Assessment of enamel remineralization and its effect on orthodontic bracket shear bond strength: an in vitro study

Assessment of enamel remineralization and its effect on orthodontic bracket shear bond strength: 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 Assessment of enamel remineralization and its effect on orthodontic bracket shear bond strength: an in vitro study Ascensión Vicente, Antonio J Ortiz-Ruiz, Clara Serna-Muñoz, Inmaculada Cabello, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9515528/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Objective This study aimed to evaluate the remineralization capacity of Orthocare™ and Regenerate™ on demineralized enamel and their effect on orthodontic bracket bond strength. Methods A total of 180 bovine teeth were divided into four groups: (1) Intact Enamel, (2) Demineralized Enamel, (3) Demineralized Enamel treated with Regenerate™, and (4) Demineralized Enamel treated with Orthocare™. Groups 2–4 were demineralized and stored in artificial saliva for 60 days. All specimens were brushed three times daily; Groups 1 and 2 without toothpaste, and Groups 3 and 4 with their respective products and serums. Shear bond strength was measured after bracket bonding, and adhesive remnant was evaluated by image analysis. Enamel characterization was performed using FE-SEM, EDX, and Raman spectroscopy. Results Demineralized enamel showed significantly lower shear bond strength and adhesive remnant than intact enamel (p < 0.05). FE-SEM and EDX analyses revealed increased porosity and reduced mineral content, while both treatments improved calcium (Ca) and phosphorus (P) levels (p < 0.001). Raman spectroscopy showed greater recovery of phosphate (I960) and carbonate (I1070) peaks in the Regenerate™ group, indicating better mineralization and crystallinity recovery than Orthocare™. Conclusions Both agents promoted partial enamel remineralization, with Regenerate™ showing better mineral recovery. However, the newly formed hydroxyapatite appeared immature, and bond strength was not significantly improved compared with demineralized enamel. Clinical Relevance: Remineralizing agents may improve demineralized enamel before orthodontic bonding without significantly affecting bracket bond strength. enamel remineralization orthodontics Raman Spectroscopy Shear bond strength Figures Figure 1 Figure 2 INTRODUCTION When a patient is scheduled to undergo orthodontic treatment, it is important to assess enamel characteristics before bracket bonding, as teeth may present with demineralized enamel. Although demineralization lesions are commonly considered a risk associated with orthodontic treatment, they may also be present in patients without previous orthodontic therapy [ 1 ]. Gorelick et al.[ 2 ] reported that 24% of patients had at least one tooth with demineralized enamel prior to orthodontic treatment. The highest incidence of these lesions was observed in maxillary incisors, mandibular molars, and second premolars. Several authors have reported lower bracket bond strength to demineralized enamel compared with intact enamel[ 3 – 5 ]. Bond failure requires removal of residual adhesive and re-etching of the enamel for rebonding. Both procedures result in tissue loss and may increase the severity of demineralization. Therefore, to achieve optimal adhesion, it is important to treat demineralized enamel before bracket bonding [ 6 ], recommending the use of remineralizing agents[ 7 ]. Recently, biomimetic materials have been introduced to induce enamel remineralization. These materials mimic the composition and structure of the mineral phase of the tooth and are capable of restoring enamel toward its native structure [ 8 ]. Among these systems, biomimetic calcium phosphate-based materials such as nano-hydroxyapatite, calcium phosphosilicates, and amorphous calcium phosphates (ACP) are particularly relevant [ 8 , 9 ]. Orthocare™ nHAp™ Toothpaste and Orthocare™ nHAp™ Boost Sponge are based on nano-hydroxyapatite. This material is considered highly biocompatible and bioactive due to its physicochemical similarity to enamel apatite crystals [ 10 , 11 ]. Owing to the nanometric size of its particles, it can fill small depressions and pores on the enamel surface [ 12 ]. The use of synthetic hydroxyapatite nanocrystals has been shown to promote enamel remineralization and repair [ 12 , 13 , 13 – 16 ]. Regenerate™ Advanced Toothpaste & Serum combines a toothpaste containing calcium silicate and sodium phosphate salts with a serum composed of sodium fluoride. This combination increases the supply and retention of calcium ions in enamel, which are subsequently transformed into hydroxyapatite [ 17 , 18 ]. Previous studies have demonstrated its effectiveness in protecting enamel subjected to stripping against demineralization [ 19 ] and in re-hardening enamel previously exposed to acid attack [ 9 ]. To our knowledge, no studies have evaluated the remineralizing capacity of these products prior to bracket bonding. Therefore, the aim of this study was to evaluate, in vitro, the effect of Orthocare™ nHAp™ Toothpaste & Boost Sponge and Regenerate™ Advanced Toothpaste & Serum on the remineralization of demineralized enamel and on bracket adhesion. MATERIALS AND METHODS The study was approved by the Biosafety Committee in Experimentation of the University XXXXX, XXXXX (CBE 332). Specimen preparation and experimental groups A total of 180 bovine teeth without signs of demineralization or enamel fractures were used. After extraction, the specimens were immersed in a 0.1% thymol solution for 24 hours. Subsequently, they were stored in distilled water (Tecnoquim S.L., Murcia, Spain) at room temperature, with daily renewal until the beginning of the experimental protocol. The apices of all teeth were sealed using a flowable composite (TetricEvoFlow, Ivoclar Vivadent, Liechtenstein). The teeth were randomly allocated into four experimental groups (n = 45): Group 1: Intact enamel Group 2: Demineralized enamel Group 3: Demineralized enamel/Regenerate™ Group 4: Demineralized enamel/Orthocare™ Teeth in groups 2, 3, and 4 were immersed in a lactic acid solution (L(+)-lactic acid, 88–92%, extrapure, pH 2.8; Scharlau, Barcelona, Spain) for 24 hours. Subsequently, the specimens were rinsed with distilled water to remove residual acid and subjected to ultrasonic cleaning (Talleres Mestraitua, S.L. MESTRA, Vizcaya, Spain) for 30 minutes. Experimental procedure All specimens were stored in artificial saliva at 37°C in a laboratory incubator (JP Selecta S.A., Barcelona, Spain) for 60 days. The artificial saliva was renewed daily. Its composition was as follows: 1% carmellose sodium, 13% sorbitol, 0.12% potassium chloride, 0.084% sodium chloride, 0.005% magnesium chloride hexahydrate, 0.015% anhydrous calcium chloride, 0.017% dipotassium phosphate, and 0.1% sodium nipagin. The pH was adjusted and maintained at 6.57. The teeth were removed from the artificial saliva three times daily, and the buccal surfaces were brushed for 15 seconds using a medium-bristle toothbrush (VITIS-DENTAID, Barcelona, Spain). In groups 1 and 2, brushing was performed without toothpaste. In group 3, brushing was performed using Regenerate™ Advanced Toothpaste. Additionally, during the first three days of each month, Regenerate™ Advanced Enamel Serum was applied after the last daily brushing and left undisturbed for 3 minutes. In group 4, brushing was performed using Orthocare™ nHAp™ Toothpaste. Orthocare™ nHAp™ Boost Sponge was applied every 5 days after the last daily brushing and left undisturbed for 3 minutes. All procedures were performed according to the manufacturers’ instructions. The composition of the products is presented in Table 1 . Table 1 Composition of the remineralizing agents evaluated. AGENT COMPOSITION Orthocare ™ nHAp ™ Tooth paste Glycerin, Xylitol, Potassium Nitrate, Hydroxyapatite (nano), Magnesium Aluminum Silicate, Mentha Piperita Oil, Sodium Lauroyl Sarcosinate, Xanthan Gum, Phenoxyethanol, Potassium Chloride, Sodium Sulfate, Sodium Saccharin, Linalool, Limonene, CI 77891 Orthocare ™ nHAp ™ Boost Sponge Aqua, Xylitol, Hydroxyapatite (nano), Potassium Chloride, Sclerotium Gum, Mentha Piperita, Oil, Linalool, Limonene Regenerate TM Advance Toothpaste Glycerin, Calcium Silicate, PEG-8, Hydrated Silica, Trisodium Phosphate, Sodium Phosphate, Aqua, PE-60, Sodium Lauryl Sulfate, Sodium Monofluorophospate (1450 ppm F), Aroma Flavour, Synthetic Fluorphlogopite, Sodium Saccharin, Polyacrylic Acid, Tin Oxide, Limonene, C177891 Regenerate ™ Advance Enamel Serum NR-5TM Serum : Glycerin, Calcium Silicate, PEG-8, Trisodium Phosphate, Sodium Phosphate, Aqua, PEG-60, Sodium Lauryl Sulfate, Sodium Monofluorophosphate, Aroma, Hydrated Silica, Synthetic Fluorphlogopite, Sodium Saccharin, Polyacrylic Acid, Tin Oxide, Limonene, CI 77891. Activator Gel : Aqua, Glycerin, Cellulose Gum, Sodium Fluoride (1450 ppm F), Benzyl Alcohol, Ethylhexylglycerin, Phenoxyethanol, CI 42090. Bond strength test and percentage of area occupied by adhesive on the tooth A total of 80 maxillary bovine incisors (n = 20 per group) were used. The specimens were mounted in cylindrical molds (3 cm internal diameter, 4 cm height), embedding the roots in type IV dental stone. Metal brackets for maxillary central incisors (3M Unitek Dental Products, California, USA) were bonded to the buccal surfaces. The enamel surfaces were polished using a prophylaxis cup and fluoride-free polishing paste (Detartrine, Septodont, France), rinsed, and air-dried. Subsequently, 37% phosphoric acid (Octacid, Laboratorios Clarben) was applied for 20 seconds, followed by rinsing and drying.The brackets were bonded using the Transbond XT adhesive system following the manufacturer's instructions. After bonding, specimens were stored in distilled water at 37°C for 24 hours. Shear bond strength was measured using a universal testing machine (Autograph AGS-1KND, Shimadzu, Kyoto, Japan) with a 1 kN load cell. A chisel-edge rod angled at 30° was applied at the bracket–adhesive interface. The crosshead speed was set at 1 mm/min, applying force parallel to the tooth surface in an incisal–cervical direction. The percentage of adhesive remaining on the tooth surface after debonding was determined using image analysis (Sony dxc 151-ap camera connected to an Olympus SZ11 microscope) and MIP software. The percentage was calculated by subtracting the adhesive-covered area on the bracket base from 100%. Field Emission-Scanning Electron Microscopy (FE-SEM) and Energy Dispersive X-ray Spectroscopy (EDX) analysis A total of 80 specimens (n = 20 per group) were analyzed. Teeth were sectioned at the cemento-enamel junction using a diamond disc (Komet Dental, Gebr. Brasseler GmbH & Co., Lemgo, Germany). Specimens were mounted on aluminum stubs and sputter-coated with a 5 nm platinum layer (Leica EM ACE 600). FE-SEM analysis was performed using an ApreoS Lovac microscope (Thermo Fisher Scientific) at 5 kV and 0.8 nA. EDX analysis was conducted using an Octane Plus EDAX microanalyzer (AMETEK, USA) at 20 kV. The elements quantified were calcium (Ca, wt%) and phosphorus (P, wt%). The Ca/P molar ratio was calculated as follows: Ca (mol)/P (mol) % = [Ca (weight %) / 40.08 (g/mol)] / [P (weight %) / 30.97 (g/mol)], where the molecular masses of Ca and P are 40.08 and 30.97, respectively. Raman Spectroscopy Raman spectra were obtain using a Jasco NRS-5100 Raman microscope (Jasco Inc., Japan). The system was equipped with a 784.79 nm diode laser and an MPLFLN 20× objective lens for laser focusing. Spectra were recorded in the range of 162–1886 cm⁻¹ with a spectral interval of 1 cm⁻¹, yielding 1725 data points per spectrum. For analysis, the spectral region between 400 and 1100 cm⁻¹ was selected. The spectral resolution was 3.22 cm⁻¹ (1.68 cm⁻¹/pixel). The laser power at the sample surface was set to 11.8 mW. Each spectrum was acquired with an exposure time of 30 seconds and 1 accumulation. A 600 lines/mm grating, a 50 × 1000 µm slit, and a DU420_OE CCD detector (cooled to − 68°C) were used. Cosmic ray reduction was enabled during acquisition. Five samples per group were analyzed. Raman measurements were performed at a single point located at the center of the enamel surface of each tooth crown. The enamel surfaces were neither sectioned nor polished, as the aim was to evaluate remineralization on previously demineralized enamel. Prior to analysis, all samples were dehydrated for 24 hours at room temperature. For each group, one