Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p Technology and Health Care -1 (2016) 1–11 DOI 10.3233/THC-161221 IOS Press Remineralization of early enamel caries lesions using different bioactive elements containing toothpastes: An in vitro study Yu Wanga , Li Meib , Lin Gonga , Jialing Lic , Shaowei Hed , Yan Jia and Weibin Sune,∗ a Department 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 n si o er fv oo pr 15 Received 22 November 2015 Accepted 28 April 2016 ed 14 Abstract BACKGROUND: Demineralization can be arrested or reversed when remineralization agents are applied to incipient carious or non-cavitated carious lesions A large number of therapeutic agents including non-fluoridated products have been developed to promote enamel remineralization OBJECTIVE: This study aims to evaluate the efficacy of different bioactive elements containing toothpastes in remineralization of artificial enamel lesions METHODS: Artificial carious lesions were created on 40 human enamel slabs, and were randomly divided into four groups: (1) control group (no treatment), (2) casein phosphopeptide-amorphous calcium phosphate group (CPP-ACP, GC Tooth Mousse), (3) 8% arginine and calcium carbonate group (ACC, Colgate Sensitive Pro-Relief), (4) calcium sodium phosphosilicate group (CSP, NovaMin R ) All samples were subjected to 15 days of pH-cycling Subsequently, a one-hour acid resistance test was carried out Surface hardness of the samples was assessed using the Knoop hardness test, and surface morphology and roughness were assessed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) Data were analyzed using one-way ANOVA, Tukey’s test and paired t test RESULTS: The three tested toothpastes exhibited a significantly higher remineralization efficacy compared with the control group (P < 0.05 for all) After pH-cycling, the specimens treated with Colgate Sensitive Pro-Relief and NovaMin R showed a significant higher surface hardness (P < 0.001 and P = 0.03, respectively) and lower surface roughness (P < 0.05 for both) compared those treated with GC Tooth Mousse While after the acid resistance test, all groups showed a significant loss of surface hardness (P < 0.001 for all) and significant increase of surface roughness (P < 0.05) The specimens treated with ct 12 13 rre 10 11 co un of Preventive Dentistry, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, China b Discipline of Orthodontics, Department of Oral Science, Faculty of Dentistry, University of Otago, Dunedin, New Zealand c Department of Orthodontics, Institute and Hospital of Stomatology, Nanjing University Medical School, Nanjing, Jiangsu, China d Department of Prosthodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, China e Department of Periodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, China ∗ Corresponding author: Weibin Sun, Department of Periodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, 30 Zhongyang Road, Nanjing, Jiangsu, China Tel.: +86 25 83620173; Fax: +86 25 82620173; E-mail: wbsun@ nju.edu.cn 0928-7329/16/$35.00 c 2016 – IOS Press and the authors All rights reserved Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements 41 Keywords: Bioactive elements, dental caries, hardness, pH-cycling, remineralization 42 Introduction 43 Dental caries is a dynamically alternative process of demineralization and remineralization Demineralization can be arrested or reversed when remineralization agents are applied to incipient carious or non-cavitated carious lesions [1,2] Therefore, it is of great importance to explore novel strategies and approaches to enhance the remineralization process Fluoride has been widely recommended as a remineralization agent for preventing early enamel carious lesions [3] The cariostatic effects of fluoride are mostly topical to precipitate calcium fluoride minerals on the surface of enamel and bacterial plaque [4] Such cariostatic therapeutic effects of fluoride are inadequate to deal with high caries risk patients [5] Promising remineralization effects are possible using high fluoride concentration agents [6] However, fluoride has a dose-response relationship, and careless handling of fluoride toothpastes may lead to adverse effects such as fluorosis [7] Considering the clinical significance of remineralization, a range of therapeutic agents including non-fluoridated products has been developed to enhance enamel remineralization A few examples of widely used remineralization