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Saforide
Saforide
Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China
Stomatology Collage, Anhui Medical Univerisity, Hefei, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Methods. Three SDF solutions with concentrations at 38%, 30% and 12% were studied. Two
4 January 2012
sodium uoride (NaF) solutions at 10% and 3% were prepared, and they had the same uoride
ion concentrations as 38% and 12% SDF, respectively. Two silver nitrate (AgNO3 ) solutions at
42% and 13% were also prepared, and they had the same silver ion concentrations as 38%
and 12% SDF, respectively. Ten samples of each experimental solution were used to study
Keywords:
their inhibitory effect on three MMPs, which were MMP-2 (gelatinase A), MMP-8 (neutrophil
Matrix metalloproteinases
collagenase) and MMP-9 (gelatinase B) using MMP assay kits. Positive control containing
Arrest
assay buffer at pH 9 and MMPs dilution was used to calculate the percentage inhibition.
Caries
Results. The percentage inhibition of 38%, 30% and 12% SDF on MMP-2 were 79%, 60% and
Dentin
17%, respectively (p < 0.001); on MMP-8 were 94%, 85% and 77%, respectively (p < 0.001); on
MMP-9 were 82%, 65% and 60%, respectively (p < 0.001). The percentage inhibition on MMP-2,
Sodium uoride
MMP-8 and MMP-9 by 38% SDF was signicantly higher than the corresponding percentage
inhibition by 10% NaF and 42% AgNO3 .
Signicance. Greater inhibitory effect on MMPs was found with higher concentration of SDF
solution. SDF had more inhibition on MMPs than solutions of NaF and AgNO3 containing
equivalent concentration of uoride and silver ions, respectively.
2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Sodium uoride (NaF) in various forms has also been used [3].
Despite there is no clinical trial reported, a case report demonstrated using NaF varnish to arrest caries in a teenager [4].
Furthermore, silver diamine uoride (SDF) was used in clinical trials to arrest dentin caries and the results were promising
[57].
Apart from clinical studies, laboratory studies have been
performed to investigate the anti-caries effect of SDF on
dentin caries [8]. Most laboratory studies have focused on
Corresponding author at: Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR, China.
Tel.: +852 2859 0287; fax: +852 2858 7874.
E-mail address: chchu@hku.hk (C.H. Chu).
0109-5641/$ see front matter 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dental.2012.04.011
904
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903908
changes in mineral content such as the calcium and phosphate level, uoride content and microhardness of dental
hard tissues [1,9,10]. However, this is only part of the picture because both demineralization of hydroxyapatite and
degradation of organic matrix are involved in dentin caries
progression. Bacterial enzymes such as collagenases are
thought to be responsible for the organic matrix destruction.
Recent studies show that matrix metalloproteinases (MMPs)
play an important part in enzymatic degradation of dentin
[11].
MMPs are metal-dependent endopeptidases commonly
known as matrixins [12]. A typical MMP consists of predomain,
prodomain, hinge, catalytic domain and hemopexin domain.
The small predomain consists of about 80 amino-acids connecting to the prodomain. MMPs are usually presented as
inactive zymogens. The prodomain holds a cysteine switch,
which is suggested to prevent intracellular degradation of
zymogen [13]. MMPs can be activated by proteinases [11],
chemical agents and, in caries lesion, the low pH of the
environment. The activation is likely to be initiated through
disturbance of the cysteineZn2+ interaction of the cysteine
switch. The prodomain links up with the catalytic domain
by a hinge. The catalytic domain contains an active Zn2+ binding site. For MMP-2 (gelatinase A) and MMP-9 (gelatinase
B), the catalytic domain holds a bronectin domain which has
a strong afnity with gelatin. The catalytic domain is connected to the hemopexin domain. For MMP-2 and MMP-9,
hemopexin is thought to mediate enzyme-tissue inhibitors of
metalloproteinases [13].