spectrum per sample was collected (n = 5), and the average spectrum was calculated to obtain representative data for each group. The laser spot size was estimated to be in the micrometer range (~ 2–5 µm). Spectral preprocessing included baseline correction and normalization using Spectragryph software (version 1.2). A spectral shift correction (− 2.47 cm⁻¹) was applied. The degree of mineralization and compositional changes were evaluated based on phosphate and carbonate vibrational bands. The analyzed parameters included: phosphate ν1 band (960 cm⁻¹; I 960 ), used as a marker of mineral content; carbonate peak (~ 1070 cm⁻¹); full width at half maximum of the band centered at 960 cm⁻¹ (FWHM 960 ), as an indicator of crystallinity; the area under the phosphate curve at 960 cm⁻¹ (A 960 ); the carbonate-to-phosphate ratio (C 1070 /P 960 ), as an indicator of carbonate substitution; and the ν2 (431 cm⁻¹; I 431 ), ν3 (582 cm⁻¹; I 582 ), and ν4 (1044 cm⁻¹; I 1044 ) bands (Lee et al., 2020; C C et al., 2020). Statistical analysis Statistical analysis was performed using jamovi project v.2.3 ( https://www.jamovi.org ). Normality and homogeneity of variance were verified for bond strength and Raman data; therefore, one-way ANOVA followed by Tukey’s post hoc test was applied (p < 0.05). Data for adhesive remnant and EDX did not meet these assumptions and were analyzed using the Kruskal–Wallis test followed by the Dwass–Steel–Critchlow–Fligner test (p < 0.05). RESULTS Shear Bond strength and adhesive remnant Shear bond strength (SBS) was significantly lower in demineralized enamel compared with intact enamel (p = 0.01). No statistically significant differences were observed among the remaining groups (p > 0.05) (Table 2 ). Table 2 Shear Bond Strength (MPa). Groups Mean ± SD Median Intact enamel 11.27 ± 2.94 10.97 Demineralized enamel 8.47 ± 2.09 7.99 a Demineralized enamel/Regenerate ™ 9.58 ± 3.4 8.85 Demineralized enamel/Orthocare ™ 9.74 ± 3.13 9.47 SD: Standard deviation Significant differences (p < 0.05) versus a: Intact enamel. The percentage of adhesive remaining on the tooth surface after debonding was significantly lower in the demineralized enamel group compared with the other groups (vs. intact enamel, p = 0.04; vs. demineralized enamel/Orthocare™, p 0.05) (Table 3 ). Table 3 Percentage of tooth area occupied by adhesive. Groups Mean ± SD Median Intact enamel 50.99 ± 18.93 58.41 Demineralized enamel 34.49 ± 9.12 32.71 a Demineralized enamel/Regenerate ™ 54.19 ± 18.08 61.97 b Demineralized enamel/Orthocare ™ 55.99 ± 13.21 58.90 b SD: Standard deviation Significant differences (p < 0.05) versus a: Intact enamel and b: Demineralized enamel. FESEM and EDX analysis Representative FE-SEM images are shown in Fig. 1 . Intact enamel (Fig. 1 A) exhibited a regular surface with characteristic wear lines. Demineralized enamel (Fig. 1 B) showed the typical demineralization pattern, characterized by increased surface porosity following lactic acid exposure. Demineralized enamel treated with Orthocare™ (Fig. 1 C) presented a slightly rough surface with non-homogeneously distributed aggregates. In contrast, demineralized enamel treated with Regenerate™ (Fig. 1 D) exhibited aggregates of varying sizes on a surface without evident signs of demineralization. EDX results are summarized in Table 4 . Table 4 Weight percentage of Ca, P and Ca/P ratio (mol/mol). Groups P Ca Ca/P Mean ± SD Median Mean ± SD Median Mean ± SD Median Intact enamel 20.72 ± 1.32 20.46 43.39 ± 4.56 44.28 1.61 ± 0.08 1.65 Demineralized enamel 18.13 ± 0.98 17.89 a 36.72 ± 2.12 36.52 a 1.57 ± 0.07 1.55 Demineralized enamel/Orthocare™ 19.22 ± 1.35 18.91 ab 42.14 ± 5.33 41.72 b 1.69 ± 0.14 1.65 b Demineralized enamel/Regenerate™ 16.89 ± 0.96 20.01 b 44.05 ± 6.15 41.64 b 1.71 ± 0.20 1.61 b SD: Standard deviation. Significant differences (p < 0.05) a: versus Intact enamel, b: versus Demineralized enamel. Phosphorus content was significantly lower in demineralized enamel and demineralized enamel/Orthocare™ compared with intact enamel (p < 0.001). Both treatment groups (Orthocare™ and Regenerate™) showed significantly higher phosphorus values than demineralized enamel (p < 0.001). Calcium content was significantly reduced in demineralized enamel compared with intact enamel (p < 0.001). Both treated groups showed significantly higher calcium values than demineralized enamel (p < 0.001). The Ca/P ratio was significantly higher in both treatment groups compared with demineralized enamel (p 0.05). Raman Spectroscopy Results are presented in Fig. 2 and Tables 5 and 6 . Table 5 Mean and standard deviation of Raman intensities (in arbitrary units) of mineral components in treated and nontreated enamel surfaces. Peaks positions are expressed in cm − 1 . Groups I 960 I 1070 A 960 FWHM I 1070 /I 960 Intact enamel 723.75 ± 58.26 59.39 ± 4.84 293.32 ± 23.88 16.44 ± 0.04 0.08 ± 0.00 Demineralized enamel 180.75 ± 31.73 a 15.64 ± 2.6 a 72.48 ± 12.96 a 16.25 ± 0.10 0.09 ± 0.00 Demineralized enamel/Orthocare™ 250.83 ± 32.62 a 22.36 ± 3.81 a 102.57 ± 12.7 a 16.58 ± 0.13 b 0.09 ± 0.00 Demineralized enamel/Regenerate™ 426.79 ± 157.18 a,b,c 36.10 ± 12.90 a,b,c 174.22 ± 63.17 a,b,c 16.61 ± 0.22 b 0.08 ± 0.01 Significant differences (p < 0.05) a: versus Intact enamel, b: versus Demineralized enamel, c: versus Demineralized enamel/ Orthocare™. Table 6 Mean and standard deviation of Raman intensities (in arbitrary units) of mineral components in treated and nontreated enamel surfaces. Peaks positions are expressed in cm − 1 . Groups V2 (I 431 ) V4 (I 582 ) V3 (I 1044 ) Intact enamel 114.63 ± 8.40 74.39 ± 5.06 38.34 ± 3.70 Demineralized enamel 37.90 ± 5.75 a 24.36 ± 3.98 a 12.44 ± 2.62 a Demineralized enamel/Orthocare™ 49.42 ± 7.56 a 29.45 ± 4.43 a 17.82 ± 3.87 a Demineralized enamel/Regenerate™ 74.48 ± 24.36 a,b 45.44 ± 13.33 a,b,c 27.66 ± 8.38 a,b,c Significant differences (p < 0.05) a: versus Intact enamel, b: versus Demineralized enamel, c: versus Demineralized enamel/ Orthocare™. The values of I 960 , I 1070 , I 431 , I 582 , I 1044 , and A 960 were significantly higher in intact enamel compared with all other groups (p < 0.05). Additionally, these parameters were significantly higher in the demineralized enamel/Regenerate™ group compared with both demineralized enamel and demineralized enamel/Orthocare™ (p 0.05). FWHM values were significantly higher in both treatment groups (Orthocare™ and Regenerate™) compared with demineralized enamel (p 0.05). The I 1070 /I 960 ratio did not differ significantly among groups (p > 0.05). Raman spectroscopy revealed clear differences in mineral content and structural organization among the experimental groups. The intensity of the ν1 phosphate band at approximately 960 cm⁻¹ (I 960 ), indicative of mineral content, was highest in intact enamel (723.75 ± 58.26; p < 0.05). A marked and significant decrease was observed after demineralization (180.75 ± 31.73; p < 0.05), confirming substantial mineral loss. Treatment with Orthocare™ resulted in a slight increase in I 960 (250.83 ± 32.62), although values remained significantly lower than those of intact enamel. In contrast, the Regenerate™ group showed a greater recovery (426.79 ± 157.18; p < 0.05), reaching intermediate values between demineralized and intact enamel. A similar pattern was observed for the integrated area of the phosphate band (A 960 ). Intact enamel showed the highest values (293.32 ± 23.88; p < 0.05), followed by a significant decrease after demineralization (72.48 ± 12.96; p < 0.05). Partial recovery was observed in both treatment groups, with higher values in the Regenerate™ group (174.22 ± 63.17; p < 0.05), indicating greater remineralization compared with Orthocare™. The carbonate band intensity (~ 1070 cm⁻¹; I 1070 ) also decreased significantly after demineralization (15.64 ± 2.60 vs. 59.39 ± 4.84 in intact enamel; p < 0.05). Both treatments increased carbonate content, with higher values observed in the Regenerate™ group (36.10 ± 12.90; p < 0.05) compared with Orthocare™ (22.36 ± 3.81). However, the carbonate-to-phosphate ratio (I 1070 /I 960 ) remained relatively stable across groups (0.08–0.09), indicating that although mineral content changed, the relative carbonate substitution was not substantially affected. FWHM values showed minimal variation between intact enamel (16.44 ± 0.04) and demineralized enamel (16.25 ± 0.10). However, both treatment groups exhibited a slight increase (16.58 ± 0.13 for Orthocare™ and 16.61 ± 0.22 for Regenerate™), suggesting subtle changes in crystal organization. Analysis of secondary phosphate vibrational modes (ν2, ν3, and ν4) supported these findings. The intensities of ν2 (431 cm⁻¹), ν4 (582 cm⁻¹), and ν3 (1044 cm⁻¹) were significantly reduced after demineralization, indicating loss of mineral structure. Both treatments resulted in partial recovery of these bands, with consistently higher values in the Regenerate™ group (74.48 ± 24.36 for ν2, 45.44 ± 13.33 for ν4, and 27.66 ± 8.38 for ν3), approaching but not reaching the values of intact enamel. DISCUSSION The aim of this study was to evaluate the ability of Orthocare™ and Regenerate™ to remineralize demineralized enamel and to assess their effect on bracket bonding. The results of the present study showed that bond strength to demineralized enamel was significantly lower than to intact enamel, in agreement with previous studies[ 1 , 3 – 7 ]. FE-SEM revealed increased porosity in demineralized enamel, consistent with previous reports [ 16 , 20 ]. In addition, EDX analysis demonstrated a significant reduction in calcium and phosphorus content compared with intact enamel, while the Ca/P ratio remained unchanged. Raman spectroscopy further confirmed these findings, showing a marked decrease in I 960 intensity and in the integrated area of the phosphate band (A 960 ), as well as reductions in the other phosphate vibrational modes (ν2, ν3, and ν4), indicating substantial loss of hydroxyapatite from the enamel surface [ 21 ]. When enamel is exposed to acidic conditions, three types of demineralization may occur: surface softening (pH 2–4, short exposure), subsurface demineralization (pH 4.5–6.5, longer exposure, associated with caries lesions), and surface etching (brief exposure to strong acids such as phosphoric acid). In the present study, the demineralization model corresponded to surface softening, in which mineral loss is limited to a depth of 1–10 µm, with gradual recovery of mineral content toward deeper enamel layers. Ultrastructurally, mineral loss occurs mainly within enamel prisms and, particularly, in interprismatic spaces [ 22 ]. A reduction in carbonate bands was also observed following demineralization. However, neither the I 1070 /I 960 ratio nor FWHM values were significantly altered, suggesting that neither the phosphate/carbonate ratio in the crystallite structure, nor the degree of crystallinity of carbonated hydroxyapatite, were modified by the demineralization to which the enamel samples were subjected. This finding differs from that of Swietlicka et al.