materials are casein phosphopeptideamorphous calcium phosphate (CPP-ACP) and bioactive glasses CPP-ACP is an amorphous calcium phosphate (ACP)-based bioactive material stabilized by casein phosphopeptide (CPP) It stabilizes free calcium and phosphate ions along with fluoride ions in a metastable solution to bind to pellicle and bacterial plaque As a reservoir of bioactive calcium and phosphate ions [8], CPP-ACP delivers ions to the enamel subsurface, and enhances its remineralization effectively [9,10] A number of studies [8,9] report the anticariogenic effects of CPP-ACP technology The formation of the CPP-ACP nanocomplex or ACPF complex takes place when CPP-ACP interacts with fluoride ions [11] NovaMin R is another biomaterial that consists of sodium-calcium-phosphate particles, which can be activated in an aqueous environment Sodium ions present in NovaMin R are rapidly released, and calcium and phosphate ions precipitate to form a calcium phosphate layer This is followed by the formation of a carbonate-enriched HCA layer [12] The incorporation of HCA and bioactive sodium-calciumphosphate particles result in the remineralization of early enamel caries lesions [9,11,12] Pro-ArginTM , a novel remineralization agent, is a saliva-based compound that contains arginine (8%) and calcium carbonate; which has been introduced to relieve dentin hypersensitivity [13] Arginine and calcium carbonate form positively charged agglomerates that readily bind to negatively charged dentin surfaces or tubules, facilitating calcium and phosphate ions to precipitate on the dentin surface [14] Arginine and calcium are two key components of toothpastes that work together to deposit a dentin-like mineral layer within dentin tubules and on the dentin surface The in vitro study was designed to evaluate the remineralization potential of various commercial toothpastes that contain different active remineralization components 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 si o er 51 fv 50 oo 49 pr 48 ed 47 ct 45 46 rre 44 co 38 39 un 37 n 40 Colgate Sensitive Pro-Relief and NovaMin R still showed a significant higher surface hardness and lower surface roughness in comparison with those treated with GC Tooth Mousse (P < 0.05 for all) No significant difference was found in surface hardness and roughness between Colgate Sensitive Pro-Relief and NovaMin R during the pH-cycling test and acid resistance test (P = 0.45 and P = 0.83, respectively) CONCLUSIONS: Colgate Sensitive Pro-Relief and NovaMin R present an advantage in enhancing remineralization and inhibiting demineralization for early enamel carious lesions in comparison with GC Tooth Mousse 35 36 Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p 3 si o n Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements oo fv er Fig Flowchart: (a) teeth were cut at the cementoenamel junction; (b) crowns were sectioned mesiodistally and buccolingually into four parts; (c) enamel windows were created in the middle one-third of the crown, and an artificial lesion was produced; (d) samples were processed for the Knoop microhardness test; (e) pH-cycling was conducted for 15 days; (f) samples were processed for the Knoop microhardness test after pH-cycling; (g) enamel slabs were subjected to one-hour acid resistance testing; (h) a Knoop microhardness test was carried out after acid resistance testing Materials and methods 77 2.1 Specimen preparation 78 88 Ten human permanent premolars extracted for orthodontic treatment were obtained from Nanjing Stomatological Hospital All selected teeth were inspected under a stereomicroscope (ACT-1, Nikon, Japan) at 100x magnification to ensure that all teeth are free of stains, decay or cracks Debris, soft tissue remnants and calculus were removed ultrasonically; and teeth were stored in athymol solution (0.01%) for 48 hours All teeth were dissected using a low-speed cutting saw (Isomet, Buheler, USA) at the cementoename junction (Fig 1a) Each tooth crown was sectioned mesiodistally and buccoling ually into four parts (Fig 1b) Each thus-prepared enamel sample was embedded in acrylic resins and polished progressively using a 600-grit wet silicon-carbide paper Enamel windows were created (4 × mm) in the middle one-third of the crown by coating the surrounding surfaces with acid-resistant nail varnish (Fig 1c) All samples were stored in distilled water at 37◦ C This research protocol was approved by the Ethical Committee