22 METHODS: Artificial carious lesions were created on 40 human enamel slabs, and were randomly divided into four groups: 1 23 control group no treatment, 2 casein phosphopeptide-amorpho
Trang 1Remineralization of early enamel caries
1
lesions using different bioactive elements
2
containing toothpastes: An in vitro study
3
Yu Wanga, Li Meib, Lin Gonga, Jialing Lic, Shaowei Hed, Yan Jiaand Weibin Sune,∗
4
aDepartment of Preventive Dentistry, Nanjing Stomatological Hospital, Medical School of Nanjing
5
University, Nanjing, Jiangsu, China
6
bDiscipline of Orthodontics, Department of Oral Science, Faculty of Dentistry, University of Otago,
7
Dunedin, New Zealand
8
cDepartment of Orthodontics, Institute and Hospital of Stomatology, Nanjing University Medical
9
School, Nanjing, Jiangsu, China
10
dDepartment of Prosthodontics, Nanjing Stomatological Hospital, Medical School of Nanjing
11
University, Nanjing, Jiangsu, China
12
eDepartment of Periodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University,
13
Nanjing, Jiangsu, China
14
Received 22 November 2015
15
Accepted 28 April 2016
16
Abstract.
17
BACKGROUND: Demineralization can be arrested or reversed when remineralization agents are applied to incipient carious
18
or non-cavitated carious lesions A large number of therapeutic agents including non-fluoridated products have been developed
19
to promote enamel remineralization.
20
OBJECTIVE: This study aims to evaluate the efficacy of different bioactive elements containing toothpastes in
remineraliza-21
tion of artificial enamel lesions.
22
METHODS: Artificial carious lesions were created on 40 human enamel slabs, and were randomly divided into four groups: (1)
23
control group (no treatment), (2) casein phosphopeptide-amorphous calcium phosphate group (CPP-ACP, GC Tooth Mousse),
24
(3) 8% arginine and calcium carbonate group (ACC, Colgate Sensitive Pro-Relief), (4) calcium sodium phosphosilicate group
25
(CSP, NovaMinR) All samples were subjected to 15 days of pH-cycling Subsequently, a one-hour acid resistance test was
26
carried out Surface hardness of the samples was assessed using the Knoop hardness test, and surface morphology and
rough-27
ness were assessed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) Data were analyzed using
28
one-way ANOVA, Tukey’s test and pairedt test.
29
RESULTS: The three tested toothpastes exhibited a significantly higher remineralization efficacy compared with the control
30
group (P < 0.05 for all) After pH-cycling, the specimens treated with Colgate Sensitive Pro-Relief and NovaMin R showed
31
a significant higher surface hardness (P < 0.001 and P = 0.03, respectively) and lower surface roughness (P < 0.05 for
32
both) compared those treated with GC Tooth Mousse While after the acid resistance test, all groups showed a significant loss
33
of surface hardness (P < 0.001 for all) and significant increase of surface roughness (P < 0.05) The specimens treated with
34
∗Corresponding author: Weibin Sun, Department of Periodontics, Nanjing Stomatological Hospital, Medical School of
Nan-jing University, 30 Zhongyang Road, NanNan-jing, 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
uncorrected
proof
version
Trang 2Colgate Sensitive Pro-Relief and NovaMinR still showed a significant higher surface hardness and lower surface roughness
35
in comparison with those treated with GC Tooth Mousse (P < 0.05 for all) No significant difference was found in surface
36
hardness and roughness between Colgate Sensitive Pro-Relief and NovaMinR during the pH-cycling test and acid resistance
37
test (P = 0.45 and P = 0.83, respectively).
38
CONCLUSIONS: Colgate Sensitive Pro-Relief and NovaMinR present an advantage in enhancing remineralization and
in-39
hibiting demineralization for early enamel carious lesions in comparison with GC Tooth Mousse.