[ 23 ], who reported a significant increase in the I 1070 /I 960 ratio in the demineralized enamel compared to the intact enamel after exposing it to 35% phosphoric acid for 15 seconds (etching). The loss of mineral content, along with increased permeability [ 4 ] and reduced microhardness[ 15 , 24 ] render demineralized enamel an unfavorable substrate for adhesion. This results in adhesive failure at the enamel/adhesive interface, leaving less remaining adhesive on the tooth than on the bracket base[ 5 , 25 , 26 ], as also observed in our study Following treatment with both remineralizing agents, a non-significant improvement in bond strength was observed, and the amount of adhesive remaining on the tooth surface became comparable to that of intact enamel. To our knowledge, no previous studies have evaluated the use of these specific products prior to bracket bonding. Scribante et al. [ 27 ] studied the efficacy of a biomimetic hydroxyapatite remineralizing solution on demineralized enamel, finding that the adhesion of brackets to remineralized enamel was significantly greater than to demineralized enamel, but significantly lower than to intact enamel. FESEM analysis revealed a reduction in enamel surface porosity after treatment, particularly in the Regenerate™ group. In agreement with these observations, EDX analysis showed a significant increase in calcium content and Ca/P ratio in the treated enamel groups, approaching values observed in intact enamel. Raman spectroscopy demonstrated an increase in phosphate band intensities after treatment, indicating mineral deposition. This effect was greater in the Regenerate™ group than in the Orthocare™ group. However, this mineral gain was at the expense of structurally immature apatite, likely due to being in the early stages of the crystallization process of the calcium phosphate phases, as indicated by the increase in FWHM in both treatment groups [ 28 ]. This incomplete remineralization may also be related to the neutral pH conditions used in the present study, as it has been suggested that complete remineralization is less likely under such conditions. In contrast, acidic environments may enhance the rate, depth, and extent of remineralization induced by nano-hydroxyapatite[ 29 ]. Thus, in a study where a nanohydroxyapatite paste was used and pH cycles were performed, microhardness and crystallinity significantly increased compared to demineralized enamel, and a decrease in the I 1070 /I 960 ratio was observed [ 20 ]. In our study, the carbonate band at I 1070 followed a similar trend to the phosphate bands, decreasing after demineralization and recovering after the application of the products. However, the I 1070 /I 960 ratio remained constant across all groups, indicating that neither demineralization nor remineralization affected the carbonate substitution in the hydroxyapatite structure, with the chemical composition of the enamel remaining unaltered. The greater remineralizing effect observed with Regenerate™ may be explained by a calcium silicate–mediated nucleation process, which promotes the organized deposition of calcium and phosphate, consistent with a biomimetic mechanism [ 17 ]. Thus, Sun et al. [ 30 ] concluded that there is evidence that with calcium silicate deposits, the formation of hydroxyapatite increases over time, with progressively more crystalline material. These authors observed, after 4 weeks of in vitro brushing, that the material deposited on the enamel was hydroxyapatite. However, the experimental conditions were different from those of our study; they used enamel blocks (not previously demineralized) which were brushed with a slurry of the calcium silicate toothpaste in water. Remineralization is often referred to as 'crystallization,' but strictly speaking, this implies the formation of a crystalline phase with internal long-range order of its constituents. However, calcium phosphate often deposits as an X-ray amorphous solid rather than as a crystal. The crystallization of calcium phosphate as a biomineral in enamel is complex, as there are multiple calcium phosphate phases, each with its own solubility and crystallization kinetics [ 31 ]. It has been shown that calcium phosphate crystallizes in a series of steps, often in the sequence of ACP to dihydrated dicalcium phosphate (DCPD) to octacalcium phosphate (OCP) to calcium-deficient hydroxyapatite (CDHA). These solid phases change until the formation of hydroxyapatite is achieved [ 28 ]. The remineralization mechanism of Orthocare™ is limited, as its main component, nanohydroxyapatite, would act more as a filling and repairing agent for the demineralized surface rather than as a biomimetic enamel regenerator from the depth of the lesion, given that the product did not supplement calcium and phosphate ions because the storage saliva pH was 6.57, relying solely on the concentrations in saliva, thus creating a superficial layer of hydroxyapatite on the enamel [ 32 , 33 ]. The enamel remineralization process involves too many unknown parameters, and the exact mechanism by which remineralization occurs is still far beyond our understanding [ 31 ]. A limitation of this study was the absence of pH cycling. Future studies should incorporate dynamic pH conditions to better simulate the oral environment. CONCLUSIONS Demineralized enamel treated with Orthocare™ and Regenerate™ exhibited signs of remineralization, which contributed to an improvement in bracket bond strength. Regenerate™ resulted in a greater recovery of mineral content compared with Orthocare™. However, the newly formed hydroxyapatite appeared to be structurally immature, as the level of crystallinity did not reach that of intact enamel. Consequently, the adhesion to remineralized enamel, although similar to that of intact enamel, was not significantly higher than that of demineralized enamel, which was the case with intact enamel. Declarations Conflict of Interest: the authors declare no conflict of interest. Acknowlegment None Funding: University of Murcia´s own funds Author Contribution AV: Conceptualization, Methodology, Investigation, Formal analisis, Writing-Original Draft, Writing-Review and Editing, Supervision. AJOR: Conceptualization, Methodology, Investigation, Formal analisis, Writing-Original Draft, Writing-Review and Editing, Supervision. CSM: Conceptualization, Methodology, Investigation, Formal análisis, Writing-Review, Supervision. ICM: Investigation, Methodology, Formal analisis, Writing-Review. YMB: Conceptualization, Methodology, Investigation, Formal analisis, Writing-Original Draft, Writing-Review and Editing, Supervision. Data availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. References Gulec A, Goymen M (2019) Assessment of the resin infiltration and CPP-ACP applications before orthodontic brackets bonding. Dent Mater J 38(5):854–860. https://doi.org/10.4012/dmj.2019-021 Gorelick L, Geiger AM, Gwinnett AJ (1982) Incidence of white spot formation after bonding and banding. Am J Orthod 81(2):93–98. https://doi.org/10.1016/0002-9416(82)90032-x Ghadirian H, Geramy A, Shallal W et al (2020) The Effect of Remineralizing Agents With/Without CO2 Laser Irradiation on Structural and Mechanical Properties of Enamel and its Shear Bond Strength to Orthodontic Brackets. J Lasers Med Sci 11(2):144–152. https://doi.org/10.34172/jlms.2020.25 Akin M, Baka ZM, Ileri Z et al (2014) Can demineralized enamel surfaces be bonded safely? Acta Odontol Scand 72(4):283–289. https://doi.org/10.3109/00016357.2013.823646 Velİ I, Akin M, Baka ZM et al (2016) Effects of different pre-treatment methods on the shear bond strength of orthodontic brackets to demineralized enamel. Acta Odontol Scand 74(1):7–13. https://doi.org/10.3109/00016357.2014.982703 Shahabi M, Moosavi H, Gholami A et al (2012) In vitro effects of several surface preparation methods on shear bond strength of orthodontic brackets to caries-like lesions of enamel. Eur J Paediatr Dent 13(3):197–202 Baka ZM, Akin M, Ileri Z et al (2016) Effects of remineralization procedures on shear bond strengths of brackets bonded to demineralized enamel surfaces with self-etch systems. Angle Orthod 86(4):661–667. https://doi.org/10.2319/041515-247.1 Iafisco M, Degli Esposti L, Ramírez-Rodríguez GB et al (2018) Fluoride-doped amorphous calcium phosphate nanoparticles as a promising biomimetic material for dental remineralization. Sci Rep 8(1):17016. https://doi.org/10.1038/s41598-018-35258-x Joiner A, Schäfer F, Naeeni MM et al (2014) Remineralisation effect of a dual-phase calcium silicate/phosphate gel combined with calcium silicate/phosphate toothpaste on acid-challenged enamel in situ. J Dent 42(Suppl 1):S53–59. https://doi.org/10.1016/S0300-5712(14)50008-5 Haghgoo R, Abbasi F, Rezvani MB (2011) Evaluation of the effect of nanohydroxyapatite on erosive lesions of the enamel of permanent teeth following exposure to soft beer in vitro. SRE ;6(28):5933–6. https://doi.org/10.5897/SRE11.1486 Tschoppe P, Zandim DL, Martus P et al (2011) Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 39(6):430–437. https://doi.org/10.1016/j.jdent.2011.03.008 Pepla E, Besharat LK, Palaia G et al (2014) Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature. Ann Stomatol (Roma) 5(3):108–114 Battistella E, Foresti E, Lelli M et al (2009) Surface Enamel Remineralization: Biomimetic Apatite Nanocrystals and Fluoride Ions Different Effects. J Nanomaterials 2009:1. https://doi.org/10.1155/2009/746383 Niwa M, Sato T, Li W et al (2001) Polishing and whitening properties of toothpaste containing hydroxyapatite. J Mater Sci Mater Med 12(3):277–281. https://doi.org/10.1023/a:1008927502523 Rajendran R, Antony SDP, Ashik PM et al (2024) Remineralization potential of strontium-doped nano-hydroxyapatite dentifrice and casein phosphopeptide-amorphous calcium phosphate cream on white spot lesions in enamel following orthodontic debonding - a randomized controlled trial. SAGE Open Med 12:20503121231221634. https://doi.org/10.1177/20503121231221634 Badiee M, Jafari N, Fatemi S et al (2020) Comparison of the effects of toothpastes containing nanohydroxyapatite and fluoride on white spot lesions in orthodontic patients: A randomized clinical trial. Dent Res J (Isfahan) 17(5):354–359 Parker AS, Patel AN, Al Botros R et al (2014) Measurement of the efficacy of calcium silicate for the protection and repair of dental enamel. J Dent 42(Suppl 1):S21–29. https://doi.org/10.1016/S0300-5712(14)50004-8 Hornby K, Ricketts SR, Philpotts CJ et al (2014) Enhanced enamel benefits from a novel toothpaste and dual phase gel containing calcium silicate and sodium phosphate salts. J Dent 42(Suppl 1):S39–45. https://doi.org/10.1016/S0300-5712(14)50006-1 Vicente A, Ortiz-Ruiz AJ, González-Paz BM et al (2021) Effectiveness of a toothpaste and a serum containing calcium silicate on protecting the enamel after interproximal reduction against demineralization. Sci Rep 11(1):834. https://doi.org/10.1038/s41598-020-80844-7 Orilisi G, Vitiello F, Notarstefano V et al (2023) Multidisciplinary evaluation of the remineralization potential of three fluoride-based toothpastes on natural white spot lesions. Clin Oral Investig 27(12):7451–7462. https://doi.org/10.1007/s00784-023-05334-2 Schulze KA, Balooch M, Balooch G et al (2004) Micro-Raman spectroscopic investigation of dental calcified tissues. J Biomed Mater Res A 69(2):286–293. https://doi.org/10.1002/jbm.a.20130 Arends J, Ten Cate JM (1981) Tooth enamel remineralization. J Cryst Growth 53(1):135–147. https://doi.org/10.1016/0022-0248(81)90060-9 Świetlicka I, Kuc D, Świetlicki M et al (2020) Near-Surface Studies of the Changes to the Structure and Mechanical Properties of Human Enamel under the Action of Fluoride Varnish Containing CPP-ACP Compound. Biomolecules 10(5):765. https://doi.org/10.3390/biom10050765 Ghadirian H, Geramy A, Shallal W et al (2020) The Effect of Remineralizing Agents With/Without CO2 Laser Irradiation on Structural and Mechanical Properties of Enamel and its Shear Bond Strength to Orthodontic Brackets. J Lasers Med Sci 11(2):144–152. https://doi.org/10.34172/jlms.2020.25 Baysal A, Uysal T (2012) Do enamel microabrasion and casein phosphopeptide-amorphous calcium phosphate affect shear bond strength of orthodontic brackets bonded to a demineralized enamel surface? Angle Orthod 82(1):36–41. https://doi.org/10.2319/041211-265.1 Uysal T, Baysal A, Uysal B et al (2011) Do fluoride and casein phosphopeptide-amorphous calcium phosphate affect shear bond strength of orthodontic brackets bonded to a demineralized enamel surface? Angle Orthod 81(3):490–495. https://doi.org/10.2319/090510-520.1 Scribante A, Dermenaki Farahani MR, Marino G et al Biomimetic Effect of Nano-Hydroxyapatite in Demineralized Enamel before Orthodontic Bonding of Brackets and Attachments: Visual, Adhesion Strength, and Hardness in In Vitro Tests. Biomed Res Int ;2020:6747498. https://doi.org/10.1155/2020/6747498 Wang L, Nancollas GH (2008) Calcium Orthophosphates: Crystallization and Dissolution. Chem Rev 108(11):4628–4669. https://doi.org/10.1021/cr0782574 Huang S, Gao S, Cheng L et al (2011) Remineralization potential of nano-hydroxyapatite on initial enamel lesions: an in vitro study. Caries Res 45(5):460–468. https://doi.org/10.1159/000331207 Sun Y, Li X, Deng Y et al (2014) Mode of action studies on the formation of enamel minerals from a novel toothpaste containing calcium silicate and sodium phosphate salts. J Dent 42(Suppl 1):S30–38. https://doi.org/10.1016/S0300-5712(14)50005-X Enax J, Fandrich P, Schulze zur Wiesche E et al (2024) The Remineralization of Enamel from Saliva: A Chemical Perspective. Dent J (Basel) 12(11):339. https://doi.org/10.3390/dj12110339 Anil A, Ibraheem WI, Meshni AA et al (2022) Nano-Hydroxyapatite (nHAp) in the Remineralization of Early Dental Caries: A Scoping Review. Int J Environ Res Public Health 19(9):5629. https://doi.org/10.3390/ijerph19095629 Meyer F, Enax J, Amaechi BT et al (2022) Hydroxyapatite as