of Nanjing Stomatological Hospital, Medical School of Nanjing University, China 89 2.2 Experimental groups 83 84 85 86 87 90 91 92 93 94 95 96 ed ct rre 82 co 80 81 un 79 pr 76 The enamel slabs (n = 40) were randomly divided into four groups (each group, n = 10): (1) Control group (no treatment); (2) CPP-ACP group (GC Tooth Mousse, GC Corporation, Japan); (3) ACC group (8% arginine and calcium carbonate; Colgate Sensitive Pro-Relief, Colgate-Palmolive Company, USA); (4) CSP group (calcium sodium phosphosilicate; NovaMin R , Beijing Bio-Tech Co Ltd, China) Slurries were prepared by stirring the above toothpastes in distilled water at a fixed ratio (1:3), as previously described [15] Fresh slurries were prepared on the day of application, and centrifuged at 4,000 rpm for 20 minutes Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements 97 2.3 Artificial lesion formation 98 101 Artificial carious lesions were created by exposing the enamel slabs to a demineralization solution (2.2 mM of calcium chloride, 2.2 mM of potassium hydrogen orthophosphate, unstirred solution of 0.05 M of acetic acid, and M of potassium hydroxide; pH4.5) at 37◦ C for 96 hours (Fig 1c) Then, specimens were washed thoroughly using a spray of water and air-dried 102 2.4 pH-cycling 99 100 109 2.5 Acid resistance testing (ART) 110 112 In order to assess acid resistance effects, all specimens were washed using deionized distilled water, and immersed in a demineralization solution at 37◦ C for one hour The demineralization solution was refreshed after microhardness testing and before acid resistance testing (Fig 1g) 113 2.6 Surface microhardness measurement 114 118 The Knoop surface hardness (KHN) of enamel specimens was measured using a digital microhardness tester (HV-1000, Shangcai Tester Manufactory, China) A Knoop diamond indenter under a 100-gload was used for 10 seconds An average of five indentations was used for analyzing each specimen (Figs 1d, 1f and 1h) Data were collected at the following stages: a) baseline (sound enamel); b) after demineralization; c) after pH-cycling; d) after acid resistance test 119 2.7 Scanning electron microscopy (SEM) 120 122 SEM observation was carried out using a scanning electron microscope (S-3400N, Hitachi, Japan) to investigate morphological changes Specimens were mounted on aluminum stubs with a carbon tape, and sputter-covered with carbon 123 2.8 Atomic force microscopy (AFM) 116 117 121 si o er fv oo pr 115 ed 111 ct 107 rre 106 co 105 un 104 n 108 Fifteen days of pH-cycling was carried out at 37◦ C, which consisted of three hours of demineralization, followed by immersion in a remineralization solution for two hours A second cycle was performed, which consisted of three hours of demineralization and an overnight remineralization Specimens were placed into the previously described dentifrice supernatant solution 60 seconds before the first demineralizing cycle, and both before and after the second demineralizing cycle (Fig 1e) All specimens were stored in distilled water at 37◦ C until required for further experimentation 103 129 AFM images of the enamel surface were obtained using a Dimension FastScan BioTM Atomic Force Microscope (Bruker Corporation, USA), which was operated in Scan Asyst mode using silicon tips Topography images were collected at a low scan rate, covering a 30 µm × 30 µm area with a 256 × 256 pixel resolution In each image, five randomized boxes of µm × µm were drawn using the Bruker NanoScope Analysis (Bruker Corporation, USA) at the enamel surface, and mean roughness (Ra) values were determined 130 2.9 Statistical analysis 124 125 126 127 128 131 Statistical analysis was performed using one-way ANOVA, Tukey’s test and Paired t test Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements Table Knoop hardness number of enamel blocks at different stages Treatment Baseline After DM After RM After ART Control 370 ± 8a1 303 ± 10b2 310 ± 8c3 206 ± 12f4 GC Tooth Mousse 372 ± 5a1 310 ± 7b2 338 ± 11d3 242 ± 9g4 a1 b2 e3 Colgate Sensitive Pro-Relief 371 ± 307 ± 355 ± 271 ± 13h4 R a1 b2 e3 Novamin 376 ± 304 ± 10 350 ± 262 ± 5h4 Same lowercase letter indicated no statistical difference in columns Same number indicated no statistical difference in rows (P < 0.05) DM, demineralization; RM, remineralization; ART, acid resistance testing Table Comparison of KHN within groups Control GC tooth mousse Colgate sensitive pro-relief T P T P T P Baseline → DM 17.03 < 