40
Keywords: Bioactive elements, dental caries, hardness, pH-cycling, remineralization
41
1 Introduction
42
Dental caries is a dynamically alternative process of demineralization and remineralization Dem-43
ineralization can be arrested or reversed when remineralization agents are applied to incipient carious 44
or non-cavitated carious lesions [1,2] Therefore, it is of great importance to explore novel strategies 45
and approaches to enhance the remineralization process Fluoride has been widely recommended as a 46
remineralization agent for preventing early enamel carious lesions [3] The cariostatic effects of fluo-47
ride are mostly topical to precipitate calcium fluoride minerals on the surface of enamel and bacterial 48
plaque [4] Such cariostatic therapeutic effects of fluoride are inadequate to deal with high caries risk 49
patients [5] Promising remineralization effects are possible using high fluoride concentration agents [6] 50
However, fluoride has a dose-response relationship, and careless handling of fluoride toothpastes may 51
lead to adverse effects such as fluorosis [7] Considering the clinical significance of remineralization, a 52
range of therapeutic agents including non-fluoridated products has been developed to enhance enamel 53
remineralization A few examples of widely used remineralization materials are casein phosphopeptide-54
amorphous calcium phosphate (CPP-ACP) and bioactive glasses
55
CPP-ACP is an amorphous calcium phosphate (ACP)-based bioactive material stabilized by casein 56
phosphopeptide (CPP) It stabilizes free calcium and phosphate ions along with fluoride ions in a 57
metastable solution to bind to pellicle and bacterial plaque As a reservoir of bioactive calcium and 58
phosphate ions [8], CPP-ACP delivers ions to the enamel subsurface, and enhances its remineralization 59
effectively [9,10] A number of studies [8,9] report the anticariogenic effects of CPP-ACP technology 60
The formation of the CPP-ACP nanocomplex or ACPF complex takes place when CPP-ACP interacts 61
with fluoride ions [11]
62
NovaMinR is another biomaterial that consists of sodium-calcium-phosphate particles, which can 63
be activated in an aqueous environment Sodium ions present in NovaMinR are rapidly released, and 64
calcium and phosphate ions precipitate to form a calcium phosphate layer This is followed by the forma-65
tion of a carbonate-enriched HCA layer [12] The incorporation of HCA and bioactive sodium-calcium-66
phosphate particles result in the remineralization of early enamel caries lesions [9,11,12]
67
Pro-ArginTM, a novel remineralization agent, is a saliva-based compound that contains arginine (8%) 68
and calcium carbonate; which has been introduced to relieve dentin hypersensitivity [13] Arginine and 69
calcium carbonate form positively charged agglomerates that readily bind to negatively charged dentin 70
surfaces or tubules, facilitating calcium and phosphate ions to precipitate on the dentin surface [14] 71
Arginine and calcium are two key components of toothpastes that work together to deposit a dentin-like 72
mineral layer within dentin tubules and on the dentin surface
73
The in vitro study was designed to evaluate the remineralization potential of various commercial
tooth-74
pastes that contain different active remineralization components
75
uncorrected
proof
version
Trang 3Fig 1 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.
2 Materials and methods
76
2.1 Specimen preparation
77
Ten human permanent premolars extracted for orthodontic treatment were obtained from Nanjing 78
Stomatological Hospital All selected teeth were inspected under a stereomicroscope (ACT-1, Nikon, 79
Japan) at 100x magnification to ensure that all teeth are free of stains, decay or cracks Debris, soft 80
tissue remnants and calculus were removed ultrasonically; and teeth were stored in athymol solution 81
(0.01%) for 48 hours All teeth were dissected using a low-speed cutting saw (Isomet, Buheler, USA) at 82
the cementoename junction (Fig 1a) Each tooth crown was sectioned mesiodistally and buccoling ually 83
into four parts (Fig 1b) Each thus-prepared enamel sample was embedded in acrylic resins and polished 84
progressively using a 600-grit wet silicon-carbide paper Enamel windows were created (4× 5 mm) in
85
the middle one-third of the crown by coating the surrounding surfaces with acid-resistant nail varnish 86
(Fig 1c) All samples were stored in distilled water at 37◦C This research protocol was approved by the
87
Ethical Committee of Nanjing Stomatological Hospital, Medical School of Nanjing University, China 88
2.2 Experimental groups
89
The enamel slabs (n = 40) were randomly divided into four groups (each group, n = 10): (1) Control
90
group (no treatment); (2) CPP-ACP group (GC Tooth Mousse, GC Corporation, Japan); (3) ACC group 91
(8% arginine and calcium carbonate; Colgate Sensitive Pro-Relief, Colgate-Palmolive Company, USA); 92
(4) CSP group (calcium sodium phosphosilicate; NovaMinR, Beijing Bio-Tech Co Ltd, China). 93
Slurries were prepared by stirring the above toothpastes in distilled water at a fixed ratio (1:3), as 94
previously described [15] Fresh slurries were prepared on the day of application, and centrifuged at 95
4,000 rpm for 20 minutes
96
uncorrected
proof
version
Trang 42.3 Artificial lesion formation
97
Artificial carious lesions were created by exposing the enamel slabs to a demineralization solution 98
(2.2 mM of calcium chloride, 2.2 mM of potassium hydrogen orthophosphate, unstirred solution of 99
0.05 M of acetic acid, and 1 M of potassium hydroxide; pH4.5) at 37◦C for 96 hours (Fig 1c) Then,
100
specimens were washed thoroughly using a spray of water and air-dried
101
2.4 pH-cycling
102
Fifteen days of pH-cycling was carried out at 37◦C, which consisted of three hours of
demineraliza-103
tion, followed by immersion in a remineralization solution for two hours A second cycle was performed, 104
which consisted of three hours of demineralization and an overnight remineralization Specimens were 105
placed into the previously described dentifrice supernatant solution 60 seconds before the first deminer-106
alizing cycle, and both before and after the second demineralizing cycle (Fig 1e) All specimens were 107
stored in distilled water at 37◦C until required for further experimentation.