Remineralization Agent for Children’s Dental Care. Front Dent Med 3. https://doi.org/10.3389/fdmed.2022.859560 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 13 May, 2026 Reviewers agreed at journal 12 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviews received at journal 06 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers invited by journal 06 May, 2026 Editor assigned by journal 30 Apr, 2026 Submission checks completed at journal 30 Apr, 2026 First submitted to journal 24 Apr, 2026 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9515528","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":639910450,"identity":"63d759f9-bf88-4cee-a6b1-81c5ca24182f","order_by":0,"name":"Ascensión Vicente","email":"","orcid":"","institution":"University of Murcia","correspondingAuthor":false,"prefix":"","firstName":"Ascensión","middleName":"","lastName":"Vicente","suffix":""},{"id":639910451,"identity":"045907a0-7945-406a-b1a1-82451a35f446","order_by":1,"name":"Antonio J Ortiz-Ruiz","email":"","orcid":"","institution":"University of Murcia","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"J","lastName":"Ortiz-Ruiz","suffix":""},{"id":639910452,"identity":"e3a37f5f-22c8-469d-bf0f-de8aa049c7f8","order_by":2,"name":"Clara Serna-Muñoz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYDACdgST8QFxWpiRmAYka2GTIEoHfzPzsw8/GOrkzGc3P6vmzWGQk28goEXiMJvxzB6Gw8Yyd46Z3ebdxmBscICAFgNmBmMGHoYDiTMkEsBaEjcQcpgBM/tnxj8MdfUzJNK/FQO11M8n5DADZh5jZh4G5gQJiRwzZqCWBAZCDpM4zFPMLGNw2HCGRE6x5NxtEoYbCGnhb2/fzPimok5eQiJ944e322zkCYYY1HkIW4lSPwpGwSgYBaOAAAAAxNYyey5JIRgAAAAASUVORK5CYII=","orcid":"","institution":"University of Murcia","correspondingAuthor":true,"prefix":"","firstName":"Clara","middleName":"","lastName":"Serna-Muñoz","suffix":""},{"id":639910453,"identity":"23e6cdb0-156a-4ccf-b8e1-27a8b172e826","order_by":3,"name":"Inmaculada Cabello","email":"","orcid":"","institution":"University of Granada","correspondingAuthor":false,"prefix":"","firstName":"Inmaculada","middleName":"","lastName":"Cabello","suffix":""},{"id":639910454,"identity":"0af96ff4-8510-4f41-90e5-4ba6d63416d7","order_by":4,"name":"Yolanda Martínez-Beneyto","email":"","orcid":"","institution":"University of Murcia","correspondingAuthor":false,"prefix":"","firstName":"Yolanda","middleName":"","lastName":"Martínez-Beneyto","suffix":""}],"badges":[],"createdAt":"2026-04-24 09:59:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9515528/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9515528/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109332223,"identity":"d96535ad-666f-40ba-8b69-1d55a67570ac","added_by":"auto","created_at":"2026-05-15 16:14:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7326487,"visible":true,"origin":"","legend":"\u003cp\u003eFE-SEM Images x1000. A) Intact enamel, B) Desmineralized enamel, C) Desmineralized enamel/ Orthocare \u003csup\u003e\u003cstrong\u003eTM\u003c/strong\u003e\u003c/sup\u003e, D) Desmineralized enamel/Regenerate \u003csup\u003e\u003cstrong\u003eTM\u003c/strong\u003e\u003c/sup\u003e .\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9515528/v1/94323c0cbecbc66a794de8e9.png"},{"id":109405824,"identity":"04244b5f-d3b2-482c-bd10-ad8f713e96ab","added_by":"auto","created_at":"2026-05-17 13:20:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":213610,"visible":true,"origin":"","legend":"\u003cp\u003eSpectra obtained from the different experimental groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9515528/v1/bbb0616646a5180e2f779bb4.png"},{"id":109406155,"identity":"88c10433-185a-4537-895a-b5aab9977008","added_by":"auto","created_at":"2026-05-17 13:25:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5746489,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9515528/v1/5073ae48-3b78-4db8-85bd-5dcc8d34e141.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of enamel remineralization and its effect on orthodontic bracket shear bond strength: an in vitro study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eWhen a patient is scheduled to undergo orthodontic treatment, it is important to assess enamel characteristics before bracket bonding, as teeth may present with demineralized enamel. Although demineralization lesions are commonly considered a risk associated with orthodontic treatment, they may also be present in patients without previous orthodontic therapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Gorelick et al.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] reported that 24% of patients had at least one tooth with demineralized enamel prior to orthodontic treatment. The highest incidence of these lesions was observed in maxillary incisors, mandibular molars, and second premolars.\u003c/p\u003e \u003cp\u003eSeveral authors have reported lower bracket bond strength to demineralized enamel compared with intact enamel[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bond failure requires removal of residual adhesive and re-etching of the enamel for rebonding. Both procedures result in tissue loss and may increase the severity of demineralization. Therefore, to achieve optimal adhesion, it is important to treat demineralized enamel before bracket bonding [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], recommending the use of remineralizing agents[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, biomimetic materials have been introduced to induce enamel remineralization. These materials mimic the composition and structure of the mineral phase of the tooth and are capable of restoring enamel toward its native structure [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among these systems, biomimetic calcium phosphate-based materials such as nano-hydroxyapatite, calcium phosphosilicates, and amorphous calcium phosphates (ACP) are particularly relevant [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOrthocare\u0026trade; nHAp\u0026trade; Toothpaste and Orthocare\u0026trade; nHAp\u0026trade; Boost Sponge are based on nano-hydroxyapatite. This material is considered highly biocompatible and bioactive due to its physicochemical similarity to enamel apatite crystals [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Owing to the nanometric size of its particles, it can fill small depressions and pores on the enamel surface [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The use of synthetic hydroxyapatite nanocrystals has been shown to promote enamel remineralization and repair [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegenerate\u0026trade; Advanced Toothpaste \u0026amp; Serum combines a toothpaste containing calcium silicate and sodium phosphate salts with a serum composed of sodium fluoride. This combination increases the supply and retention of calcium ions in enamel, which are subsequently transformed into hydroxyapatite [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Previous studies have demonstrated its effectiveness in protecting enamel subjected to stripping against demineralization [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and in re-hardening enamel previously exposed to acid attack [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, no studies have evaluated the remineralizing capacity of these products prior to bracket bonding. Therefore, the aim of this study was to evaluate, in vitro, the effect of Orthocare\u0026trade; nHAp\u0026trade; Toothpaste \u0026amp; Boost Sponge and Regenerate\u0026trade; Advanced Toothpaste \u0026amp; Serum on the remineralization of demineralized enamel and on bracket adhesion.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eThe study was approved by the Biosafety Committee in Experimentation of the University XXXXX, XXXXX (CBE 332).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSpecimen preparation and experimental groups\u003c/h2\u003e \u003cp\u003eA total of 180 bovine teeth without signs of demineralization or enamel fractures were used. After extraction, the specimens were immersed in a 0.1% thymol solution for 24 hours. Subsequently, they were stored in distilled water (Tecnoquim S.L., Murcia, Spain) at room temperature, with daily renewal until the beginning of the experimental protocol. The apices of all teeth were sealed using a flowable composite (TetricEvoFlow, Ivoclar Vivadent, Liechtenstein).\u003c/p\u003e \u003cp\u003eThe teeth were randomly allocated into four experimental groups (n\u0026thinsp;=\u0026thinsp;45):\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eGroup 1: Intact enamel\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup 2: Demineralized enamel\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup 3: Demineralized enamel/Regenerate\u0026trade;\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup 4: Demineralized enamel/Orthocare\u0026trade;\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eTeeth in groups 2, 3, and 4 were immersed in a lactic acid solution (L(+)-lactic acid, 88\u0026ndash;92%, extrapure, pH 2.8; Scharlau, Barcelona, Spain) for 24 hours. Subsequently, the specimens were rinsed with distilled water to remove residual acid and subjected to ultrasonic cleaning (Talleres Mestraitua, S.L. MESTRA, Vizcaya, Spain) for 30 minutes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental procedure\u003c/h3\u003e\n\u003cp\u003eAll specimens were stored in artificial saliva at 37\u0026deg;C in a laboratory incubator (JP Selecta S.A., Barcelona, Spain) for 60 days. The artificial saliva was renewed daily. Its composition was as follows: 1% carmellose sodium, 13% sorbitol, 0.12% potassium chloride, 0.084% sodium chloride, 0.005% magnesium chloride hexahydrate, 0.015% anhydrous calcium chloride, 0.017% dipotassium phosphate, and 0.1% sodium nipagin. The pH was adjusted and maintained at 6.57.\u003c/p\u003e \u003cp\u003eThe teeth were removed from the artificial saliva three times daily, and the buccal surfaces were brushed for 15 seconds using a medium-bristle toothbrush (VITIS-DENTAID, Barcelona, Spain). In groups 1 and 2, brushing was performed without toothpaste.\u003c/p\u003e \u003cp\u003eIn group 3, brushing was performed using Regenerate\u0026trade; Advanced Toothpaste. Additionally, during the first three days of each month, Regenerate\u0026trade; Advanced Enamel Serum was applied after the last daily brushing and left undisturbed for 3 minutes.\u003c/p\u003e \u003cp\u003eIn group 4, brushing was performed using Orthocare\u0026trade; nHAp\u0026trade; Toothpaste. Orthocare\u0026trade; nHAp\u0026trade; Boost Sponge was applied every 5 days after the last daily brushing and left undisturbed for 3 minutes.\u003c/p\u003e \u003cp\u003eAll procedures were performed according to the manufacturers\u0026rsquo; instructions. The composition of the products is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of the remineralizing agents evaluated.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAGENT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCOMPOSITION\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\u003eOrthocare\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003enHAp\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eTooth paste\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycerin, Xylitol, Potassium Nitrate, Hydroxyapatite (nano), Magnesium Aluminum Silicate, Mentha Piperita Oil, Sodium Lauroyl Sarcosinate, Xanthan Gum, Phenoxyethanol, Potassium Chloride, Sodium Sulfate, Sodium Saccharin, Linalool, Limonene, CI 77891\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOrthocare\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003enHAp\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eBoost Sponge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAqua, Xylitol, Hydroxyapatite (nano), Potassium Chloride, Sclerotium Gum, Mentha Piperita, Oil, Linalool, Limonene\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRegenerate\u003c/b\u003e \u003csup\u003e\u003cb\u003eTM\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eAdvance Toothpaste\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycerin, Calcium Silicate, PEG-8, Hydrated Silica, Trisodium Phosphate, Sodium Phosphate, Aqua, PE-60, Sodium Lauryl Sulfate, Sodium Monofluorophospate (1450 ppm F), Aroma Flavour, Synthetic Fluorphlogopite, Sodium Saccharin, Polyacrylic Acid, Tin Oxide, Limonene, C177891\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRegenerate\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eAdvance Enamel Serum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eNR-5TM Serum\u003c/span\u003e: Glycerin, Calcium Silicate, PEG-8, Trisodium Phosphate, Sodium Phosphate, Aqua, PEG-60, Sodium Lauryl Sulfate, Sodium Monofluorophosphate, Aroma, Hydrated Silica, Synthetic Fluorphlogopite, Sodium Saccharin, Polyacrylic Acid, Tin Oxide, Limonene, CI 77891.