0.001 18.21 < 0.001 32.63 < 0.001 DM → RM −1.71 0.13 −7.90 < 0.001 −11.66 < 0.001 RM → ART 29.72 < 0.001 25.67 < 0.001 23.989 < 0.001 DM, demineralization; RM, remineralization; ART, acid resistance testing NovaMin R T P 16.67 < 0.001 −12.62 < 0.001 37.298 < 0.001 si o n Phase er Table Multiple comparison of surface hardness (KHN) among groups at different stages Baseline After DM After RM After ART MD P MD P MD P MD P Group1 vs Group2 −1.99 0.93 −6.64 0.34 −28.05 < 0.001 −36.68 < 0.001 Group1 vs Group3 −1.07 0.99 −4.03 0.73 −45.67 < 0.001 −65.59 < 0.001 Group1 vs Group4 −6.38 0.24 −0.82 1.00 −39.78 < 0.001 −61.64 < 0.001 Group2 vs Group3 0.92 0.99 2.61 0.91 −17.62 < 0.001 −28.91 < 0.001 Group2 vs Group4 −4.39 0.56 5.82 0.46 −11.73 0.03 −24.96 < 0.001 Group3 vs Group4 −5.32 0.40 3.22 0.84 5.89 0.45 3.95 0.83 Group 1, control group; group 2,casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) group; group 3, 8% arginine and calcium carbonate (ACC, Colgate Sensitive Pro-Relief) group; group 4, calcium sodium phosphosilicate (CSP, NovaMin R ) group; MD, mean difference rre ct ed pr oo fv Groups Results 133 3.1 Knoop hardness (KHN) 135 136 137 138 139 140 141 142 143 144 un 134 co 132 The baseline surface hardness (KHN) was similar among all groups (P > 0.05) (Table 1) After the application of different dentifrices, the KHN values in the treatment groups (GC Tooth Mousse, Colgate Sensitive Pro-Relief, and NovaMin R groups) were all significantly increased (P < 0.001) (338 ± 11,355 ± 8, and 350 ± 7, respectively) Following the acid resistance tests (ART), a significant reduction of KHN values was observed in all groups (Table 1) (P < 0.001) The changes of surface hardness within each group are summarized in Table The multiple comparison of surface hardness among different groups (Table 3) revealed that surface hardness were significantly higher in the Colgate Sensitive Pro-Relief and NovaMin R groups compared with the GC Tooth Mousse group after pH-cycling test as well as acid resistance test (P < 0.05 for all) No significant difference of surface hardness was found between Colgate Sensitive Pro-Relief and NovaMin R during the pH-cycling test and acid resistance test (P = 0.45 and P = 0.83, respectively) Galley Proof File: thc–1-thc1221.tex; BOKCTP/ljl p Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements n 20/05/2016; 16:00 fv er si o Fig Representative SEM micrographs of specimens after demineralization and remineralization: (a) after demineralization, the enamel showed a typical prismatic structure; (b) remineralization by CPP-ACP; (c) remineralization by Colgate Sensitive Pro-Relief; (d) remineralization by NovaMin R The enamel surface was covered by an irregular deposition layer in all dentifrice groups, the honeycomb-like structures were not evident (Figs 2b, 2c, and 2d) The layer generated by ACC (Fig 2c) and CSP (Fig 2d) seemed to be more thick and uniform than CPP-ACP (Fig 2b) 3.2 Scanning electron microscopy (SEM) 146 152 Demineralization affected the surface structure of the enamel, showing demineralized regions with a honeycomb-like appearance and a rough surface without a protective layer (Fig 2a) After 15 days of pH-cycling, the enamel surfaces were remineralized and covered by an irregular deposition layer in all dentifrice groups, sealing most of their regularities (Figs 2b, 2c, and 2d) After an acid attack, some depositions were dissolved; and partially porous enamel was evidenced in the GC Tooth Mousse group (Fig 3a) However, a non-homogeneous thin film with precipitated agents remained visible on the enamel surface in the Colgate Sensitive Pro-Relief and NovaMin R groups (Figs 3b and 3c) 153 3.3 Atomic force microscopic observations 154 AFM micrographs of the demineralized specimens displayed a significant depression with prismatic structures of HCA and loss of enamel materials from the surfaces (Fig 4a) The surface roughness (Ra) in all groups significantly increased almost three times after demineralization, averagely from 45 ± nm to 123 ± nm (P < 0.05 for all) (Table 4) The remineralization treatments by different dentifrices significantly reduced the Ra values to 125 ± nm, 112 ± nm, 106 ± nm and 102 ± nm in the control, GC Tooth Mousse, Colgate Sentsitive Pro-Relief and NovaMin R groups, respectively In addition, surface mineral layers formed by Colgate Sentsitive Pro-Relief (Fig 4c) and NovaMin R (Fig 4d) treatments were relatively uniform and homogeneous