-four specimens were made by isolating 8 mm of the cervical part centered at the cementoenamel junction of extracted premolars. Twelve specimens were not treated and considered as control. The experimental material was applied to the other twelve specimens by brushing for 10,000 strokes. Each group was randomly divided into four subgroups according to the period of immersion in phosphate buffer saline (PBS) for 1, 30, 60, and 90 days each. The specimens were sectioned longitudinally and examined with scanning electron microscopy and energy dispersion X-ray spectroscopy. The intratubular crystal were formed in PBS and densely filled the dentinal tubules over time. The crystal formation occurred at a depth of more than 50 μm from the dentin surface. The Ca/P ratio of formed intratubular crystals was 1.68 after three months. The experimental material consisting of C 2 S and C 3 S with a nanoscale particle size can form hydroxyapatite-like crystals in dentinal tubules in PBS, and there is a possibility of reducing dentin hypersensitivity by blocking the dentinal fluid flow. Health sciences/Diseases/Dental diseases Physical sciences/Materials science/Biomaterials/Biomineralization Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Dentin hypersensitivity (DH) is defined as “pain arising from exposed dentin in response to stimuli, typically thermal, evaporative, tactile, osmotic or chemical, which cannot be ascribed to any other form of dental defect or pathology” 1 . According to hydrodynamic theory, teeth with dentin hypersensitivity have opened dentinal tubules with pulpal patency 2 , a channel for stimulation transmission by moving fluids within the tubules 3 . There are several approaches to reduce discomfort from DH. Potassium nitrate could decrease the excitability of the intradental nerve fibers and achieve pain relief 4 . Although the effect of relief dentin hypersensitivity appears immediately, it does not last long 5 , and it is no substantial evidence supporting its efficacy 6 . Another approach is occluding the tubules by hindering the tubular fluid movement. The concept of tubule occlusion is a logical extension of the hydrodynamic theory 7 . Products including strontium, bioactive glass particles, amorphous calcium phosphate, arginine, oxalate, and fluoride can be found on the market using ions and salt. The limitations of these desensitizers are related to lack of intratubular occlusion due to large particle size and high solubility of the occluding materials, and short duration of effective time due to poor resistance to acid attacks 8-10 . Moreover, the desensitizing effects depend significantly on the individual’s sensory threshold 11 . Effective occlusion of dentinal tubules is difficult due to the small dimensions of dentinal tubules (0.5-4 μm in diameter) 2 , the complex structure of a pulp-dentin system, the outward hydraulic pressure of dental pulp (0.15 kg/cm 2 ) 12 . For effective management of dentine hypersensitivity, new materials are required to penetrate deep enough into dentinal tubules, last a long time, and not easily wash out with the oral environment 13 . Various types of materials, such as bioceramic, synthetic polymer, and peptides, were attempted as an alternative desensitizer. However, these alternative materials did not completely overcome the disadvantages of the existing desensitizers mentioned above 14,15 . Calcium silicate, can form calcium phosphate precipitation when contacted with physiological fluid containing phosphate 16,17 . A tag-like structure in the dentinal tubule was observed after calcium silicate based sealer application during the root canal filling procedure 18 . However, no studies have used calcium silicate as a desensitizer and applied it to outer dentin surface over a period time. The aim of this study was to evaluate that intratubular crystals were formed in dentinal tubules when experimental material consisting of dicalcium silicate (2CaO.SiO 2 , C 2 S) and tricalcium silicate (3CaO.SiO 2 , C 3 S) was applied to the exposed outer dentin surface under PBS for three months. Results Figure 1 represented SEM images of a longitudinally sectioned specimen immersed for two months in PBS after applying the experimental material consisting of C 2 S and C 3 S. There were plug shape precipitates below the dentinal tubule orifice. The lower surface of the occluding plug became rough, and plate-shaped intratubular crystals were formed below the occluding plug. The intratubular crystals were formed at a depth of more than 50 µm from the surface of the exposed dentin surface (Fig. 1 white arrows). Figure 2 shows the SEM images of longitudinally sectioned specimens. There was no intratubular crystal formation in all specimens for the entire observation period in the control group (white arrows in a). In the experimental group, the roughening of the inner surface of the dentinal tubules was observed after 1 day, and plate-shaped crystals were formed in the dentinal tubules after two months. As the period stored in the media gets longer, the plate-shaped crystals are linked together to form a denser collection (white arrows in b). The composition of the intratubular crystals in PBS was evaluated using EDS (Table 1 ). After immersion in PBS for 1 day, the Ca/P ratio of the crystal was 1.57. Then, the Ca/P ratio increased to 1.68 after 90 days, which meant the crystals from C 2 S/C 3 S in PBS were supposed to be the hydroxyapatite-like crystal, of which the Ca/P ratio was 1.67. Table 1 Ca/P ratio of the intratubular crystals. Time 1d 30d 60d 90d Ca/P 1.57 1.59 1.64 1.68 * Hydroxyapatite (HAp), Ca 10 (PO 4 ) 6 (OH) 2 , Ca/P = 1.67 Discussion According to hydrodynamic theory, blocking the opened dentinal tubules could reduce discomfort from dentin hypersensitivity by reducing fluid movement through dentinal tubules 3 . In this study, the intratubular crystals were formed in PBS after the experimental material was applied to the exposed dentin surface and filled the dentinal tubules more densely as the period of storage in PBS increased. In the case of intratubular crystal formation in previous study 18 , when using a calcium silicate based sealer for root canal treatment, calcium silicate was continuously present inside the root canal and in contact with the dentinal tubules. For dentin hypersensitivity treatment, a desensitizer should be applied to the dentin surface exposed to the outside, which is not in contact with the surface of dentin continuously exposed due to physical and chemical factors in the oral cavity. Therefore, in order to improve intratubular occlusion, desensitizer must efficiently penetrate the dentinal tubules. In this study, two efforts were made to achieve efficient penetration of experimental particles as greater penetration of particles into the dentinal tubules would have occurred. First, the experimental used in this study consisted of particles with a smaller diameter than in other studies using calcium silicate (lower than 85 µm or 0.5 mm) 19 , 20 or calcium silicate in commercially available mineral trioxide aggregate (MTA) products 21 . The diameter of dentinal tubules of the sensitive dentin is larger than that of the non-sensitive dentin but is only 0.83 µm on average 2 . The small dimension of dentinal tubules makes it difficult for desensitizers to be efficiently penetrated 12 . According to a study by Kim et al. 22 , comparing the degree of absorption according to the particle size of the desensitizer, the desensitizer with small-sized particles had more opportunities to penetrate the dental tubules. Therefore, the experimental materials consisting of specially designed nano-scale particles used in this study would effectively penetrate the dentinal tubules and form occluding plugs, as in other studies 23 . Second, C 2 S/C 3 S were applied as a brushing motion to the dentin surface. This simulated that calcium silicate was contained in toothpaste and applied to the surface of the dentin when brushing. A total of 10,000 repeated strokes (1 