\u003c/p\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eActivator Gel\u003c/span\u003e: Aqua, Glycerin, Cellulose Gum, Sodium Fluoride (1450 ppm F), Benzyl Alcohol, Ethylhexylglycerin, Phenoxyethanol, CI 42090.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eBond strength test and percentage of area occupied by adhesive on the tooth\u003c/h3\u003e\n\u003cp\u003eA total of 80 maxillary bovine incisors (n\u0026thinsp;=\u0026thinsp;20 per group) were used. The specimens were mounted in cylindrical molds (3 cm internal diameter, 4 cm height), embedding the roots in type IV dental stone.\u003c/p\u003e \u003cp\u003eMetal brackets for maxillary central incisors (3M Unitek Dental Products, California, USA) were bonded to the buccal surfaces. The enamel surfaces were polished using a prophylaxis cup and fluoride-free polishing paste (Detartrine, Septodont, France), rinsed, and air-dried. Subsequently, 37% phosphoric acid (Octacid, Laboratorios Clarben) was applied for 20 seconds, followed by rinsing and drying.The brackets were bonded using the Transbond XT adhesive system following the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eAfter bonding, specimens were stored in distilled water at 37\u0026deg;C for 24 hours.\u003c/p\u003e \u003cp\u003eShear bond strength was measured using a universal testing machine (Autograph AGS-1KND, Shimadzu, Kyoto, Japan) with a 1 kN load cell. A chisel-edge rod angled at 30\u0026deg; was applied at the bracket\u0026ndash;adhesive interface. The crosshead speed was set at 1 mm/min, applying force parallel to the tooth surface in an incisal\u0026ndash;cervical direction.\u003c/p\u003e \u003cp\u003eThe percentage of adhesive remaining on the tooth surface after debonding was determined using image analysis (Sony dxc 151-ap camera connected to an Olympus SZ11 microscope) and MIP software. The percentage was calculated by subtracting the adhesive-covered area on the bracket base from 100%.\u003c/p\u003e\n\u003ch3\u003eField Emission-Scanning Electron Microscopy (FE-SEM) and Energy Dispersive X-ray Spectroscopy (EDX) analysis\u003c/h3\u003e\n\u003cp\u003eA total of 80 specimens (n\u0026thinsp;=\u0026thinsp;20 per group) were analyzed. Teeth were sectioned at the cemento-enamel junction using a diamond disc (Komet Dental, Gebr. Brasseler GmbH \u0026amp; Co., Lemgo, Germany). Specimens were mounted on aluminum stubs and sputter-coated with a 5 nm platinum layer (Leica EM ACE 600).\u003c/p\u003e \u003cp\u003eFE-SEM analysis was performed using an ApreoS Lovac microscope (Thermo Fisher Scientific) at 5 kV and 0.8 nA. EDX analysis was conducted using an Octane Plus EDAX microanalyzer (AMETEK, USA) at 20 kV.\u003c/p\u003e \u003cp\u003eThe elements quantified were calcium (Ca, wt%) and phosphorus (P, wt%). The Ca/P molar ratio was calculated as follows: Ca (mol)/P (mol) % = [Ca (weight %) / 40.08 (g/mol)] / [P (weight %) / 30.97 (g/mol)], where the molecular masses of Ca and P are 40.08 and 30.97, respectively.\u003c/p\u003e\n\u003ch3\u003eRaman Spectroscopy\u003c/h3\u003e\n\u003cp\u003eRaman spectra were obtain using a Jasco NRS-5100 Raman microscope (Jasco Inc., Japan). The system was equipped with a 784.79 nm diode laser and an MPLFLN 20\u0026times; objective lens for laser focusing.\u003c/p\u003e \u003cp\u003eSpectra were recorded in the range of 162\u0026ndash;1886 cm⁻\u0026sup1; with a spectral interval of 1 cm⁻\u0026sup1;, yielding 1725 data points per spectrum. For analysis, the spectral region between 400 and 1100 cm⁻\u0026sup1; was selected. The spectral resolution was 3.22 cm⁻\u0026sup1; (1.68 cm⁻\u0026sup1;/pixel). The laser power at the sample surface was set to 11.8 mW.\u003c/p\u003e \u003cp\u003eEach spectrum was acquired with an exposure time of 30 seconds and 1 accumulation. A 600 lines/mm grating, a 50 \u0026times; 1000 \u0026micro;m slit, and a DU420_OE CCD detector (cooled to \u0026minus;\u0026thinsp;68\u0026deg;C) were used. Cosmic ray reduction was enabled during acquisition.\u003c/p\u003e \u003cp\u003eFive samples per group were analyzed. Raman measurements were performed at a single point located at the center of the enamel surface of each tooth crown. The enamel surfaces were neither sectioned nor polished, as the aim was to evaluate remineralization on previously demineralized enamel. Prior to analysis, all samples were dehydrated for 24 hours at room temperature.\u003c/p\u003e \u003cp\u003eFor each group, one spectrum per sample was collected (n\u0026thinsp;=\u0026thinsp;5), and the average spectrum was calculated to obtain representative data for each group. The laser spot size was estimated to be in the micrometer range (~\u0026thinsp;2\u0026ndash;5 \u0026micro;m).\u003c/p\u003e \u003cp\u003eSpectral preprocessing included baseline correction and normalization using Spectragryph software (version 1.2). A spectral shift correction (\u0026minus;\u0026thinsp;2.47 cm⁻\u0026sup1;) was applied.\u003c/p\u003e \u003cp\u003eThe degree of mineralization and compositional changes were evaluated based on phosphate and carbonate vibrational bands. The analyzed parameters included: phosphate ν1 band (960 cm⁻\u0026sup1;; I\u003csub\u003e960\u003c/sub\u003e), used as a marker of mineral content; carbonate peak (~\u0026thinsp;1070 cm⁻\u0026sup1;); full width at half maximum of the band centered at 960 cm⁻\u0026sup1; (FWHM\u003csub\u003e960\u003c/sub\u003e), as an indicator of crystallinity; the area under the phosphate curve at 960 cm⁻\u0026sup1; (A\u003csub\u003e960\u003c/sub\u003e); the carbonate-to-phosphate ratio (C\u003csub\u003e1070\u003c/sub\u003e/P\u003csub\u003e960\u003c/sub\u003e), as an indicator of carbonate substitution; and the ν2 (431 cm⁻\u0026sup1;; I\u003csub\u003e431\u003c/sub\u003e), ν3 (582 cm⁻\u0026sup1;; I\u003csub\u003e582\u003c/sub\u003e), and ν4 (1044 cm⁻\u0026sup1;; I\u003csub\u003e1044\u003c/sub\u003e) bands (Lee et al., 2020; C C et al., 2020).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using jamovi project v.2.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jamovi.org\u003c/span\u003e\u003cspan address=\"https://www.jamovi.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNormality and homogeneity of variance were verified for bond strength and Raman data; therefore, one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was applied (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eData for adhesive remnant and EDX did not meet these assumptions and were analyzed using the Kruskal\u0026ndash;Wallis test followed by the Dwass\u0026ndash;Steel\u0026ndash;Critchlow\u0026ndash;Fligner test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eShear Bond strength and adhesive remnant\u003c/h2\u003e \u003cp\u003eShear bond strength (SBS) was significantly lower in demineralized enamel compared with intact enamel (p\u0026thinsp;=\u0026thinsp;0.01). No statistically significant differences were observed among the remaining groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\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\u003eShear Bond Strength (MPa).\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=\"char\" char=\"\u0026plusmn;\" 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\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian\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\u003eIntact enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.99 \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Regenerate\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Orthocare\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSD: Standard deviation\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSignificant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) versus a: Intact enamel.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe percentage of adhesive remaining on the tooth surface after debonding was significantly lower in the demineralized enamel group compared with the other groups (vs. intact enamel, p\u0026thinsp;=\u0026thinsp;0.04; vs. demineralized enamel/Orthocare\u0026trade;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; vs. demineralized enamel/Regenerate\u0026trade;, p\u0026thinsp;=\u0026thinsp;0.006). No statistically significant differences were found among the other groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage of tooth area occupied by adhesive.\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=\"char\" char=\"\u0026plusmn;\" 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\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian\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\u003eIntact enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e50.99\u0026thinsp;\u0026plusmn;\u0026thinsp;18.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e34.49\u0026thinsp;\u0026plusmn;\u0026thinsp;9.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.71 \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Regenerate\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e54.19\u0026thinsp;\u0026plusmn;\u0026thinsp;18.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.97 \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Orthocare\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026trade;\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e55.99\u0026thinsp;\u0026plusmn;\u0026thinsp;13.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.90 \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSD: Standard deviation\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSignificant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) versus a: Intact enamel and b: Demineralized enamel.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFESEM and EDX analysis\u003c/h2\u003e \u003cp\u003eRepresentative FE-SEM images are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Intact enamel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) exhibited a regular surface with characteristic wear lines. Demineralized enamel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) showed the typical demineralization pattern, characterized by increased surface porosity following lactic acid exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDemineralized enamel treated with Orthocare\u0026trade; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) presented a slightly rough surface with non-homogeneously distributed aggregates. In contrast, demineralized enamel treated with Regenerate\u0026trade; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) exhibited aggregates of varying sizes on a surface without evident signs of demineralization.\u003c/p\u003e \u003cp\u003eEDX results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWeight percentage of Ca, P and Ca/P ratio (mol/mol).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" 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=\"char\" char=\"\u0026plusmn;\" 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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eCa/P\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMedian\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eMedian\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eMedian\u003c/b\u003e\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\u003eIntact enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e43.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.89 \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e36.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.52 \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Orthocare\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.91 \u003cb\u003eab\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e42.14\u0026thinsp;\u0026plusmn;\u0026thinsp;5.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.72 \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.65 \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Regenerate\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.01 \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e44.05\u0026thinsp;\u0026plusmn;\u0026thinsp;6.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.64 \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.61 \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eSD: Standard deviation.