After the acid resistance test, the deposited crystals on the enamel surfaces were dissolved at the varying degrees (Fig 5); and the Ra value of GC Tooth Mousse group (152 ± nm) was significantly higher than Colgate Sensitive Pro-Relief (144 ± 4) and NovaMin R (145 ± 5) group No significant difference of Ra was found between Colgate Sensitive Pro-Relief and NovaMin R during the pH-cycling test and acid resistance test (P > 0.05 for both) 155 156 157 158 159 160 161 162 163 164 165 166 pr ed ct 151 rre 149 150 co 148 un 147 oo 145 Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements Table Surface roughness (Ra, nm) of enamel at different stages ed pr oo fv er si o n Treatment Baseline After DM After RM After ART Control 44 ± 2a1 122 ± 4b2 125 ± 3c3 175 ± 2f4 GC Tooth Mousse 45 ± 2a1 126 ± 8b2 112 ± 6d3 152 ± 5g4 a1 b2 e3 Colgate Sensitive Pro-Relief 45 ± 120 ± 106 ± 144 ± 4h4 R a1 b2 e3 Novamin 46 ± 123 ± 102 ± 145 ± 5h4 Same lowercase letter indicated no statistical difference in columns Same number indicated no statistical difference in rows (P < 0.05) DM, demineralization; RM, remineralization; ART, acid resistance testing co rre ct Fig Representative SEM micrographs of specimens after acid resistance test: (a) CPP-ACP; (b) Colgate Sensitive Pro-Relief; (c) NovaMin R The deposited crystals in enamel surfaces were partially dissolved, in CPP-ACP group the Enamel prism and interprism structures became slightly evident (Fig 3a) However, a film with precipitated agents remained visible on the enamel surface in the ACC and CSP groups (Figs 3b, 3c) Discussion 168 The present study demonstrates that Colgate Sensitive Pro-Relief and NovaMin R are superior in promoting remineralization and inhibiting demineralization over CPP-ACP containing toothpastes These present results were somehow in line with the results of other studies [15,16] Mehta A [15] compared the remineralization efficiency of NovaMin R and CPP-ACP containing dentifrices After remineralization the mean microhardness in NovaMin R group was 371.76 and in CPP-ACP group was 357.07, the difference between the two groups was found to be statistatically significant, indicating NovaMin R dentifrices demonstrated a better remineralization potential than CPP-ACP Elizabeta G and John W [16] observed morphological changes in remineralized enamel surfces by Scanning Electron Microscope, and found that deposits formed by NovaMin R were larger and angular than deposits created by CPP-ACP The high KNH values for NovaMin R treatments may be due to its firm attachment to the enamel surface by means of a layer that has a composition similar to enamel, unlike the amorphous nature of CPP-ACP 169 170 171 172 173 174 175 176 177 178 un 167 Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements (b) (c) (d) si o n (a) 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 oo pr 184 185 ed 183 ct 182 rre 181 co 180 Petrou et al [17] proposed that the combination of arginine and calcium carbonate favors calcium and phosphate ions to deposit dentin-like minerals within dentin tubules and on the dentin surface This combination works on the principle of organic-inorganic interactions similar to the mineralization of natural tissues (biomimetic approaches) The role of arginine is similar to the organic matrix in the mineralization process [18] Organic molecules (such as arginine) are positively charged on the surface or hidden inside structural folds [17] These organic molecules act as a nucleation center for mineralization, as a result of strong electrostatic interactions and precipitating inorganic minerals Depositions that contained calcium, phosphate and arginine were revealed after exposure to an acid challenge for two minutes, which was performed to simulate the consumption of an acidic beverage Acid resistance of the deposition ensures the lasting remineralization of toothpastes In the present study, specimens treated with arginine (8%) and calcium carbonate revealed less reduction of surface hardness after the acid resistance test, compared with the specimens in the CPP-ACP and control groups Remineralization occurs when calcium and phosphate ions are redeposited on the effected enamel surface crystals The primary source of calcium and phosphate ions is saliva The low concentration of calcium and phosphateions in saliva limits its remineralization capacity [19] The three different toothpastes used in the study include calcium