strokes/second) of brushing simulated about 18.5 days assuming brushing three minutes/time and three times a day 24 . It can have a chance to push desensitizing materials into dentinal tubules compared with just dropping or rubbing with a micro-brush on the specimen surface. As a result, the occluding plug was formed below the dentinal tubule orifice in applying experimental material to the brushing motion (Fig. 1 ). The occluding plugs can act as a reservoir of calcium ions, continuously dissolving calcium ions and making the inside of the dentinal tubules into a supersaturation state 25 . The supersaturation condition was expected to cause local aggregation of calcium ions and phosphate ions due to interaction with ions present on the inner surface of the dentinal tubules, which caused the growth of the intratubular crystals 23 . This reaction can be inferred that the lower part of the occluding plug was rough, and plate-shaped crystals were formed below (Fig. 1 b white arrowheads). The intratubular crystal formation reaction by diffusion according to the concentration gradient of ions can occur at a deep point in the dentinal tubules. In this study, the intratubular crystals were formed at a depth of more than 50 µm from the exposed dentin surface (Fig. 1 a white arrows). In a clinical situation, superficial occlusion of dentinal tubules has a short-term effect as the precipitates can be easily removed due to daily tooth brushing, dissolution by saliva, and acidic beverages 26 . For the long-term effect of desensitizer, the material blocking the dentinal tubules should be deep enough 27 . Crystal formation reaction continuously takes place in dentinal tubules forming denser crystal complex as the period of storage in PBS increased (Fig. 2 ). The crystal complex is expected to contribute to preventing the movement of the pulpal fluid through the dentinal tubules and reduce discomfort from dentin hypersensitivity more effectively 7 , 28 . Further research will be needed on the clinical effectiveness of the experimental material. When C 2 S and C 3 S are in contact with water, they are hydroxylated, and the surface dissolves according to below 29 ; $$2(2CaO\bullet Si{O}_{2})+4{H}_{2}O\to 3CaO\bullet 2Si{O}_{2}\bullet 3{H}_{2}O+Ca{\left(OH\right)}_{2}$$ 1 $$2(3CaO\bullet Si{O}_{2})+6{H}_{2}O\to 3CaO\bullet 2Si{O}_{2}\bullet 3{H}_{2}O+3Ca{\left(OH\right)}_{2}$$ 2 As a result of these reactions, calcium and hydroxyl ions are released, resulting in a highly alkaline environment 30 . After the hydration reaction of calcium silicate, the hydroxyapatite-like crystals were formed under contact with PBS 31 , 32 . A previous study showed calcium-deficient and B-type carbonated apatite with a 1.4–1.5 Ca/P ratio formed from Portland cement in PBS 32 . Other studies showed calcium silicate in PBS could make hydroxyapatites after hydration 33 – 35 . In this study, the Ca/P ratio of formed intratubular crystals was 1.68 after three months. Considering that the Ca/P ratio of hydroxyapatite is 1.67 36 , it can be expected that the intratubular crystals made in this experiment would be hydroxyapatite-like crystals. However, this experimental design could not simulate an oral environment, such as an acid challenge from the diet. Therefore, further studies need to be performed to evaluate the effect of the acid-neutral cycle on intratubular crystal formation. This study demonstrated that the experimental material could form intratubular crystals in PBS after being applied to the exposed dentin surface. The crystal formation occurred at more than 50 µm from the dentin surface, and the crystals more densely filled the dentinal tubules over time. The experimental material consisting of C 2 S and C 3 S with nano-scaled particle size used in this study allowed for effective penetration to dentinal tubules and the formation of intratubular crystals, which makes the material a promising alternative for clinical use to reduce discomfort from dentin hypersensitivity. Methods Specimen preparation. This study was approved by the Ethics Committee of the Seoul National University, Graduate School of Dentistry (IRB number: S-D20190010). Total twenty-four recently extracted human premolars for orthodontic treatment with intact coronal and root surfaces were prepared. They were stored in 0.1% thymol solution to inhibit microbial growth for no longer than three months prior to use. Debris on the surface of all teeth was removed by perio-curette and examined whether there were crack lines under a microscope (200×) (Carl Zeiss Surgical GmbH, Oberkochen, Germany). A specimen with a total length of 8 mm was made by cutting the 4 mm above and below the cemento-enamel junction with a low-speed diamond saw (Isomet™, Buehler, Lake Bluff, IL, USA) under constant water cooling. Flat and fresh dentin was exposed on the occlusal side surface without perforation at the pulp horn area. Remained pulp tissue was removed carefully with small forceps without touching the inner part of the pulpal space. Then, the sectioned tooth was mounted in the ring-shaped acrylic mold with self-cured acrylic resin (Bosworth Fastray, Keystone Industries GmbH, Singen, Germany) (Fig. 3 ). The exposed upper dentin surface of each specimen was treated with 17% ethylenediaminetetraacetic acid (EDTA) for 1 minute, followed by 2.5 mL of 5.25% sodium hypochlorite to open dentinal tubules and remove the smear layer on the exposed dentin surface and rinsed with 10 mL of distilled water twice. C 2 S/C 3 S preparation and application. The experimental material consisting of dicalcium silicate and tricalcium silicate (C 2 S/C 3 S) was prepared by a sol-gel method as Zhao W and Chang J 37 using CaCO3, SiO2, Al2O3 as the raw materials and the obtained material calcined at 1,450 ℃ for six hours. The resultant powders were ground at 300 rpm using a Disk Mill (Disk Mill, KM tech, Icheon, Republic of Korea) and then at 200 rpm using a Ball Mill (BML-2, DAITHAN SCIENTIFIC GROUP, Wonju-si, Republic of Korea) for 48 hours. Table 2 shows the main component and content of the experimental material. The particle size of the experimental material was distributed between 0.052 µm (D0.1) – 1.267 µm (D0.9), and the median value (D0.5) is 0.184µm (Fig. 4 ). Table 2 Experimental material of the study. Groups (n = 12) Component Content (wt%) C 2 S/C 3 S Dicalcium silicate (2CaO.SiO 2 , C 2 S) 10 ~ 15 Tricalcium silicate (3CaO.SiO 2 , C 3 S) 70 ~ 80 Others 5 After preparation of the specimens, 0.5g of C 2 S/C 3 S was mixed with 5 ml of distilled water and applied on the exposed dentin surface of the specimen by tooth brushing motion according to ISO 11609 standards for dentin wear test, abrasive in the dentifrice is about 10% of dentifrice and water mixture. The concentration of tricalcium silicate applied is followed this standard. A total of 10,000 repeated strokes (1 strokes/second) were applied using the toothbrush onto each specimen under a 150 g-load continuously being touched among test material mixtures and the exposed dentin surface. The specimens were randomly divided into four subgroups according to the period of immersion in PBS (D8662, Sigma-Aldrich, St. Louis, MO, U.S.A.) media for 1, 30, 60, and 90 days each at 37 ℃ (n = 3). The PBS solution was replaced every seven days. The composition (in g/L) of used PBS was CaCl 2 •2H 2 O 0.133, MgCl 2 •6H 2 O 0.1, KCI 0.2, KH 2 PO 4 0.2, NaCl 8.0, Na 2 HPO 4 (anhydrous) 1.15, and the pH was 7.4. Scanning Electron Microscope analysis and EDS analysis. The specimens were longitudinally sectioned, and six sectioned surfaces in each group were examined to assess crystal formation in dentinal tubules after experimental material application on exposed upper dentin surfaces. All specimens were mounted on aluminum stubs and sputter-coated with a 30 nm layer of gold and examined using field emission scanning electron microscopy (FE-SEM, Apreo S; Thermo Fisher SCIENTIFIC, Waltham, MA, U.S.A.). The intratubular crystals were examined by energy dispersive spectroscopy (EDS, XFlash 6160, Bruker, Germany) to analyze the components. Declarations Ethics declarations This study was approved by the Ethics Committee of the Seoul National University, Graduate School of Dentistry (IRB number: S-D20190010). All biological samples were included after obtaining the informed consent from all subjects. All methods were conducted in accordance with Declarations of Helsinki. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2020R1F1A1076307). Author contributions M.-J.J. designed the analysis, collected the data, performed the analysis, and wrote the main manuscript text. J.-W.P. designed and performed the analysis. D.-G.S. designed experiment, performed the analysis, contributed analysis method and tool. All authors reviewed and edited the manuscript. Additional information Competing interests The authors declare no competing interests. Correspondence and requests for materials should be addressed to D.-G.S. Reference Hypersensitivity, C. A. B. o. D. Consensus-based recommendations for the diagnosis and management of dentin hypersensitivity. J. Can. Dent. Assoc. 69 , 221-226 (2003). Absi, E. G., Addy, M. & Adams, D. Dentine hypersensitivity: a study of the patency of dentinal tubules in sensitive and non‐sensitive cervical dentine. J. Clin. Periodontol. 14 , 280-284, doi:10.1111/j.1600-051X.1987.tb01533.x (1987). Brannstrom, M. A hydrodynamic mechanism in the transmission of pain-producing stimuli through the dentine. Sensory mechanisms in dentine , 73-79 (1963). Schmidlin, P. R. & Sahrmann, P. Current management of dentin hypersensitivity. Clin. Oral Investig. 17 Suppl 1 , S55-59, doi:10.1007/s00784-012-0912-0 (2013). Solati, M. et al. Dentinal tubule blockage using nanobioglass in the presence of diode (980 nm) and Nd: YAG lasers: an in vitro study. Clin. Oral Investig. 26 , 2975-2981, doi:10.1007/s00784-021-04279-8 (2022). Poulsen, S., Errboe, M., Lescay Mevil, Y. & Glenny, A. M. Potassium containing toothpastes for dentine hypersensitivity. Cochrane Database Syst. Rev. 2006 , Cd001476, doi:10.1002/14651858.CD001476.pub2 (2006). Pashley, D. H. Dentin permeability, dentin sensitivity, and treatment through tubule occlusion. J. Endod. 12 , 465-474, doi:10.1016/S0099-2399(86)80201-1 (1986). Suge, T. et al. Duration of dentinal tubule occlusion formed by calcium phosphate precipitation method: in vitro evaluation using synthetic saliva. J. Dent. Res. 74 , 1709-1714, doi:10.1177/00220345950740101301 (1995). Orchardson, R. & Gillam, D. G. Managing dentin hypersensitivity. J. Am. Dent. Assoc. 137 , 990-998; quiz 1028-1029, doi:10.14219/jada.archive.2006.0321 (2006). Al-Sabbagh, M., Brown, A. & Thomas, M. V. In-office treatment of dentinal hypersensitivity. Dent. Clin. North Am. 53 , 47-60, viii, doi:10.1016/j.cden.2008.11.003 (2009). Bartold, P. M. Dentinal hypersensitivity: a review. Aust. Dent. J. 51 , 212-218, doi:10.1111/j.1834-7819.2006.tb00431.x (2006). Chiang, Y.-C. et al. A mesoporous silica biomaterial for dental biomimetic crystallization. ACS nano 8 , 12502-12513, doi:10.1021/nn5053487 (2014). Addy, M., Embery, G., Edgar, W. M. & Orchardson, R. Tooth wear and sensitivity. London. Martin Dunitz , 339-362 (2000). Mrinalini, U. B., Hegde, M. N. & Bhat, G. S. An update on dentinal hypersensitivity-aetiology to management–a review. J Evolution Med Dent Sci 10 , 3289-3293, doi:10.14260/jemds/2021/667 (2021). Miglani, S., Aggarwal, V. & Ahuja, B. Dentin hypersensitivity: Recent trends in management. J Conserv Dent 13 , 218-224, doi:10.4103/0972-0707.73385 (2010). Gou, Z., Chang, J., Zhai, W. & Wang, J. Study on the self-setting property and the in vitro bioactivity of beta-Ca2SiO4. J. Biomed. Mater. Res. B Appl. Biomater. 73 , 244-251, doi:10.1002/jbm.b.30203 (2005). Zhao, W., Wang, J., Zhai, W., Wang, Z. & Chang, J. The self-setting properties and in vitro bioactivity of tricalcium silicate. Biomaterials 26 , 6113-6121, doi:10.1016/j.biomaterials.2005.04.025 (2005). Yoo, Y. J. et al. Dynamic intratubular biomineralization following root canal obturation with pozzolan-based mineral trioxide aggregate sealer cement. Scanning 38 , 50-56, doi:10.1002/sca.21240 (2016). Dong, Z., Chang, J., Deng, Y. & Joiner, A. Tricalcium silicate induced mineralization for occlusion of dentinal tubules. Aust. Dent. J. 56 , 175-180, doi:10.1111/j.1834-7819.2011.01321.x (2011). Gandolfi, M. G., Silvia, F., H, P. D., Gasparotto, G. & Carlo, P. Calcium silicate coating derived from Portland cement as treatment for hypersensitive dentine. J. Dent. 36 , 565-578, doi:10.1016/j.jdent.2008.03.012 (2008). Komabayashi, T. & Spångberg, L. S. Comparative analysis of the particle size and shape of commercially available mineral trioxide aggregates and Portland cement: a study with a flow particle image analyzer. J. Endod. 34 , 94-98, doi:10.1016/j.joen.2007.10.013 (2008). Yuan, P. et al. Effect of a dentifrice containing different particle sizes of hydroxyapatite on dentin tubule occlusion and aqueous Cr (VI) sorption. Int J Nanomedicine 14 , 5243-5256, doi:10.2147/ijn.S205804 (2019). Berg, C., Unosson, E., Engqvist, H. & Xia, W. Amorphous Calcium Magnesium Phosphate Particles for Treatment of Dentin Hypersensitivity: A Mode of Action Study. ACS Biomater Sci Eng 6 , 3599-3607, doi:10.1021/acsbiomaterials.0c00262 (2020). Jang, Y. et al. Dentin wear after simulated toothbrushing with water, a liquid dentifrice or a standard toothpaste. Am. J. Dent. 28 , 333-336 (2015). Reynolds, E. C. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J. Dent. Res. 76 , 1587-1595, doi:10.1177/00220345970760091101 (1997). Kuroiwa, M., Kodaka, T., Kuroiwa, M. & Abe, M. Dentin hypersensitivity. Occlusion of dentinal tubules by brushing with and without an abrasive dentifrice. J. Periodontol. 65 , 291-296, doi:10.1902/jop.1994.65.4.291 (1994). Kunam, D., Manimaran, S., Sampath, V. & 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. J Conserv Dent 19 , 239-244, doi:10.4103/0972-0707.181940 (2016). Mehta, D., Gowda, V., Finger, W. J. & Sasaki, K. Randomized, placebo-controlled study of the efficacy of a calcium phosphate containing paste on dentin hypersensitivity. Dent. Mater. 31 , 1298-1303, doi:10.1016/j.dental.2015.08.162 (2015). Camilleri, J. Characterization of hydration products of mineral trioxide aggregate. Int. Endod. J. 41 , 408-417, doi:10.1111/j.1365-2591.2007.01370.x (2008). Camilleri, J. Hydration mechanisms of mineral trioxide aggregate. Int. Endod. J. 40 , 462-470, doi:10.1111/j.1365-2591.2007.01248.x (2007). Sarkar, N. K., Caicedo, R., Ritwik, P., Moiseyeva, R. & Kawashima, I. Physicochemical basis of the biologic properties of mineral trioxide aggregate. J. Endod. 31 , 97-100, doi:10.1097/01.don.0000133155.04468.41 (2005). Tay, F. R., Pashley, D. H., Rueggeberg, F. A., Loushine, R. J. & Weller, R. N. Calcium phosphate phase transformation produced by the interaction of the portland cement component of white mineral trioxide aggregate with a phosphate-containing fluid. J. Endod. 33 , 1347-1351, doi:10.1016/j.joen.2007.07.008 (2007). Martin, R. L. et al. Sealing properties of mineral trioxide aggregate orthograde apical plugs and root fillings in an in vitro apexification model. J. Endod. 33 , 272-275, doi:10.1016/j.joen.2006.11.002 (2007). Reyes-Carmona, J. F., Felippe, M. S. & Felippe, W. T. Biomineralization ability and interaction of mineral trioxide aggregate and white portland cement with dentin in a phosphate-containing fluid. J. Endod. 35 , 731-736, doi:10.1016/j.joen.2009.02.011 (2009). Reyes-Carmona, J. F., Felippe, M. S. & Felippe, W. T. The biomineralization ability of mineral trioxide aggregate and Portland cement on dentin enhances the push-out strength. J. Endod. 36 , 286-291, doi:10.1016/j.joen.2009.10.009 (2010). Guo, H., Wei, J., Yuan, Y. & Liu, C. Development of calcium silicate/calcium phosphate cement for bone regeneration. Biomedical Materials 2 , S153, doi:10.1088/1748-6041/2/3/S13 (2007). Zhao, W. & Chang, J. Sol–gel synthesis and in vitro bioactivity of tricalcium silicate powders. Materials Letters 58 , 2350-2353, doi:10.1016/j.matlet.2004.02.045 (2004). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Aug, 2023 Read the published version in Scientific Reports → Version 2 posted Editorial decision: Major revision 23 Jan, 2023 Reviews received at journal 16 Jan, 2023 Reviewers agreed at journal 06 Jan, 2023 Reviewers invited by journal 28 Dec, 2022 Editor assigned by journal 28 Dec, 2022 Editor invited by journal 12 Dec, 2022 Submission checks completed at journal 12 Dec, 2022 First submitted to journal 05 Dec, 2022 You are reading this latest preprint version Show more versions 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. We do this by developing innovative software and high quality services for the global research community. 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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-2093584","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-10-24 20:42:35","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}}],"articleType":"Article","associatedPublications":[],"authors":[{"id":170436725,"identity":"88206fa8-2ece-424a-add9-e3767fc354d4","order_by":0,"name":"Mi-Jeong Jeon","email":"","orcid":"","institution":"Department of Conservative Dentistry, School of Dentistry and Dental Research Institute, Seoul National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mi-Jeong","middleName":"","lastName":"Jeon","suffix":""},{"id":170436726,"identity":"8cc0f064-f0ec-497a-a1bd-a563d1d0e53a","order_by":1,"name":"Jeong-Won Park","email":"","orcid":"","institution":"Department of Conservative Dentistry, College of Dentistry, Gangnam Severance Hospital, Yonsei University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeong-Won","middleName":"","lastName":"Park","suffix":""},{"id":170436727,"identity":"d776f929-29b6-441a-9f85-69ac78bcdd75","order_by":2,"name":"Deog-Gyu Seo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYDCCAyCiAoVHlJYzJGthbCNFC9/55Q8ffJxXl2dwgPnhB4Yz9whrkbzxxthw5rbDxQYH2IwlGG4UE9ZicOMMmzTvtgOJGw4wmDEwfEggRsvx579559QBtbB/I1LL+QYzZt4GZqAWHqAtN4jQInmDx1hyxrHDiTMP8xRLJJwhQgvf+eMPP3yoqUvsO96+8cOHY0RoYZCAKWIGYmI0MDDwHyBK2SgYBaNgFIxkAAD6Y0FrhSfeBgAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Conservative Dentistry, School of Dentistry and Dental Research Institute, Seoul National University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Deog-Gyu","middleName":"","lastName":"Seo","suffix":""}],"badges":[],"createdAt":"2022-09-22 16:29:24","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-2093584/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2093584/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-30351-2","type":"published","date":"2023-08-30T15:09:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":32065991,"identity":"9e467bf5-11fe-4a2d-8753-217e45be849d","added_by":"auto","created_at":"2023-01-25 23:44:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1983513,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of longitudinally sectioned specimens immersed for two months in PBS. (a) The white arrows show that the intratubular crystals were formed to a depth of more than 50 μm from the exposed dentin surface. (b) Plug-shaped precipitate below occluding plug (white arrowheads). (c) plate-shaped crystals in the dentinal tubule (white arrowheads).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2093584/v2/2ac91ac88b4a0271121141aa.png"},{"id":32065994,"identity":"9845665a-23f2-48da-9426-51f2c9e3f3f6","added_by":"auto","created_at":"2023-01-25 23:44:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10376899,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of longitudinally sectioned specimens (x50,000). (a) Control group and (b) C\u003csub\u003e2\u003c/sub\u003eS/C\u003csub\u003e3\u003c/sub\u003eS group of 1, 30, 60, and 90 days after immersion in PBS, respectively. For the entire observation period, no intratubular crystal formation in the control group (a, white arrowheads). Crystal formation was observed in dentinal tubules forming a denser crystal complex as the period of storage in PBS increased (b, white arrowheads).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2093584/v2/b7b1b2417c0f996f24cf50ed.png"},{"id":32065992,"identity":"5acbd7eb-fea5-413b-acb3-0c61376b314e","added_by":"auto","created_at":"2023-01-25 23:44:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":252469,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of specimen preparation. Total 8 mm of cervical part centered at the cementoenamel junction was isolated. Remained pulp tissue was removed and sectioned tooth was mounted in the ring-shape acrylic mold.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2093584/v2/10d2b1ac929f287961568cb9.png"},{"id":32066151,"identity":"094d574b-aa8a-4713-b81b-db3707877e60","added_by":"auto","created_at":"2023-01-25 23:52:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146639,"visible":true,"origin":"","legend":"\u003cp\u003eParticle distribution in diameter of experimental material (C\u003csub\u003e2\u003c/sub\u003eS/C\u003csub\u003e3\u003c/sub\u003eS). The particle size of the experimental material was distributed between 0.052μm (D0.1) – 1.267μm (D0.9), and the median value (D0.5) is 0.184μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2093584/v2/9e5f295e6e0e69e338ca3955.png"},{"id":42782771,"identity":"40522fe6-5bb4-47e4-8af0-aba8966c8948","added_by":"auto","created_at":"2023-09-07 15:17:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2283086,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2093584/v2/4ea27ae4-aeb7-401b-9ce9-bb42e4d7a177.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intratubular crystal formation in the exposed dentin from nano-sized calcium silicate for dentin hypersensitivity treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDentin hypersensitivity (DH) is defined as \u0026ldquo;pain arising from exposed dentin in response to stimuli, typically thermal, evaporative, tactile, osmotic or chemical, which cannot be ascribed to any other form of dental defect or pathology\u0026rdquo;\u003csup\u003e1\u003c/sup\u003e. According to hydrodynamic theory, teeth with dentin hypersensitivity have opened dentinal tubules with pulpal patency\u003csup\u003e2\u003c/sup\u003e, a channel for stimulation transmission by moving fluids within the tubules\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThere are several approaches to reduce discomfort from DH. Potassium nitrate could decrease the excitability of the intradental nerve fibers and achieve pain relief\u003csup\u003e4\u003c/sup\u003e. Although the effect of relief dentin hypersensitivity appears immediately, it does not last long\u003csup\u003e5\u003c/sup\u003e, and it is no substantial evidence supporting its efficacy\u003csup\u003e6\u003c/sup\u003e. Another approach is occluding the tubules by hindering the tubular fluid movement. The concept of tubule occlusion is a logical extension of the hydrodynamic theory\u003csup\u003e7\u003c/sup\u003e. Products including strontium, bioactive glass particles, amorphous calcium phosphate, arginine, oxalate, and fluoride can be found on the market using ions and salt. The limitations of these desensitizers are related to lack of intratubular occlusion due to large particle size and high solubility of the occluding materials, and short duration of effective time due to poor resistance to acid attacks\u003csup\u003e8-10\u003c/sup\u003e. Moreover, the desensitizing effects depend significantly on the individual\u0026rsquo;s sensory threshold\u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEffective occlusion of dentinal tubules is difficult due to the small dimensions of dentinal tubules (0.5-4 \u0026mu;m in diameter)\u003csup\u003e2\u003c/sup\u003e, the complex structure of a pulp-dentin system, the outward hydraulic pressure of dental pulp (0.15 kg/cm\u003csup\u003e2\u003c/sup\u003e)\u003csup\u003e12\u003c/sup\u003e. For effective management of dentine hypersensitivity, new materials are required to penetrate deep enough into dentinal tubules, last a long time, and not easily wash out with the oral environment\u003csup\u003e13\u003c/sup\u003e. Various types of materials, such as bioceramic, synthetic polymer, and peptides, were attempted as an alternative desensitizer. However, these alternative materials did not completely overcome the disadvantages of the existing desensitizers mentioned above\u003csup\u003e14,15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCalcium silicate, can form calcium phosphate precipitation when contacted with physiological fluid containing phosphate\u003csup\u003e16,17\u003c/sup\u003e. A tag-like structure in the dentinal tubule was observed after calcium silicate based sealer application during the root canal filling procedure\u003csup\u003e18\u003c/sup\u003e. However, no studies have used calcium silicate as a desensitizer and applied it to outer dentin surface over a period time.