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eSignificant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) a: versus Intact enamel, b: versus Demineralized enamel.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePhosphorus content was significantly lower in demineralized enamel and demineralized enamel/Orthocare\u0026trade; compared with intact enamel (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Both treatment groups (Orthocare\u0026trade; and Regenerate\u0026trade;) showed significantly higher phosphorus values than demineralized enamel (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eCalcium content was significantly reduced in demineralized enamel compared with intact enamel (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Both treated groups showed significantly higher calcium values than demineralized enamel (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eThe Ca/P ratio was significantly higher in both treatment groups compared with demineralized enamel (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No statistically significant differences were observed among the remaining comparisons (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRaman Spectroscopy\u003c/h2\u003e \u003cp\u003eResults are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Tables\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean and standard deviation of Raman intensities (in arbitrary units) of mineral components in treated and nontreated enamel surfaces. Peaks positions are expressed in cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003csub\u003e960\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003csub\u003e1070\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003csub\u003e960\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFWHM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eI\u003csub\u003e1070\u003c/sub\u003e/I\u003csub\u003e960\u003c/sub\u003e\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\u003eIntact enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e723.75\u0026thinsp;\u0026plusmn;\u0026thinsp;58.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e293.32\u0026thinsp;\u0026plusmn;\u0026thinsp;23.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e180.75\u0026thinsp;\u0026plusmn;\u0026thinsp;31.73 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.48\u0026thinsp;\u0026plusmn;\u0026thinsp;12.96 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Orthocare\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250.83\u0026thinsp;\u0026plusmn;\u0026thinsp;32.62 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e102.57\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Regenerate\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e426.79\u0026thinsp;\u0026plusmn;\u0026thinsp;157.18 \u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.10\u0026thinsp;\u0026plusmn;\u0026thinsp;12.90 \u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e174.22\u0026thinsp;\u0026plusmn;\u0026thinsp;63.17 \u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eSignificant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) a: versus Intact enamel, b: versus Demineralized enamel, c: versus Demineralized enamel/ Orthocare\u0026trade;.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean and standard deviation of Raman intensities (in arbitrary units) of mineral components in treated and nontreated enamel surfaces. Peaks positions are expressed in cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eV2 (I\u003csub\u003e431\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV4 (I\u003csub\u003e582\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eV3 (I\u003csub\u003e1044\u003c/sub\u003e)\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\u003eIntact enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e114.63\u0026thinsp;\u0026plusmn;\u0026thinsp;8.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.39\u0026thinsp;\u0026plusmn;\u0026thinsp;5.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.90\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.98 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Orthocare\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.42\u0026thinsp;\u0026plusmn;\u0026thinsp;7.56 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87 \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDemineralized enamel/Regenerate\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74.48\u0026thinsp;\u0026plusmn;\u0026thinsp;24.36 \u003csup\u003e\u003cb\u003ea,b\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.44\u0026thinsp;\u0026plusmn;\u0026thinsp;13.33 \u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.66\u0026thinsp;\u0026plusmn;\u0026thinsp;8.38 \u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eSignificant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) a: versus Intact enamel, b: versus Demineralized enamel, c: versus Demineralized enamel/ Orthocare\u0026trade;.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe values of I\u003csub\u003e960\u003c/sub\u003e, I\u003csub\u003e1070\u003c/sub\u003e, I\u003csub\u003e431\u003c/sub\u003e, I\u003csub\u003e582\u003c/sub\u003e, I\u003csub\u003e1044\u003c/sub\u003e, and A\u003csub\u003e960\u003c/sub\u003e were significantly higher in intact enamel compared with all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, these parameters were significantly higher in the demineralized enamel/Regenerate\u0026trade; group compared with both demineralized enamel and demineralized enamel/Orthocare\u0026trade; (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No statistically significant differences were observed among the remaining groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFWHM values were significantly higher in both treatment groups (Orthocare\u0026trade; and Regenerate\u0026trade;) compared with demineralized enamel (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no significant differences among the other groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe I\u003csub\u003e1070\u003c/sub\u003e/I\u003csub\u003e960\u003c/sub\u003e ratio did not differ significantly among groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eRaman spectroscopy revealed clear differences in mineral content and structural organization among the experimental groups.\u003c/p\u003e \u003cp\u003eThe intensity of the ν1 phosphate band at approximately 960 cm⁻\u0026sup1; (I\u003csub\u003e960\u003c/sub\u003e), indicative of mineral content, was highest in intact enamel (723.75\u0026thinsp;\u0026plusmn;\u0026thinsp;58.26; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A marked and significant decrease was observed after demineralization (180.75\u0026thinsp;\u0026plusmn;\u0026thinsp;31.73; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming substantial mineral loss.\u003c/p\u003e \u003cp\u003eTreatment with Orthocare\u0026trade; resulted in a slight increase in I\u003csub\u003e960\u003c/sub\u003e (250.83\u0026thinsp;\u0026plusmn;\u0026thinsp;32.62), although values remained significantly lower than those of intact enamel. In contrast, the Regenerate\u0026trade; group showed a greater recovery (426.79\u0026thinsp;\u0026plusmn;\u0026thinsp;157.18; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reaching intermediate values between demineralized and intact enamel.\u003c/p\u003e \u003cp\u003eA similar pattern was observed for the integrated area of the phosphate band (A\u003csub\u003e960\u003c/sub\u003e). Intact enamel showed the highest values (293.32\u0026thinsp;\u0026plusmn;\u0026thinsp;23.88; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by a significant decrease after demineralization (72.48\u0026thinsp;\u0026plusmn;\u0026thinsp;12.96; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Partial recovery was observed in both treatment groups, with higher values in the Regenerate\u0026trade; group (174.22\u0026thinsp;\u0026plusmn;\u0026thinsp;63.17; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating greater remineralization compared with Orthocare\u0026trade;.\u003c/p\u003e \u003cp\u003eThe carbonate band intensity (~\u0026thinsp;1070 cm⁻\u0026sup1;; I\u003csub\u003e1070\u003c/sub\u003e) also decreased significantly after demineralization (15.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60 vs. 59.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.84 in intact enamel; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both treatments increased carbonate content, with higher values observed in the Regenerate\u0026trade; group (36.10\u0026thinsp;\u0026plusmn;\u0026thinsp;12.90; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with Orthocare\u0026trade; (22.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81).\u003c/p\u003e \u003cp\u003eHowever, the carbonate-to-phosphate ratio (I\u003csub\u003e1070\u003c/sub\u003e/I\u003csub\u003e960\u003c/sub\u003e) remained relatively stable across groups (0.08\u0026ndash;0.09), indicating that although mineral content changed, the relative carbonate substitution was not substantially affected.\u003c/p\u003e \u003cp\u003eFWHM values showed minimal variation between intact enamel (16.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04) and demineralized enamel (16.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10). However, both treatment groups exhibited a slight increase (16.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 for Orthocare\u0026trade; and 16.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 for Regenerate\u0026trade;), suggesting subtle changes in crystal organization.\u003c/p\u003e \u003cp\u003eAnalysis of secondary phosphate vibrational modes (ν2, ν3, and ν4) supported these findings. The intensities of ν2 (431 cm⁻\u0026sup1;), ν4 (582 cm⁻\u0026sup1;), and ν3 (1044 cm⁻\u0026sup1;) were significantly reduced after demineralization, indicating loss of mineral structure. Both treatments resulted in partial recovery of these bands, with consistently higher values in the Regenerate\u0026trade; group (74.48\u0026thinsp;\u0026plusmn;\u0026thinsp;24.36 for ν2, 45.44\u0026thinsp;\u0026plusmn;\u0026thinsp;13.33 for ν4, and 27.66\u0026thinsp;\u0026plusmn;\u0026thinsp;8.38 for ν3), approaching but not reaching the values of intact enamel.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe aim of this study was to evaluate the ability of Orthocare\u0026trade; and Regenerate\u0026trade; to remineralize demineralized enamel and to assess their effect on bracket bonding.\u003c/p\u003e \u003cp\u003eThe results of the present study showed that bond strength to demineralized enamel was significantly lower than to intact enamel, in agreement with previous studies[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFE-SEM revealed increased porosity in demineralized enamel, consistent with previous reports [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, EDX analysis demonstrated a significant reduction in calcium and phosphorus content compared with intact enamel, while the Ca/P ratio remained unchanged. Raman spectroscopy further confirmed these findings, showing a marked decrease in I\u003csub\u003e960\u003c/sub\u003e intensity and in the integrated area of the phosphate band (A\u003csub\u003e960\u003c/sub\u003e), as well as reductions in the other phosphate vibrational modes (ν2, ν3, and ν4), indicating substantial loss of hydroxyapatite from the enamel surface [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen enamel is exposed to acidic conditions, three types of demineralization may occur: surface softening (pH 2\u0026ndash;4, short exposure), subsurface demineralization (pH 4.5\u0026ndash;6.5, longer exposure, associated with caries lesions), and surface etching (brief exposure to strong acids such as phosphoric acid). In the present study, the demineralization model corresponded to surface softening, in which mineral loss is limited to a depth of 1\u0026ndash;10 \u0026micro;m, with gradual recovery of mineral content toward deeper enamel layers. Ultrastructurally, mineral loss occurs mainly within enamel prisms and, particularly, in interprismatic spaces [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA reduction in carbonate bands was also observed following demineralization. However, neither the I\u003csub\u003e1070\u003c/sub\u003e/I\u003csub\u003e960\u003c/sub\u003e ratio nor FWHM values were significantly altered, suggesting that neither the phosphate/carbonate ratio in the crystallite structure, nor the degree of crystallinity of carbonated hydroxyapatite, were modified by the demineralization to which the enamel samples were subjected. This finding differs from that of Swietlicka et al.