phosphate-based systems; and their remineralization capacities were mainly based on the enhancement of the natural remineralization by salivas [20] PH-cycling models have been broadly used in laboratories to evaluate the progress of carious lesions [21] The pH-cycling protocol carried out in the present study was in accordance to the protocol used by Ten Cate and Duijsters [22] In order to simulate the daily routine of oral environment, this model was designed to apply toothpastes three times a day, i.e early morning, midday and before bed-time NovaMin R is a calcium-phosphate based bioactive glass that releases sodium, calcium, phosphorous and silica ions to form HCAdirectlywithout the intermediate ACP phase [15] It has been found that ions were released and transformed to HCA in up to two weeks [12] And the average time for CPP-ACP to remineralize after an acid challenge is also approximately two weeks [23] Considering this fact, the un 179 fv er Fig Representative AFM images of specimens after demineralization and remineralization: (a) after demineralization; (b) treated with CPP-ACP; (c) treated with Colgate Sensitive Pro-Relief; (d) treated with NovaMin R Figure 4a showed the honeycomb-like structures after demineralization The enamel surface was covered with globular mineral particles (Figs 4b, 4c, and 4d) (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/THC-161221) Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements (b) (c) fv er si o n (a) un co rre ct ed pr oo Fig Representative AFM images of specimens after acid resistance test: (a) CPP-ACP; (b) Colgate Sensitive Pro-Relief; (c) NovaMin R After an acid attack, some depositions were dissolved, the prismatic structure of hydroxyapatite in ACC (Fig 5b) and CSP (Fig 5c) group were less visible than CPP-ACP group (Fig 5a) (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/THC-161221) Fig Surface roughens (Ra) of enamel blocks at different stages measured by Atomic force microscopy (AFM) (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/THC-161221) Galley Proof 10 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p 10 Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements 230 Conclusion 231 234 Despite the limitations of in vitro studies, all tested bioactive toothpastes were found to be effective in enhancing remineralization, Colgate Sensitive Pro-Relief and NovaMin R present a significant advantage in promoting remineralization and inhibiting the demineralization of early enamel carious lesions in comparison with GC Tooth Mousse 235 Acknowledgement 236 237 This study was supported by the grants from the Key Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (No YKK14111) 238 References 218 219 220 221 222 223 224 225 226 227 228 232 233 239 si o 217 er 216 fv 215 oo 213 214 pr 212 [1] 240 241 ed 211 ct 210 rre 208 209 co 207 un 206 n 229 current study was designed to carry out pH-cycling for 15 days Surface hardness testing has been widely used to assess the changes of surface mechanical properties during the demineralization/remineralization process since it is a relatively simple, rapid, noninvasive and reproducible technique [24] The hardness of mineralized hard tissues is directly related to its mineral content Loss of minerals from enamel (for example, in cases of caries or acid etching) affects surface mechanical properties including hardness The microhardness of demineralized specimens was observed to decrease in all groups, but increased after pH-cycling Colgate Sensitive Pro-Relief and NovaMin R exhibited a significantly higher efficacy in enhancing remineralization after pH-cycling Both the ACC and CSP groups continued to present significantly less microhardness loss compared to the other groups Both SEM and AFM were used in the study to evaluate the changes of enamel surface morphology and roughness at different stages AFM has been reported offered relatively better resolution and high-contrast images than SEM [25] Furthermore, AFM can display a more intuitive three-dimensional picture of the outermost layer of the enamel surface, even at a nanometer scale The characterization of SEM and AFM for dentifrice-treated specimens revealed that enamel surfaces were covered by a superficial layer of precipitated crystals to repair the erosive enamel, suggesting the remineralizing ability of these three treatments These findings were consistent with microhardness test results After the acid resistance