108
2.5 Acid resistance testing (ART)
109
In order to assess acid resistance effects, all specimens were washed using deionized distilled water, 110
and immersed in a demineralization solution at 37◦C for one hour The demineralization solution was
111
refreshed after microhardness testing and before acid resistance testing (Fig 1g)
112
2.6 Surface microhardness measurement
113
The Knoop surface hardness (KHN) of enamel specimens was measured using a digital microhardness 114
tester (HV-1000, Shangcai Tester Manufactory, China) A Knoop diamond indenter under a 100-gload 115
was used for 10 seconds An average of five indentations was used for analyzing each specimen (Figs 1d, 116
1f and 1h) Data were collected at the following stages: a) baseline (sound enamel); b) after demineral-117
ization; c) after pH-cycling; d) after acid resistance test
118
2.7 Scanning electron microscopy (SEM)
119
SEM observation was carried out using a scanning electron microscope (S-3400N, Hitachi, Japan) to 120
investigate morphological changes Specimens were mounted on aluminum stubs with a carbon tape, 121
and sputter-covered with carbon
122
2.8 Atomic force microscopy (AFM)
123
AFM images of the enamel surface were obtained using a Dimension FastScan BioTMAtomic Force 124
Microscope (Bruker Corporation, USA), which was operated in Scan Asyst mode using silicon tips 125
Topography images were collected at a low scan rate, covering a 30µm × 30 µm area with a 256 × 256
126
pixel resolution In each image, five randomized boxes of 2µm × 2 µm were drawn using the Bruker
127
NanoScope Analysis (Bruker Corporation, USA) at the enamel surface, and mean roughness (Ra) values 128
were determined
129
2.9 Statistical analysis
130
Statistical analysis was performed using one-way ANOVA, Tukey’s test and Pairedt test.
131
uncorrected
proof
version
Trang 5Table 1 Knoop hardness number of enamel blocks at different stages
310± 7b2
338± 11d3
242± 9g4
Colgate Sensitive Pro-Relief 371± 6a1
307± 7b2
355± 8e3
271± 13h4
304± 10b2
350± 7e3
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 2 Comparison of KHN within groups
Baseline→ DM 17.03 < 0.001 18.21 < 0.001 32.63 < 0.001 16.67 < 0.001
DM, demineralization; RM, remineralization; ART, acid resistance testing.
Table 3 Multiple comparison of surface hardness (KHN) among groups at different stages
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, NovaMinR) group; MD, mean difference.
3 Results
132
3.1 Knoop hardness (KHN)
133
The baseline surface hardness (KHN) was similar among all groups (P > 0.05) (Table 1) After the
134
application of different dentifrices, the KHN values in the treatment groups (GC Tooth Mousse, Col-135
gate Sensitive Pro-Relief, and NovaMinR groups) were all significantly increased (P < 0.001) (338 ±
136
11,355± 8, and 350 ± 7, respectively) Following the acid resistance tests (ART), a significant
reduc-137
tion of KHN values was observed in all groups (Table 1) (P < 0.001) The changes of surface hardness
138
within each group are summarized in Table 2 The multiple comparison of surface hardness among dif-139
ferent groups (Table 3) revealed that surface hardness were significantly higher in the Colgate Sensitive 140
Pro-Relief and NovaMinR groups compared with the GC Tooth Mousse group after pH-cycling test as 141
well as acid resistance test (P < 0.05 for all) No significant difference of surface hardness was found 142
between Colgate Sensitive Pro-Relief and NovaMinR during the pH-cycling test and acid resistance test 143
(P = 0.45 and P = 0.83, respectively).