\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to evaluate that intratubular crystals were formed in dentinal tubules when experimental material consisting of dicalcium silicate (2CaO.SiO\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003eS)\u0026nbsp;and\u0026nbsp;tricalcium silicate (3CaO.SiO\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003eS) was applied to the exposed outer dentin surface under PBS for three months.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e represented SEM images of a longitudinally sectioned specimen immersed for two months in PBS after applying the experimental material consisting of C\u003csub\u003e2\u003c/sub\u003eS and C\u003csub\u003e3\u003c/sub\u003eS. There were plug shape precipitates below the dentinal tubule orifice. The lower surface of the occluding plug became rough, and plate-shaped intratubular crystals were formed below the occluding plug. The intratubular crystals were formed at a depth of more than 50 \u0026micro;m from the surface of the exposed dentin surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e white arrows).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the SEM images of longitudinally sectioned specimens. There was no intratubular crystal formation in all specimens for the entire observation period in the control group (white arrows in a). In the experimental group, the roughening of the inner surface of the dentinal tubules was observed after 1 day, and plate-shaped crystals were formed in the dentinal tubules after two months. As the period stored in the media gets longer, the plate-shaped crystals are linked together to form a denser collection (white arrows in b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe composition of the intratubular crystals in PBS was evaluated using EDS (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After immersion in PBS for 1 day, the Ca/P ratio of the crystal was 1.57. Then, the Ca/P ratio increased to 1.68 after 90 days, which meant the crystals from C\u003csub\u003e2\u003c/sub\u003eS/C\u003csub\u003e3\u003c/sub\u003eS in PBS were supposed to be the hydroxyapatite-like crystal, of which the Ca/P ratio was 1.67.\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\u003eCa/P ratio of the intratubular crystals.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90d\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCa/P\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e* Hydroxyapatite (HAp), Ca\u003csub\u003e10\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e, Ca/P\u0026thinsp;=\u0026thinsp;1.67\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccording to hydrodynamic theory, blocking the opened dentinal tubules could reduce discomfort from dentin hypersensitivity by reducing fluid movement through dentinal tubules\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In this study, the intratubular crystals were formed in PBS after the experimental material was applied to the exposed dentin surface and filled the dentinal tubules more densely as the period of storage in PBS increased.\u003c/p\u003e \u003cp\u003eIn the case of intratubular crystal formation in previous study\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, when using a calcium silicate based sealer for root canal treatment, calcium silicate was continuously present inside the root canal and in contact with the dentinal tubules. For dentin hypersensitivity treatment, a desensitizer should be applied to the dentin surface exposed to the outside, which is not in contact with the surface of dentin continuously exposed due to physical and chemical factors in the oral cavity. Therefore, in order to improve intratubular occlusion, desensitizer must efficiently penetrate the dentinal tubules.\u003c/p\u003e \u003cp\u003eIn this study, two efforts were made to achieve efficient penetration of experimental particles as greater penetration of particles into the dentinal tubules would have occurred. First, the experimental used in this study consisted of particles with a smaller diameter than in other studies using calcium silicate (lower than 85 \u0026micro;m or 0.5 mm)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e or calcium silicate in commercially available mineral trioxide aggregate (MTA) products \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The diameter of dentinal tubules of the sensitive dentin is larger than that of the non-sensitive dentin but is only 0.83 \u0026micro;m on average\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The small dimension of dentinal tubules makes it difficult for desensitizers to be efficiently penetrated\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. According to a study by Kim et al.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, comparing the degree of absorption according to the particle size of the desensitizer, the desensitizer with small-sized particles had more opportunities to penetrate the dental tubules. Therefore, the experimental materials consisting of specially designed nano-scale particles used in this study would effectively penetrate the dentinal tubules and form occluding plugs, as in other studies\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Second, C\u003csub\u003e2\u003c/sub\u003eS/C\u003csub\u003e3\u003c/sub\u003eS were applied as a brushing motion to the dentin surface. This simulated that calcium silicate was contained in toothpaste and applied to the surface of the dentin when brushing. A total of 10,000 repeated strokes (1 strokes/second) of brushing simulated about 18.5 days assuming brushing three minutes/time and three times a day\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. It can have a chance to push desensitizing materials into dentinal tubules compared with just dropping or rubbing with a micro-brush on the specimen surface. As a result, the occluding plug was formed below the dentinal tubule orifice in applying experimental material to the brushing motion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe occluding plugs can act as a reservoir of calcium ions, continuously dissolving calcium ions and making the inside of the dentinal tubules into a supersaturation state\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The supersaturation condition was expected to cause local aggregation of calcium ions and phosphate ions due to interaction with ions present on the inner surface of the dentinal tubules, which caused the growth of the intratubular crystals\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This reaction can be inferred that the lower part of the occluding plug was rough, and plate-shaped crystals were formed below (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb white arrowheads).