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], who reported a significant increase in the I\u003csub\u003e1070\u003c/sub\u003e/I\u003csub\u003e960\u003c/sub\u003e ratio in the demineralized enamel compared to the intact enamel after exposing it to 35% phosphoric acid for 15 seconds (etching).\u003c/p\u003e \u003cp\u003eThe loss of mineral content, along with increased permeability [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and reduced microhardness[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] render demineralized enamel an unfavorable substrate for adhesion. This results in adhesive failure at the enamel/adhesive interface, leaving less remaining adhesive on the tooth than on the bracket base[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], as also observed in our study\u003c/p\u003e \u003cp\u003eFollowing treatment with both remineralizing agents, a non-significant improvement in bond strength was observed, and the amount of adhesive remaining on the tooth surface became comparable to that of intact enamel. To our knowledge, no previous studies have evaluated the use of these specific products prior to bracket bonding. Scribante et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] studied the efficacy of a biomimetic hydroxyapatite remineralizing solution on demineralized enamel, finding that the adhesion of brackets to remineralized enamel was significantly greater than to demineralized enamel, but significantly lower than to intact enamel.\u003c/p\u003e \u003cp\u003eFESEM analysis revealed a reduction in enamel surface porosity after treatment, particularly in the Regenerate\u0026trade; group. In agreement with these observations, EDX analysis showed a significant increase in calcium content and Ca/P ratio in the treated enamel groups, approaching values observed in intact enamel.\u003c/p\u003e \u003cp\u003eRaman spectroscopy demonstrated an increase in phosphate band intensities after treatment, indicating mineral deposition. This effect was greater in the Regenerate\u0026trade; group than in the Orthocare\u0026trade; group. However, this mineral gain was at the expense of structurally immature apatite, likely due to being in the early stages of the crystallization process of the calcium phosphate phases, as indicated by the increase in FWHM in both treatment groups [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This incomplete remineralization may also be related to the neutral pH conditions used in the present study, as it has been suggested that complete remineralization is less likely under such conditions. In contrast, acidic environments may enhance the rate, depth, and extent of remineralization induced by nano-hydroxyapatite[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, in a study where a nanohydroxyapatite paste was used and pH cycles were performed, microhardness and crystallinity significantly increased compared to demineralized enamel, and a decrease in the I\u003csub\u003e1070\u003c/sub\u003e/I\u003csub\u003e960\u003c/sub\u003e ratio was observed [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In our study, the carbonate band at I\u003csub\u003e1070\u003c/sub\u003e followed a similar trend to the phosphate bands, decreasing after demineralization and recovering after the application of the products. However, the I\u003csub\u003e1070\u003c/sub\u003e/I\u003csub\u003e960\u003c/sub\u003e ratio remained constant across all groups, indicating that neither demineralization nor remineralization affected the carbonate substitution in the hydroxyapatite structure, with the chemical composition of the enamel remaining unaltered.\u003c/p\u003e \u003cp\u003eThe greater remineralizing effect observed with Regenerate\u0026trade; may be explained by a calcium silicate\u0026ndash;mediated nucleation process, which promotes the organized deposition of calcium and phosphate, consistent with a biomimetic mechanism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thus, Sun et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] concluded that there is evidence that with calcium silicate deposits, the formation of hydroxyapatite increases over time, with progressively more crystalline material. These authors observed, after 4 weeks of in vitro brushing, that the material deposited on the enamel was hydroxyapatite. However, the experimental conditions were different from those of our study; they used enamel blocks (not previously demineralized) which were brushed with a slurry of the calcium silicate toothpaste in water.\u003c/p\u003e \u003cp\u003eRemineralization is often referred to as 'crystallization,' but strictly speaking, this implies the formation of a crystalline phase with internal long-range order of its constituents. However, calcium phosphate often deposits as an X-ray amorphous solid rather than as a crystal. The crystallization of calcium phosphate as a biomineral in enamel is complex, as there are multiple calcium phosphate phases, each with its own solubility and crystallization kinetics [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It has been shown that calcium phosphate crystallizes in a series of steps, often in the sequence of ACP to dihydrated dicalcium phosphate (DCPD) to octacalcium phosphate (OCP) to calcium-deficient hydroxyapatite (CDHA). These solid phases change until the formation of hydroxyapatite is achieved [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe remineralization mechanism of Orthocare\u0026trade; is limited, as its main component, nanohydroxyapatite, would act more as a filling and repairing agent for the demineralized surface rather than as a biomimetic enamel regenerator from the depth of the lesion, given that the product did not supplement calcium and phosphate ions because the storage saliva pH was 6.57, relying solely on the concentrations in saliva, thus creating a superficial layer of hydroxyapatite on the enamel [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe enamel remineralization process involves too many unknown parameters, and the exact mechanism by which remineralization occurs is still far beyond our understanding [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A limitation of this study was the absence of pH cycling. Future studies should incorporate dynamic pH conditions to better simulate the oral environment.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eDemineralized enamel treated with Orthocare\u0026trade; and Regenerate\u0026trade; exhibited signs of remineralization, which contributed to an improvement in bracket bond strength.\u003c/p\u003e \u003cp\u003eRegenerate\u0026trade; resulted in a greater recovery of mineral content compared with Orthocare\u0026trade;. However, the newly formed hydroxyapatite appeared to be structurally immature, as the level of crystallinity did not reach that of intact enamel. Consequently, the adhesion to remineralized enamel, although similar to that of intact enamel, was not significantly higher than that of demineralized enamel, which was the case with intact enamel.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest:\u003c/h2\u003e \u003cp\u003ethe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAcknowlegment\u003c/strong\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eUniversity of Murcia\u0026acute;s own funds\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAV: Conceptualization, Methodology, Investigation, Formal analisis, Writing-Original Draft, Writing-Review and Editing, Supervision. AJOR: Conceptualization, Methodology, Investigation, Formal analisis, Writing-Original Draft, Writing-Review and Editing, Supervision. CSM: Conceptualization, Methodology, Investigation, Formal an\u0026aacute;lisis, Writing-Review, Supervision. ICM: Investigation, Methodology, Formal analisis, Writing-Review. YMB: Conceptualization, Methodology, Investigation, Formal analisis, Writing-Original Draft, Writing-Review and Editing, Supervision.\u003c/p\u003e\u003ch2\u003eData availability statement:\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGulec A, Goymen M (2019) Assessment of the resin infiltration and CPP-ACP applications before orthodontic brackets bonding. Dent Mater J 38(5):854\u0026ndash;860. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4012/dmj.2019-021\u003c/span\u003e\u003cspan address=\"10.4012/dmj.2019-021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorelick L, Geiger AM, Gwinnett AJ (1982) Incidence of white spot formation after bonding and banding. Am J Orthod 81(2):93\u0026ndash;98. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0002-9416(82)90032-x\u003c/span\u003e\u003cspan address=\"10.1016/0002-9416(82)90032-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhadirian H, Geramy A, Shallal W et al (2020) The Effect of Remineralizing Agents With/Without CO2 Laser Irradiation on Structural and Mechanical Properties of Enamel and its Shear Bond Strength to Orthodontic Brackets. J Lasers Med Sci 11(2):144\u0026ndash;152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.34172/jlms.2020.25\u003c/span\u003e\u003cspan address=\"10.34172/jlms.2020.25\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkin M, Baka ZM, Ileri Z et al (2014) Can demineralized enamel surfaces be bonded safely? Acta Odontol Scand 72(4):283\u0026ndash;289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/00016357.2013.823646\u003c/span\u003e\u003cspan address=\"10.3109/00016357.2013.823646\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelİ I, Akin M, Baka ZM et al (2016) Effects of different pre-treatment methods on the shear bond strength of orthodontic brackets to demineralized enamel. Acta Odontol Scand 74(1):7\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/00016357.2014.982703\u003c/span\u003e\u003cspan address=\"10.3109/00016357.2014.982703\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahabi M, Moosavi H, Gholami A et al (2012) In vitro effects of several surface preparation methods on shear bond strength of orthodontic brackets to caries-like lesions of enamel. Eur J Paediatr Dent 13(3):197\u0026ndash;202\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaka ZM, Akin M, Ileri Z et al (2016) Effects of remineralization procedures on shear bond strengths of brackets bonded to demineralized enamel surfaces with self-etch systems. Angle Orthod 86(4):661\u0026ndash;667. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2319/041515-247.1\u003c/span\u003e\u003cspan address=\"10.2319/041515-247.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIafisco M, Degli Esposti L, Ram\u0026iacute;rez-Rodr\u0026iacute;guez GB et al (2018) Fluoride-doped amorphous calcium phosphate nanoparticles as a promising biomimetic material for dental remineralization. Sci Rep 8(1):17016. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-018-35258-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-35258-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoiner A, Sch\u0026auml;fer F, Naeeni MM et al (2014) Remineralisation effect of a dual-phase calcium silicate/phosphate gel combined with calcium silicate/phosphate toothpaste on acid-challenged enamel in situ. J Dent 42(Suppl 1):S53\u0026ndash;59. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0300-5712(14)50008-5\u003c/span\u003e\u003cspan address=\"10.1016/S0300-5712(14)50008-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaghgoo R, Abbasi F, Rezvani MB (2011) Evaluation of the effect of nanohydroxyapatite on erosive lesions of the enamel of permanent teeth following exposure to soft beer in vitro. \u003cem\u003eSRE\u003c/em\u003e ;6(28):5933\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5897/SRE11.1486\u003c/span\u003e\u003cspan address=\"10.5897/SRE11.1486\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTschoppe P, Zandim DL, Martus P et al (2011) Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 39(6):430\u0026ndash;437. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jdent.2011.03.008\u003c/span\u003e\u003cspan address=\"10.1016/j.jdent.2011.03.