144
uncorrected
proof
version
Trang 6Fig 2 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 NovaMinR 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)
145
Demineralization affected the surface structure of the enamel, showing demineralized regions with 146
a honeycomb-like appearance and a rough surface without a protective layer (Fig 2a) After 15 days 147
of pH-cycling, the enamel surfaces were remineralized and covered by an irregular deposition layer 148
in all dentifrice groups, sealing most of their regularities (Figs 2b, 2c, and 2d) After an acid attack, 149
some depositions were dissolved; and partially porous enamel was evidenced in the GC Tooth Mousse 150
group (Fig 3a) However, a non-homogeneous thin film with precipitated agents remained visible on the 151
enamel surface in the Colgate Sensitive Pro-Relief and NovaMinR groups (Figs 3b and 3c).
152
3.3 Atomic force microscopic observations
153
AFM micrographs of the demineralized specimens displayed a significant depression with prismatic 154
structures of HCA and loss of enamel materials from the surfaces (Fig 4a) The surface roughness (Ra) 155
in all groups significantly increased almost three times after demineralization, averagely from 45 ±
156
2 nm to 123 ± 5 nm (P < 0.05 for all) (Table 4) The remineralization treatments by different
den-157
tifrices significantly reduced the Ra values to 125± 2 nm, 112 ± nm, 106 ± 3 nm and 102 ± 3 nm
158
in the control, GC Tooth Mousse, Colgate Sentsitive Pro-Relief and NovaMinR groups, respectively. 159
In addition, surface mineral layers formed by Colgate Sentsitive Pro-Relief (Fig 4c) and NovaMinR 160
(Fig 4d) treatments were relatively uniform and homogeneous After the acid resistance test, the de-161
posited crystals on the enamel surfaces were dissolved at the varying degrees (Fig 5); and the Ra value 162
of GC Tooth Mousse group (152± 5 nm) was significantly higher than Colgate Sensitive Pro-Relief
163
(144± 4) and NovaMin R (145± 5) group No significant difference of Ra was found between Colgate
164
Sensitive Pro-Relief and NovaMinR during the pH-cycling test and acid resistance test (P > 0.05 for 165
both)
166
uncorrected
proof
version
Trang 7Table 4 Surface roughness (Ra, nm) of enamel at different stages
126± 8b2
112± 6d3
152± 5g4
Colgate Sensitive Pro-Relief 45± 2a1
120± 3b2
106± 3e3
144± 4h4
123± 3b2
102± 3e3
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.
Fig 3 Representative SEM micrographs of specimens after acid resistance test: (a) CPP-ACP; (b) Colgate Sensitive Pro-Relief; (c) NovaMinR 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).
4 Discussion
167
The present study demonstrates that Colgate Sensitive Pro-Relief and NovaMinR are superior in pro-168
moting remineralization and inhibiting demineralization over CPP-ACP containing toothpastes These 169
present results were somehow in line with the results of other studies [15,16] Mehta A [15] compared 170
the remineralization efficiency of NovaMinR and CPP-ACP containing dentifrices After remineraliza-171
tion the mean microhardness in NovaMinR group was 371.76 and in CPP-ACP group was 357.07, the 172
difference between the two groups was found to be statistatically significant, indicating NovaMinR den-173
tifrices demonstrated a better remineralization potential than CPP-ACP Elizabeta G and John W [16] 174
observed morphological changes in remineralized enamel surfces by Scanning Electron Microscope, and 175
found that deposits formed by NovaMinR were larger and angular than deposits created by CPP-ACP. 176
The high KNH values for NovaMinR treatments may be due to its firm attachment to the enamel surface 177
by means of a layer that has a composition similar to enamel, unlike the amorphous nature of CPP-ACP 178
uncorrected
proof
version
Trang 8(a) (b) (c) (d)
Fig 4 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 NovaMinR Figure 4a showed the honey-comb-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)
Petrou et al [17] proposed that the combination of arginine and calcium carbonate favors calcium 179
and phosphate ions to deposit dentin-like minerals within dentin tubules and on the dentin surface This 180
combination works on the principle of organic-inorganic interactions similar to the mineralization of 181
natural tissues (biomimetic approaches) The role of arginine is similar to the organic matrix in the 182
mineralization process [18] Organic molecules (such as arginine) are positively charged on the surface 183
or hidden inside structural folds [17] These organic molecules act as a nucleation center for mineral-184
ization, as a result of strong electrostatic interactions and precipitating inorganic minerals Depositions 185
that contained calcium, phosphate and arginine were revealed after exposure to an acid challenge for 186
two minutes, which was performed to simulate the consumption of an acidic beverage Acid resistance 187
of the deposition ensures the lasting remineralization of toothpastes In the present study, specimens 188
treated with arginine (8%) and calcium carbonate revealed less reduction of surface hardness after the 189