\u003c/p\u003e \u003cp\u003eThe intratubular crystal formation reaction by diffusion according to the concentration gradient of ions can occur at a deep point in the dentinal tubules. In this study, the intratubular crystals were formed at a depth of more than 50 \u0026micro;m from the exposed dentin surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea white arrows). In a clinical situation, superficial occlusion of dentinal tubules has a short-term effect as the precipitates can be easily removed due to daily tooth brushing, dissolution by saliva, and acidic beverages\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. For the long-term effect of desensitizer, the material blocking the dentinal tubules should be deep enough\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCrystal formation reaction continuously takes place in dentinal tubules forming denser crystal complex as the period of storage in PBS increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The crystal complex is expected to contribute to preventing the movement of the pulpal fluid through the dentinal tubules and reduce discomfort from dentin hypersensitivity more effectively\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Further research will be needed on the clinical effectiveness of the experimental material.\u003c/p\u003e \u003cp\u003eWhen C\u003csub\u003e2\u003c/sub\u003eS and C\u003csub\u003e3\u003c/sub\u003eS are in contact with water, they are hydroxylated, and the surface dissolves according to below\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e;\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$2(2CaO\\bullet Si{O}_{2})+4{H}_{2}O\\to 3CaO\\bullet 2Si{O}_{2}\\bullet 3{H}_{2}O+Ca{\\left(OH\\right)}_{2}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$2(3CaO\\bullet Si{O}_{2})+6{H}_{2}O\\to 3CaO\\bullet 2Si{O}_{2}\\bullet 3{H}_{2}O+3Ca{\\left(OH\\right)}_{2}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAs a result of these reactions, calcium and hydroxyl ions are released, resulting in a highly alkaline environment\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. After the hydration reaction of calcium silicate, the hydroxyapatite-like crystals were formed under contact with PBS\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A previous study showed calcium-deficient and B-type carbonated apatite with a 1.4\u0026ndash;1.5 Ca/P ratio formed from Portland cement in PBS\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Other studies showed calcium silicate in PBS could make hydroxyapatites after hydration\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In this study, the Ca/P ratio of formed intratubular crystals was 1.68 after three months. Considering that the Ca/P ratio of hydroxyapatite is 1.67\u003csup\u003e36\u003c/sup\u003e, it can be expected that the intratubular crystals made in this experiment would be hydroxyapatite-like crystals.\u003c/p\u003e \u003cp\u003eHowever, this experimental design could not simulate an oral environment, such as an acid challenge from the diet. Therefore, further studies need to be performed to evaluate the effect of the acid-neutral cycle on intratubular crystal formation.\u003c/p\u003e \u003cp\u003eThis study demonstrated that the experimental material could form intratubular crystals in PBS after being applied to the exposed dentin surface. The crystal formation occurred at more than 50 \u0026micro;m from the dentin surface, and the crystals more densely filled the dentinal tubules over time. The experimental material consisting of C\u003csub\u003e2\u003c/sub\u003eS and C\u003csub\u003e3\u003c/sub\u003eS with nano-scaled particle size used in this study allowed for effective penetration to dentinal tubules and the formation of intratubular crystals, which makes the material a promising alternative for clinical use to reduce discomfort from dentin hypersensitivity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eSpecimen preparation.\u003c/b\u003e This study was approved by the Ethics Committee of the Seoul National University, Graduate School of Dentistry (IRB number: S-D20190010). Total twenty-four recently extracted human premolars for orthodontic treatment with intact coronal and root surfaces were prepared. They were stored in 0.1% thymol solution to inhibit microbial growth for no longer than three months prior to use. Debris on the surface of all teeth was removed by perio-curette and examined whether there were crack lines under a microscope (200\u0026times;) (Carl Zeiss Surgical GmbH, Oberkochen, Germany).\u003c/p\u003e \u003cp\u003eA specimen with a total length of 8 mm was made by cutting the 4 mm above and below the cemento-enamel junction with a low-speed diamond saw (Isomet\u0026trade;, Buehler, Lake Bluff, IL, USA) under constant water cooling. Flat and fresh dentin was exposed on the occlusal side surface without perforation at the pulp horn area. Remained pulp tissue was removed carefully with small forceps without touching the inner part of the pulpal space. Then, the sectioned tooth was mounted in the ring-shaped acrylic mold with self-cured acrylic resin (Bosworth Fastray, Keystone Industries GmbH, Singen, Germany) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe exposed upper dentin surface of each specimen was treated with 17% ethylenediaminetetraacetic acid (EDTA) for 1 minute, followed by 2.5 mL of 5.25% sodium hypochlorite to open dentinal tubules and remove the smear layer on the exposed dentin surface and rinsed with 10 mL of distilled water twice.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eS/C\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eS preparation and application.\u003c/b\u003e The experimental material consisting of dicalcium silicate and tricalcium silicate (C\u003csub\u003e2\u003c/sub\u003eS/C\u003csub\u003e3\u003c/sub\u003eS) was prepared by a sol-gel method as Zhao W and Chang J \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e using CaCO3, SiO2, Al2O3 as the raw materials and the obtained material calcined at 1,450 ℃ for six hours. The resultant powders were ground at 300 rpm using a Disk Mill (Disk Mill, KM tech, Icheon, Republic of Korea) and then at 200 rpm using a Ball Mill (BML-2, DAITHAN SCIENTIFIC GROUP, Wonju-si, Republic of Korea) for 48 hours. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the main component and content of the experimental material. The particle size of the experimental material was distributed between 0.052 \u0026micro;m (D0.1) \u0026ndash; 1.267 \u0026micro;m (D0.9), and the median value (D0.5) is 0.184\u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental material of the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComponent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContent (wt%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eS/C\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDicalcium silicate (2CaO.SiO\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003eS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026thinsp;~\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTricalcium silicate (3CaO.SiO\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003eS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u0026thinsp;~\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\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\u003e \u003c/p\u003e \u003cp\u003eAfter preparation of the specimens, 0.5g of C\u003csub\u003e2\u003c/sub\u003eS/C\u003csub\u003e3\u003c/sub\u003eS was mixed with 5 ml of distilled water and applied on the exposed dentin surface of the specimen by tooth brushing motion according to ISO 11609 standards for dentin wear test, abrasive in the dentifrice is about 10% of dentifrice and water mixture. The concentration of tricalcium silicate applied is followed this standard. A total of 10,000 repeated strokes (1 strokes/second) were applied using the toothbrush onto each specimen under a 150 g-load continuously being touched among test material mixtures and the exposed dentin surface.\u003c/p\u003e \u003cp\u003eThe specimens were randomly divided into four subgroups according to the period of immersion in PBS (D8662, Sigma-Aldrich, St. Louis, MO, U.S.A.) media for 1, 30, 60, and 90 days each at 37 ℃ (n\u0026thinsp;=\u0026thinsp;3). The PBS solution was replaced every seven days. The composition (in g/L) of used PBS was CaCl\u003csub\u003e2\u003c/sub\u003e\u0026bull;2H\u003csub\u003e2\u003c/sub\u003eO 0.133, MgCl\u003csub\u003e2\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO 0.1, KCI 0.2, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.2, NaCl 8.0, Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (anhydrous) 1.15, and the pH was 7.4.