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePepla E, Besharat LK, Palaia G et al (2014) Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature. Ann Stomatol (Roma) 5(3):108\u0026ndash;114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBattistella E, Foresti E, Lelli M et al (2009) Surface Enamel Remineralization: Biomimetic Apatite Nanocrystals and Fluoride Ions Different Effects. J Nanomaterials 2009:1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2009/746383\u003c/span\u003e\u003cspan address=\"10.1155/2009/746383\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiwa M, Sato T, Li W et al (2001) Polishing and whitening properties of toothpaste containing hydroxyapatite. J Mater Sci Mater Med 12(3):277\u0026ndash;281. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/a:1008927502523\u003c/span\u003e\u003cspan address=\"10.1023/a:1008927502523\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajendran R, Antony SDP, Ashik PM et al (2024) Remineralization potential of strontium-doped nano-hydroxyapatite dentifrice and casein phosphopeptide-amorphous calcium phosphate cream on white spot lesions in enamel following orthodontic debonding - a randomized controlled trial. SAGE Open Med 12:20503121231221634. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/20503121231221634\u003c/span\u003e\u003cspan address=\"10.1177/20503121231221634\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadiee M, Jafari N, Fatemi S et al (2020) Comparison of the effects of toothpastes containing nanohydroxyapatite and fluoride on white spot lesions in orthodontic patients: A randomized clinical trial. Dent Res J (Isfahan) 17(5):354\u0026ndash;359\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParker AS, Patel AN, Al Botros R et al (2014) Measurement of the efficacy of calcium silicate for the protection and repair of dental enamel. J Dent 42(Suppl 1):S21\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0300-5712(14)50004-8\u003c/span\u003e\u003cspan address=\"10.1016/S0300-5712(14)50004-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHornby K, Ricketts SR, Philpotts CJ et al (2014) Enhanced enamel benefits from a novel toothpaste and dual phase gel containing calcium silicate and sodium phosphate salts. J Dent 42(Suppl 1):S39\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0300-5712(14)50006-1\u003c/span\u003e\u003cspan address=\"10.1016/S0300-5712(14)50006-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVicente A, Ortiz-Ruiz AJ, Gonz\u0026aacute;lez-Paz BM et al (2021) Effectiveness of a toothpaste and a serum containing calcium silicate on protecting the enamel after interproximal reduction against demineralization. Sci Rep 11(1):834. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-80844-7\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-80844-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrilisi G, Vitiello F, Notarstefano V et al (2023) Multidisciplinary evaluation of the remineralization potential of three fluoride-based toothpastes on natural white spot lesions. Clin Oral Investig 27(12):7451\u0026ndash;7462. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00784-023-05334-2\u003c/span\u003e\u003cspan address=\"10.1007/s00784-023-05334-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulze KA, Balooch M, Balooch G et al (2004) Micro-Raman spectroscopic investigation of dental calcified tissues. J Biomed Mater Res A 69(2):286\u0026ndash;293. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jbm.a.20130\u003c/span\u003e\u003cspan address=\"10.1002/jbm.a.20130\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArends J, Ten Cate JM (1981) Tooth enamel remineralization. J Cryst Growth 53(1):135\u0026ndash;147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0022-0248(81)90060-9\u003c/span\u003e\u003cspan address=\"10.1016/0022-0248(81)90060-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŚwietlicka I, Kuc D, Świetlicki M et al (2020) Near-Surface Studies of the Changes to the Structure and Mechanical Properties of Human Enamel under the Action of Fluoride Varnish Containing CPP-ACP Compound. Biomolecules 10(5):765. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom10050765\u003c/span\u003e\u003cspan address=\"10.3390/biom10050765\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhadirian H, Geramy A, Shallal W et al (2020) The Effect of Remineralizing Agents With/Without CO2 Laser Irradiation on Structural and Mechanical Properties of Enamel and its Shear Bond Strength to Orthodontic Brackets. J Lasers Med Sci 11(2):144\u0026ndash;152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.34172/jlms.2020.25\u003c/span\u003e\u003cspan address=\"10.34172/jlms.2020.25\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaysal A, Uysal T (2012) Do enamel microabrasion and casein phosphopeptide-amorphous calcium phosphate affect shear bond strength of orthodontic brackets bonded to a demineralized enamel surface? Angle Orthod 82(1):36\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2319/041211-265.1\u003c/span\u003e\u003cspan address=\"10.2319/041211-265.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUysal T, Baysal A, Uysal B et al (2011) Do fluoride and casein phosphopeptide-amorphous calcium phosphate affect shear bond strength of orthodontic brackets bonded to a demineralized enamel surface? Angle Orthod 81(3):490\u0026ndash;495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2319/090510-520.1\u003c/span\u003e\u003cspan address=\"10.2319/090510-520.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScribante A, Dermenaki Farahani MR, Marino G et al Biomimetic Effect of Nano-Hydroxyapatite in Demineralized Enamel before Orthodontic Bonding of Brackets and Attachments: Visual, Adhesion Strength, and Hardness in In Vitro Tests. Biomed Res Int ;2020:6747498. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2020/6747498\u003c/span\u003e\u003cspan address=\"10.1155/2020/6747498\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Nancollas GH (2008) Calcium Orthophosphates: Crystallization and Dissolution. Chem Rev 108(11):4628\u0026ndash;4669. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/cr0782574\u003c/span\u003e\u003cspan address=\"10.1021/cr0782574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang S, Gao S, Cheng L et al (2011) Remineralization potential of nano-hydroxyapatite on initial enamel lesions: an in vitro study. Caries Res 45(5):460\u0026ndash;468. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1159/000331207\u003c/span\u003e\u003cspan address=\"10.1159/000331207\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Li X, Deng Y et al (2014) Mode of action studies on the formation of enamel minerals from a novel toothpaste containing calcium silicate and sodium phosphate salts. J Dent 42(Suppl 1):S30\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0300-5712(14)50005-X\u003c/span\u003e\u003cspan address=\"10.1016/S0300-5712(14)50005-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnax J, Fandrich P, Schulze zur Wiesche E et al (2024) The Remineralization of Enamel from Saliva: A Chemical Perspective. Dent J (Basel) 12(11):339. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/dj12110339\u003c/span\u003e\u003cspan address=\"10.3390/dj12110339\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnil A, Ibraheem WI, Meshni AA et al (2022) Nano-Hydroxyapatite (nHAp) in the Remineralization of Early Dental Caries: A Scoping Review. Int J Environ Res Public Health 19(9):5629. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijerph19095629\u003c/span\u003e\u003cspan address=\"10.3390/ijerph19095629\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer F, Enax J, Amaechi BT et al (2022) Hydroxyapatite as Remineralization Agent for Children\u0026rsquo;s Dental Care. Front Dent Med 3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fdmed.2022.859560\u003c/span\u003e\u003cspan address=\"10.3389/fdmed.2022.859560\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"enamel remineralization, orthodontics, Raman Spectroscopy, Shear bond strength","lastPublishedDoi":"10.21203/rs.3.rs-9515528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9515528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aimed to evaluate the remineralization capacity of Orthocare\u0026trade; and Regenerate\u0026trade; on demineralized enamel and their effect on orthodontic bracket bond strength.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 180 bovine teeth were divided into four groups: (1) Intact Enamel, (2) Demineralized Enamel, (3) Demineralized Enamel treated with Regenerate\u0026trade;, and (4) Demineralized Enamel treated with Orthocare\u0026trade;. Groups 2\u0026ndash;4 were demineralized and stored in artificial saliva for 60 days. All specimens were brushed three times daily; Groups 1 and 2 without toothpaste, and Groups 3 and 4 with their respective products and serums. Shear bond strength was measured after bracket bonding, and adhesive remnant was evaluated by image analysis. Enamel characterization was performed using FE-SEM, EDX, and Raman spectroscopy.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDemineralized enamel showed significantly lower shear bond strength and adhesive remnant than intact enamel (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). FE-SEM and EDX analyses revealed increased porosity and reduced mineral content, while both treatments improved calcium (Ca) and phosphorus (P) levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Raman spectroscopy showed greater recovery of phosphate (I960) and carbonate (I1070) peaks in the Regenerate\u0026trade; group, indicating better mineralization and crystallinity recovery than Orthocare\u0026trade;.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBoth agents promoted partial enamel remineralization, with Regenerate\u0026trade; showing better mineral recovery. However, the newly formed hydroxyapatite appeared immature, and bond strength was not significantly improved compared with demineralized enamel.\u003c/p\u003e\u003ch2\u003eClinical Relevance:\u003c/h2\u003e \u003cp\u003eRemineralizing agents may improve demineralized enamel before orthodontic bonding without significantly affecting bracket bond strength.\u003c/p\u003e","manuscriptTitle":"Assessment of enamel remineralization and its effect on orthodontic bracket shear bond strength: an in vitro study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 16:14:27","doi":"10.21203/rs.3.rs-9515528/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"100838016671772212382666085335260026208","date":"2026-05-13T18:45:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285752300641950612412276481413073704826","date":"2026-05-12T13:48:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17274174307779361571722782883681795146","date":"2026-05-08T12:48:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"340186939147300594851975464378565582391","date":"2026-05-08T12:10:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T16:02:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183285177967490229861217966996648299396","date":"2026-05-06T15:48:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T15:18:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T06:24:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T06:24:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Oral Investigations","date":"2026-04-24T09:36:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"19f7b56c-0bd0-4047-a92a-bf966760ad25","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"100838016671772212382666085335260026208","date":"2026-05-13T18:45:37+00:00","index":36,"fulltext":""},{"type":"reviewerAgreed","content":"285752300641950612412276481413073704826","date":"2026-05-12T13:48:51+00:00","index":34,"fulltext":""},{"type":"reviewerAgreed","content":"17274174307779361571722782883681795146","date":"2026-05-08T12:48:06+00:00","index":33,"fulltext":""},{"type":"reviewerAgreed","content":"340186939147300594851975464378565582391","date":"2026-05-08T12:10:45+00:00","index":31,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T16:02:03+00:00","index":21,"fulltext":""},{"type":"reviewerAgreed","content":"183285177967490229861217966996648299396","date":"2026-05-06T15:48:43+00:00","index":20,"fulltext":""},{"type":"reviewersInvited","content":"19","date":"2026-05-06T15:18:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T06:24:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T06:24:39+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T16:14:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 16:14:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9515528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9515528","identity":"rs-9515528","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}