acid resistance test, compared with the specimens in the CPP-ACP and control groups
190
Remineralization occurs when calcium and phosphate ions are redeposited on the effected enamel 191
surface crystals The primary source of calcium and phosphate ions is saliva The low concentration of 192
calcium and phosphateions in saliva limits its remineralization capacity [19] The three different tooth-193
pastes used in the study include calcium phosphate-based systems; and their remineralization capacities 194
were mainly based on the enhancement of the natural remineralization by salivas [20]
195
PH-cycling models have been broadly used in laboratories to evaluate the progress of carious le-196
sions [21] The pH-cycling protocol carried out in the present study was in accordance to the protocol 197
used by Ten Cate and Duijsters [22] In order to simulate the daily routine of oral environment, this model 198
was designed to apply toothpastes three times a day, i.e early morning, midday and before bed-time 199
NovaMinR is a calcium-phosphate based bioactive glass that releases sodium, calcium, phosphorous 200
and silica ions to form HCAdirectlywithout the intermediate ACP phase [15] It has been found that ions 201
were released and transformed to HCA in up to two weeks [12] And the average time for CPP-ACP 202
to remineralize after an acid challenge is also approximately two weeks [23] Considering this fact, the 203
uncorrected
proof
version
Trang 9(a) (b) (c)
Fig 5 Representative AFM images of specimens after acid resistance test: (a) CPP-ACP; (b) Colgate Sensitive Pro-Relief; (c) NovaMinR 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 6 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)
uncorrected
proof
version
Trang 10current study was designed to carry out pH-cycling for 15 days.
204
Surface hardness testing has been widely used to assess the changes of surface mechanical properties 205
during the demineralization/remineralization process since it is a relatively simple, rapid, noninvasive 206
and reproducible technique [24] The hardness of mineralized hard tissues is directly related to its min-207
eral content Loss of minerals from enamel (for example, in cases of caries or acid etching) affects 208
surface mechanical properties including hardness The microhardness of demineralized specimens was 209
observed to decrease in all groups, but increased after pH-cycling Colgate Sensitive Pro-Relief and 210
NovaMinR exhibited a significantly higher efficacy in enhancing remineralization after pH-cycling. 211
Both the ACC and CSP groups continued to present significantly less microhardness loss compared to 212
the other groups
213
Both SEM and AFM were used in the study to evaluate the changes of enamel surface morphol-214
ogy and roughness at different stages AFM has been reported offered relatively better resolution and 215
high-contrast images than SEM [25] Furthermore, AFM can display a more intuitive three-dimensional 216
picture of the outermost layer of the enamel surface, even at a nanometer scale The characterization 217
of SEM and AFM for dentifrice-treated specimens revealed that enamel surfaces were covered by a 218
superficial layer of precipitated crystals to repair the erosive enamel, suggesting the remineralizing abil-219
ity of these three treatments These findings were consistent with microhardness test results After the 220
acid resistance test, SEM and AFM images revealed that partial depositions were dissolved, and that 221
some cavities were covered by a protective layer before the test, which was observed in the CPP-ACP 222
group (Fig 3a) Enamel prism and interprism structures became slightly evident (Fig 5a) A homoge-223
neous layer covering the erosive enamel surfaces was observed in the Colgate Sensitive Pro-Relief and 224
NovaMinR groups, which was supported by results of the surface hardness tests.
225
This study demonstrates a promising potential of topical agents for remineralization of early enamel 226
caries lesions Further researches, especially in vivo studies, are still needed to provide quality evidences
227
for clinicians to treat early enamel caries lesions in a relatively non-invasive way to conserve tooth 228
structure and underlying pulp tissues
229
5 Conclusion
230
Despite the limitations of in vitro studies, all tested bioactive toothpastes were found to be effective in
231
enhancing remineralization, Colgate Sensitive Pro-Relief and NovaMinR present a significant advantage 232
in promoting remineralization and inhibiting the demineralization of early enamel carious lesions in 233
comparison with GC Tooth Mousse
234
Acknowledgement
235
This study was supported by the grants from the Key Project supported by Medical Science and 236
Technology Development Foundation, Nanjing Department of Health (No YKK14111)
237
References
238
[1] Lynch E, Baysan A Reversal of primary root caries using a dentifrice with a high fluoride content Caries Res 2001; 35:
239
60-64.
240
[2] Backer Dirks O Posteruptive changes in dental enamel J Dent Res 1966; 45: 503-511.
241
uncorrected
proof
version