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScanning Electron Microscope analysis and EDS analysis.\u003c/b\u003e The specimens were longitudinally sectioned, and six sectioned surfaces in each group were examined to assess crystal formation in dentinal tubules after experimental material application on exposed upper dentin surfaces. All specimens were mounted on aluminum stubs and sputter-coated with a 30 nm layer of gold and examined using field emission scanning electron microscopy (FE-SEM, Apreo S; Thermo Fisher SCIENTIFIC, Waltham, MA, U.S.A.). The intratubular crystals were examined by energy dispersive spectroscopy (EDS, XFlash 6160, Bruker, Germany) to analyze the components.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Seoul National University, Graduate School of Dentistry (IRB number: S-D20190010). All biological samples were included after obtaining the informed consent from all subjects. All methods were conducted in accordance with Declarations of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2020R1F1A1076307).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.-J.J. designed the analysis, collected the data, performed the analysis, and wrote the main manuscript text. J.-W.P. designed and performed the analysis. D.-G.S. designed experiment, performed the analysis, contributed analysis method and tool. All authors reviewed and edited the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to D.-G.S.\u003c/p\u003e"},{"header":"Reference","content":"\u003col\u003e\n\u003cli\u003eHypersensitivity, C. A. B. o. D. Consensus-based recommendations for the diagnosis and management of dentin hypersensitivity. \u003cem\u003eJ. Can. Dent. Assoc.\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 221-226 (2003).\u003c/li\u003e\n\u003cli\u003eAbsi, E. G., Addy, M. \u0026amp; Adams, D. Dentine hypersensitivity: a study of the patency of dentinal tubules in sensitive and non‐sensitive cervical dentine. \u003cem\u003eJ. Clin. Periodontol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 280-284, doi:10.1111/j.1600-051X.1987.tb01533.x (1987).\u003c/li\u003e\n\u003cli\u003eBrannstrom, M. A hydrodynamic mechanism in the transmission of pain-producing stimuli through the dentine. \u003cem\u003eSensory mechanisms in dentine\u003c/em\u003e, 73-79 (1963).\u003c/li\u003e\n\u003cli\u003eSchmidlin, P. R. \u0026amp; Sahrmann, P. Current management of dentin hypersensitivity. \u003cem\u003eClin. Oral Investig.\u003c/em\u003e \u003cstrong\u003e17 Suppl 1\u003c/strong\u003e, S55-59, doi:10.1007/s00784-012-0912-0 (2013).\u003c/li\u003e\n\u003cli\u003eSolati, M.\u003cem\u003e et al.\u003c/em\u003e Dentinal tubule blockage using nanobioglass in the presence of diode (980 nm) and Nd: YAG lasers: an in vitro study. \u003cem\u003eClin. Oral Investig.\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 2975-2981, doi:10.1007/s00784-021-04279-8 (2022).\u003c/li\u003e\n\u003cli\u003ePoulsen, S., Errboe, M., Lescay Mevil, Y. \u0026amp; Glenny, A. M. Potassium containing toothpastes for dentine hypersensitivity. \u003cem\u003eCochrane Database Syst. Rev.\u003c/em\u003e \u003cstrong\u003e2006\u003c/strong\u003e, Cd001476, doi:10.1002/14651858.CD001476.pub2 (2006).\u003c/li\u003e\n\u003cli\u003ePashley, D. H. Dentin permeability, dentin sensitivity, and treatment through tubule occlusion. \u003cem\u003eJ. Endod.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 465-474, doi:10.1016/S0099-2399(86)80201-1 (1986).\u003c/li\u003e\n\u003cli\u003eSuge, T.\u003cem\u003e et al.\u003c/em\u003e Duration of dentinal tubule occlusion formed by calcium phosphate precipitation method: in vitro evaluation using synthetic saliva. \u003cem\u003eJ. Dent. Res.\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 1709-1714, doi:10.1177/00220345950740101301 (1995).\u003c/li\u003e\n\u003cli\u003eOrchardson, R. \u0026amp; Gillam, D. G. Managing dentin hypersensitivity. \u003cem\u003eJ. Am. Dent. Assoc.\u003c/em\u003e \u003cstrong\u003e137\u003c/strong\u003e, 990-998; quiz 1028-1029, doi:10.14219/jada.archive.2006.0321 (2006).\u003c/li\u003e\n\u003cli\u003eAl-Sabbagh, M., Brown, A. \u0026amp; Thomas, M. V. In-office treatment of dentinal hypersensitivity. \u003cem\u003eDent. Clin. North Am.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 47-60, viii, doi:10.1016/j.cden.2008.11.003 (2009).\u003c/li\u003e\n\u003cli\u003eBartold, P. M. Dentinal hypersensitivity: a review. \u003cem\u003eAust. Dent. J.\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 212-218, doi:10.1111/j.1834-7819.2006.tb00431.x (2006).\u003c/li\u003e\n\u003cli\u003eChiang, Y.-C.\u003cem\u003e et al.\u003c/em\u003e A mesoporous silica biomaterial for dental biomimetic crystallization. \u003cem\u003eACS nano\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 12502-12513, doi:10.1021/nn5053487 (2014).\u003c/li\u003e\n\u003cli\u003eAddy, M., Embery, G., Edgar, W. M. \u0026amp; Orchardson, R. Tooth wear and sensitivity. \u003cem\u003eLondon. Martin Dunitz\u003c/em\u003e, 339-362 (2000).\u003c/li\u003e\n\u003cli\u003eMrinalini, U. B., Hegde, M. N. \u0026amp; Bhat, G. S. An update on dentinal hypersensitivity-aetiology to management\u0026ndash;a review. \u003cem\u003eJ Evolution Med Dent Sci\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 3289-3293, doi:10.14260/jemds/2021/667 (2021).\u003c/li\u003e\n\u003cli\u003eMiglani, S., Aggarwal, V. \u0026amp; Ahuja, B. Dentin hypersensitivity: Recent trends in management. \u003cem\u003eJ Conserv Dent\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 218-224, doi:10.4103/0972-0707.73385 (2010).\u003c/li\u003e\n\u003cli\u003eGou, Z., Chang, J., Zhai, W. \u0026amp; Wang, J. Study on the self-setting property and the in vitro bioactivity of beta-Ca2SiO4. \u003cem\u003eJ. Biomed. Mater. Res. B Appl. Biomater.\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 244-251, doi:10.1002/jbm.b.30203 (2005).\u003c/li\u003e\n\u003cli\u003eZhao, W., Wang, J., Zhai, W., Wang, Z. \u0026amp; Chang, J. The self-setting properties and in vitro bioactivity of tricalcium silicate. \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 6113-6121, doi:10.1016/j.biomaterials.2005.04.025 (2005).\u003c/li\u003e\n\u003cli\u003eYoo, Y. J.\u003cem\u003e et al.\u003c/em\u003e Dynamic intratubular biomineralization following root canal obturation with pozzolan-based mineral trioxide aggregate sealer cement. \u003cem\u003eScanning\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 50-56, doi:10.1002/sca.21240 (2016).\u003c/li\u003e\n\u003cli\u003eDong, Z., Chang, J., Deng, Y. \u0026amp; Joiner, A. Tricalcium silicate induced mineralization for occlusion of dentinal tubules. \u003cem\u003eAust. Dent. J.\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 175-180, doi:10.1111/j.1834-7819.2011.01321.x (2011).\u003c/li\u003e\n\u003cli\u003eGandolfi, M. G., Silvia, F., H, P. D., Gasparotto, G. \u0026amp; Carlo, P. Calcium silicate coating derived from Portland cement as treatment for hypersensitive dentine. \u003cem\u003eJ. Dent.\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 565-578, doi:10.1016/j.jdent.2008.03.012 (2008).\u003c/li\u003e\n\u003cli\u003eKomabayashi, T. \u0026amp; Sp\u0026aring;ngberg, L. S. Comparative analysis of the particle size and shape of commercially available mineral trioxide aggregates and Portland cement: a study with a flow particle image analyzer. \u003cem\u003eJ. Endod.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 94-98, doi:10.1016/j.joen.2007.10.013 (2008).\u003c/li\u003e\n\u003cli\u003eYuan, P.\u003cem\u003e et al.\u003c/em\u003e Effect of a dentifrice containing different particle sizes of hydroxyapatite on dentin tubule occlusion and aqueous Cr (VI) sorption. \u003cem\u003eInt J Nanomedicine\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 5243-5256, doi:10.2147/ijn.S205804 (2019).