test, SEM and AFM images revealed that partial depositions were dissolved, and that some cavities were covered by a protective layer before the test, which was observed in the CPP-ACP group (Fig 3a) Enamel prism and interprism structures became slightly evident (Fig 5a) A homogeneous layer covering the erosive enamel surfaces was observed in the Colgate Sensitive Pro-Relief and NovaMin R groups, which was supported by results of the surface hardness tests This study demonstrates a promising potential of topical agents for remineralization of early enamel caries lesions Further researches, especially in vivo studies, are still needed to provide quality evidences for clinicians to treat early enamel caries lesions in a relatively non-invasive way to conserve tooth structure and underlying pulp tissues 204 205 [2] Lynch E, Baysan A Reversal of primary root caries using a dentifrice with a high fluoride content Caries Res 2001; 35: 60-64 Backer Dirks O Posteruptive changes in dental enamel J Dent Res 1966; 45: 503-511 Galley Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p 11 Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 n 258 si o 256 257 er 254 255 fv 252 253 oo 250 251 pr 249 ed 247 248 ct 245 246 Featherstone JD, Preventation and reversal of dental caries: role of low level fluoride Community Dental Oral Epidemiol 1999; 27: 31-40 [4] Ten Cate JM Current concepts on the theories of the mechanism of action of fluoride ActaOdontolScand 1999; 57: 325-329 [5] Kata Rošin-Grget, Kristina Peroš, Ivana Šutej The cariostatic mechanisms of fluoride Acta Medica Academica 2013; 42: 179-188 [6] Larsen VM, Richards A Fluoride is unable to reduce dental erosion from soft drinks Caries Res 2002; 36: 75-80 [7] Lagerweij MD, Ten Cate JM Remineralization of enamel lesions with daily applications of a high-concentration fluoride gel and a fluoridated toothpaste: An in suit study Caries Res 2002; 36: 270-274 [8] Reynolds EC Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions JDent Res 1997; 76: 1587-1595 [9] Zhou C, Zhang D, Bai Y, Li S Casein phosphopeptide-amorphous calcium phosphate remineralization of primary teeth early enamel lesions J Dent Res 2014; 42: 21-29 [10] Walker G, Cai F, Shen P, Reynolds C, Ward B, Fone C Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate J Dairy Res 2006; 73; 74-78 [11] Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker GD, Morgan MV et al Fluoride and casein phosphpeptite-amorphous calcium phosphate J Dent Res 2008; 87: 344-348 [12] Burwell AK, Litkowski LJ, Greenspan DC Calcium sodium phosphosilicate (Novamin R ): remineralization potential Adv Dent Res 2009; 21: 35-39 [13] Wang Z, Jiang T, Sauro S, Pashley DH, Toledano M, Osorio R et al The dentine remineralization activity of a desensitizing bioactive glass-containing toothpaste: an in vitro study Aust Dent J 2011; 56: 372-381 [14] Tirapelli C, Panzeri H, Lara EH, Soares RG, Peitl O, et al The effect of a novel crystallised bioactive glass-ceramic powder on dentine hypersensitivity: a long-term clinical study J Oral Rehabil 2011; 38: 253-262 [15] Mehta AB, Kumari V, Jose R, et al Remineralization potential of bioactive glass and casein casein phosphopeptideamorphous calcium phosphate on initial carious lesion: An in-vitro pH-cycling study J Conserv Dent 2014; 17: 3-7 [16] Elizabeta S, john W A preliminary study of enamel remineralization by dentifrices based on recaldentTM (CPP-ACP) and NovaMin R (calcium-sodium-phosphosilicate) Acta Odontol Latinoam 2010; 23: 234-239 [17] Petrou I, Heu R, Stranick M, et al A breakthrough therapy for dentin hypersensitivity: how dental products containing 8% arginine and calcium carbonate work to deliver effective relief of sensitive teeth J Clin Dent 2009; 20: 23-31 [18] White DJ The application of in vitro models to research on demineralization and remineralization of the teeth Adv Dent Res 1995; 9: 175-193 [19] Winston AE, Bhaskar SN Caries prevention in the 21st century J Am Dent Assoc 1998; 129: 1579-1587 [20] Meurman JH, Ten Cate, Pathogenesis and modifying factors of dental erosion Eur J Oral Sci 1996; 104: 199-206 [21] Azrak B, Callaway A, Knözinger S, Willershausen B Reduction of the pH-values of whole saliva after the intake of apple juice containing beverages in children and adults Oral Health Prev Dent 2003; 1: 229-236 [22] Ten Cate JM, Duijsters PP Alternating demineralization and remineralization of artificial lesion Caries Res 1982; 16: 201-210 [23] Reynolds EC Casein Phosphopeptide-amorphous calcium phosphate: the scientific evidence Adv Dent Res 2009; 21: 25-29 [24] Heshmat H, Ganjkar MH, Jaberi S, et al The effect of Remin Pro and MI paste plus on bleached enamel surface roughness J Dent (Tehran) 2014; 11: 131-136 [25] Cheng ZJ, Wang XM, Cui FZ, Ge J, Yan JX The enamel softening and loss during early erosion studies by AFM, SEM and nanoindentation Biomed Mater 2009; 4: 015020 rre 244 [3] co 243 un 242 11 [...]