\u003c/li\u003e\n\u003cli\u003eBerg, C., Unosson, E., Engqvist, H. \u0026amp; Xia, W. Amorphous Calcium Magnesium Phosphate Particles for Treatment of Dentin Hypersensitivity: A Mode of Action Study. \u003cem\u003eACS Biomater Sci Eng\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 3599-3607, doi:10.1021/acsbiomaterials.0c00262 (2020).\u003c/li\u003e\n\u003cli\u003eJang, Y.\u003cem\u003e et al.\u003c/em\u003e Dentin wear after simulated toothbrushing with water, a liquid dentifrice or a standard toothpaste. \u003cem\u003eAm. J. Dent.\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 333-336 (2015).\u003c/li\u003e\n\u003cli\u003eReynolds, E. C. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. \u003cem\u003eJ. Dent. Res.\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 1587-1595, doi:10.1177/00220345970760091101 (1997).\u003c/li\u003e\n\u003cli\u003eKuroiwa, M., Kodaka, T., Kuroiwa, M. \u0026amp; Abe, M. Dentin hypersensitivity. Occlusion of dentinal tubules by brushing with and without an abrasive dentifrice. \u003cem\u003eJ. Periodontol.\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 291-296, doi:10.1902/jop.1994.65.4.291 (1994).\u003c/li\u003e\n\u003cli\u003eKunam, D., Manimaran, S., Sampath, V. \u0026amp; 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. \u003cem\u003eJ Conserv Dent\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 239-244, doi:10.4103/0972-0707.181940 (2016).\u003c/li\u003e\n\u003cli\u003eMehta, D., Gowda, V., Finger, W. J. \u0026amp; Sasaki, K. Randomized, placebo-controlled study of the efficacy of a calcium phosphate containing paste on dentin hypersensitivity. \u003cem\u003eDent. Mater.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 1298-1303, doi:10.1016/j.dental.2015.08.162 (2015).\u003c/li\u003e\n\u003cli\u003eCamilleri, J. Characterization of hydration products of mineral trioxide aggregate. \u003cem\u003eInt. Endod. J.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 408-417, doi:10.1111/j.1365-2591.2007.01370.x (2008).\u003c/li\u003e\n\u003cli\u003eCamilleri, J. Hydration mechanisms of mineral trioxide aggregate. \u003cem\u003eInt. Endod. J.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 462-470, doi:10.1111/j.1365-2591.2007.01248.x (2007).\u003c/li\u003e\n\u003cli\u003eSarkar, N. K., Caicedo, R., Ritwik, P., Moiseyeva, R. \u0026amp; Kawashima, I. Physicochemical basis of the biologic properties of mineral trioxide aggregate. \u003cem\u003eJ. Endod.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 97-100, doi:10.1097/01.don.0000133155.04468.41 (2005).\u003c/li\u003e\n\u003cli\u003eTay, F. R., Pashley, D. H., Rueggeberg, F. A., Loushine, R. J. \u0026amp; Weller, R. N. Calcium phosphate phase transformation produced by the interaction of the portland cement component of white mineral trioxide aggregate with a phosphate-containing fluid. \u003cem\u003eJ. Endod.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 1347-1351, doi:10.1016/j.joen.2007.07.008 (2007).\u003c/li\u003e\n\u003cli\u003eMartin, R. L.\u003cem\u003e et al.\u003c/em\u003e Sealing properties of mineral trioxide aggregate orthograde apical plugs and root fillings in an in vitro apexification model. \u003cem\u003eJ. Endod.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 272-275, doi:10.1016/j.joen.2006.11.002 (2007).\u003c/li\u003e\n\u003cli\u003eReyes-Carmona, J. F., Felippe, M. S. \u0026amp; Felippe, W. T. Biomineralization ability and interaction of mineral trioxide aggregate and white portland cement with dentin in a phosphate-containing fluid. \u003cem\u003eJ. Endod.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 731-736, doi:10.1016/j.joen.2009.02.011 (2009).\u003c/li\u003e\n\u003cli\u003eReyes-Carmona, J. F., Felippe, M. S. \u0026amp; Felippe, W. T. The biomineralization ability of mineral trioxide aggregate and Portland cement on dentin enhances the push-out strength. \u003cem\u003eJ. Endod.\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 286-291, doi:10.1016/j.joen.2009.10.009 (2010).\u003c/li\u003e\n\u003cli\u003eGuo, H., Wei, J., Yuan, Y. \u0026amp; Liu, C. Development of calcium silicate/calcium phosphate cement for bone regeneration. \u003cem\u003eBiomedical Materials\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, S153, doi:10.1088/1748-6041/2/3/S13 (2007).\u003c/li\u003e\n\u003cli\u003eZhao, W. \u0026amp; Chang, J. Sol\u0026ndash;gel synthesis and in vitro bioactivity of tricalcium silicate powders. \u003cem\u003eMaterials Letters\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 2350-2353, doi:10.1016/j.matlet.2004.02.045 (2004).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2093584/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2093584/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study is to evaluate intratubular crystal formation from the experimental material consisting of dicalcium silicate (C\u003csub\u003e2\u003c/sub\u003eS) and tricalcium silicate (C\u003csub\u003e3\u003c/sub\u003eS) with nano-scaled particle size. A total of twenty-four specimens were made by isolating 8 mm of the cervical part centered at the cementoenamel junction of extracted premolars. Twelve specimens were not treated and considered as control. The experimental material was applied to the other twelve specimens by brushing for 10,000 strokes. Each group was randomly divided into four subgroups according to the period of immersion in phosphate buffer saline (PBS) for 1, 30, 60, and 90 days each. The specimens were sectioned longitudinally and examined with scanning electron microscopy and energy dispersion X-ray spectroscopy. The intratubular crystal were formed in PBS and densely filled the dentinal tubules over time. The crystal formation occurred at a depth of more than 50 μm from the dentin surface. The Ca/P ratio of formed intratubular crystals was 1.68 after three months. The experimental material consisting of C\u003csub\u003e2\u003c/sub\u003eS and C\u003csub\u003e3\u003c/sub\u003eS with a nanoscale particle size can form hydroxyapatite-like crystals in dentinal tubules in PBS, and there is a possibility of reducing dentin hypersensitivity by blocking the dentinal fluid flow.\u003c/p\u003e","manuscriptTitle":"Intratubular crystal formation in the exposed dentin from nano-sized calcium silicate for dentin hypersensitivity treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2023-01-25 23:44:50","doi":"10.21203/rs.3.rs-2093584/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-01-23T17:42:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-01-16T15:46:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"bfada53a-71b5-4af7-8622-edc4c1bdc9e2","date":"2023-01-06T07:14:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-12-28T17:14:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-12-28T17:07:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-12-12T13:55:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-12-12T13:48:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-12-05T13:14:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b2f6b72-e696-4fda-a91c-17dad0e9816c","owner":[],"postedDate":"January 25th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":18674750,"name":"Health sciences/Diseases/Dental diseases"},{"id":18674751,"name":"Physical sciences/Materials science/Biomaterials/Biomineralization"}],"tags":[],"updatedAt":"2023-09-07T15:16:56+00:00","versionOfRecord":{"articleIdentity":"rs-2093584","link":"https://doi.org/10.1038/s41598-023-30351-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-08-30 15:09:23","publishedOnDateReadable":"August 30th, 2023"},"versionCreatedAt":"2023-01-25 23:44:50","video":"","vorDoi":"10.1038/s41598-023-30351-2","vorDoiUrl":"https://doi.org/10.1038/s41598-023-30351-2","workflowStages":[]},"version":"v2","identity":"rs-2093584","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2093584","identity":"rs-2093584","version":["v2"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}