... Pathogenesis and modifying factors of dental erosion Eur J Oral Sci 1996; 104: 199-206 [21] Azrak B, Callaway A, Knözinger S, Willershausen B Reduction of the pH-values of whole saliva after the intake of apple juice containing beverages in children and adults Oral Health Prev Dent 2003; 1: 229-236 [22] Ten Cate JM, Duijsters PP Alternating demineralization and remineralization of artificial lesion Caries. .. Morgan MV et al Fluoride and casein phosphpeptite-amorphous calcium phosphate J Dent Res 2008; 87: 344-348 [12] Burwell AK, Litkowski LJ, Greenspan DC Calcium sodium phosphosilicate (Novamin R ): remineralization potential Adv Dent Res 2009; 21: 35-39 [13] Wang Z, Jiang T, Sauro S, Pashley DH, Toledano M, Osorio R et al The dentine remineralization activity of a desensitizing bioactive glass -containing. .. products containing 8% arginine and calcium carbonate work to deliver effective relief of sensitive teeth J Clin Dent 2009; 20: 23-31 [18] White DJ The application of in vitro models to research on demineralization and remineralization of the teeth Adv Dent Res 1995; 9: 175-193 [19] Winston AE, Bhaskar SN Caries prevention in the 21st century J Am Dent Assoc 1998; 129: 1579-1587 [20] Meurman JH, Ten Cate,... Fluoride is unable to reduce dental erosion from soft drinks Caries Res 2002; 36: 75-80 [7] Lagerweij MD, Ten Cate JM Remineralization of enamel lesions with daily applications of a high-concentration fluoride gel and a fluoridated toothpaste: An in suit study Caries Res 2002; 36: 270-274 [8] Reynolds EC Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions... Zhou C, Zhang D, Bai Y, Li S Casein phosphopeptide-amorphous calcium phosphate remineralization of primary teeth early enamel lesions J Dent Res 2014; 42: 21-29 [10] Walker G, Cai F, Shen P, Reynolds C, Ward B, Fone C Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate J Dairy Res 2006; 73; 74-78 [11] Reynolds EC, Cai F, Cochrane NJ, Shen... on initial carious lesion: An in- vitro pH-cycling study J Conserv Dent 2014; 17: 3-7 [16] Elizabeta S, john W A preliminary study of enamel remineralization by dentifrices based on recaldentTM (CPP-ACP) and NovaMin R (calcium-sodium-phosphosilicate) Acta Odontol Latinoam 2010; 23: 234-239 [17] Petrou I, Heu R, Stranick M, et al A breakthrough therapy for dentin hypersensitivity: how dental products containing. .. Reynolds EC Casein Phosphopeptide-amorphous calcium phosphate: the scientific evidence Adv Dent Res 2009; 21: 25-29 [24] Heshmat H, Ganjkar MH, Jaberi S, et al The effect of Remin Pro and MI paste plus on bleached enamel surface roughness J Dent (Tehran) 2014; 11: 131-136 [25] Cheng ZJ, Wang XM, Cui FZ, Ge J, Yan JX The enamel softening and loss during early erosion studies by AFM, SEM and nanoindentation... Proof 20/05/2016; 16:00 File: thc–1-thc1221.tex; BOKCTP/ljl p 11 Y Wang et al / Remineralization of early enamel caries lesions using different bioactive elements 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 n 258 si o 256 257 er 254 255 fv 252 253 oo 250 251 pr 249 ed 247 248 ct 245 246 Featherstone JD, Preventation and reversal of dental caries: ... glass -containing toothpaste: an in vitro study Aust Dent J 2011; 56: 372-381 [14] Tirapelli C, Panzeri H, Lara EH, Soares RG, Peitl O, et al The effect of a novel crystallised bioactive glass-ceramic powder on dentine hypersensitivity: a long-term clinical study J Oral Rehabil 2011; 38: 253-262 [15] Mehta AB, Kumari V, Jose R, et al Remineralization potential of bioactive glass and casein casein phosphopeptideamorphous... role of low level fluoride Community Dental Oral Epidemiol 1999; 27: 31-40 [4] Ten Cate JM Current concepts on the theories of the mechanism of action of fluoride ActaOdontolScand 1999; 57: 325-329 [5] Kata Ro in- Grget, Kristina Peroš, Ivana Šutej The cariostatic mechanisms of fluoride Acta Medica Academica 2013; 42: 179-188 [6] Larsen VM, Richards A Fluoride is unable to reduce dental erosion from soft