In the presence of zinc ion (Zn2+ ) which acts as a co-factor,
MMPs mediate the degradation of practically all extracellular
matrix molecules, including native and denatured collagen
[11]. It is found that MMPs are present in dentin matrix
[14,15] or in saliva [6]. They can be activated in an acidic
environment or by lactate released by cariogenic bacteria
[11]. MMP-8 (neutrophil collagenase) is capable of degrading
triple-helical brillar collagens into distinctive 3/4 and 1/4
fragments. MMP-2 and MMP-9 are gelatinase, which degrades
type IV collagen. The activation of MMP-2, MMP-8 and MMP-9
has been shown to have a crucial role in collagen breakdown
in dentin caries lesions [11]. Hence, inhibition of MMP activities may contribute to caries arrest. So far, there is no study in
the literature on the effect of SDF on MMPs. Thus, this study
aimed to investigate the inhibitory effect on MMPs by SDF
solutions at various commercially available concentrations.
2.
2.1.
Commercially available SDF solutions at 12% (Cariostop, Biodinamica, Brazil), 30% (Cariostop, Biodinamica, Brazil) and 38%
(Saforide, Toyo Seiyaku Kasei, Japan) were selected for this
in vitro study. Solutions of AgNO3 at 42% and 13%, and NaF at
10% and 3% were prepared, which contained equivalent concentrations of silver (Ag+ ) and uoride (F ) ions of the 38% and
12% SDF, respectively. The commercially available SDF solutions had high pH values (pH = 1213) which could affect MMP
activities. Three reference solutions of SDF at 38%, 30% and
12% buffered with 10% HNO3 to lower the pH value to 9 were
prepared. Ten samples of each test solution were used in this
study. Totally 10 test solutions were assessed; and they were
assigned to be Group 110 as shown in Table 1.
2.2.
Inhibition solution reacts with MMP enzymatic
assays
The inhibitory effects of the 10 experimental solutions on
MMP-2, MMP-8 and MMP-9 were assessed by using commercially available puried recombinant human MMPs (MMP-2,
MMP-8 and MMP-9) and Sensolyte MMP uorimetric assay
kits (MMP-2, MMP-8 and MMP-9) from AnaSpec, Inc. (San Jose,
CA, USA). The Sensolyte MMP kit is a complete assay system
designed to continuously analyze MMP activities or to screen
MMP inhibitors by using uorogenic MMP substrate. In this
study protocol, pro-MMP was incubated for 1 h at 37 C with
1 mM 4-aminophenylmetcuric acetate (APMA) solution which
activates MMPs [16]. According to protocol suggested by the
manufacturer, the volume ratio of pro-MMP to APMA was 1:9
for MMP-2 and MMP-9; and 1:4 for MMP-8. The pro-MMP was
activated immediately before the experiment. Then, 1 L activated MMP and 10 L of experimental solution were added
to each well of a black uorometric 96-well microtiter plate
(Fisher Scientic, Gainesville, USA). All wells of the microtiter
Table 1 Fluoride and silver content and acidity (pH) of the 10 experimental solutions.
Group
1
2
3
4
5
6
7
8
9
10
Product
Chemicals
F (ppm)
Ag+ (ppm)
pH
Saforide 38%
Buffered Saforide
Cariestop 30%
Buffered Cariestop 30%
Cariestop 12%
Buffered Cariestop 12%
Fluoride solution A
Fluoride solution B
Ag solution A
Ag solution B
38% SDF
38% SDF + 10% HNO3
30% SDF
30% SDF + 10% HNO3
12% SDF
12% SDF + 10% HNO3
10% NaF
3% NaF
42% AgNO3
13% AgNO3
44,800
44,800
35,400
35,400
14,150
14,150
44,800
14,150
255,000
255,000
200,000
200,000
80,000
80,000
255,000
80,000
13
9
12
9
12
9
9
9
10
10
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903908
2.3.
Briey, 1 mM 5-((2-aminoethyl) amino) naphthalene-1sulfonic acid (EDANS) was diluted to 5 M with deionized
water. Multiple 1:2 serial dilutions were performed to get
concentrations of 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078 and
0 M. Add 50 L of the diluted EDANS of the above 8 concentrations from 5 to 0 M to the well. A volume of 50 L substrate
was added to the EDANS reference standard to correct the
absorptive quenching by the uorescence resonance energy
transfer (FRET) peptide.
2.4.
End-point reading
The reagents were then mixed gently in the dark for 10 s and
uorescence intensity of the assay was measured at excitation/emission (340 nm/490 nm) using multitask plate reader
http://www.perkinelmer.com/Catalog/Family/ID/VICTOR X M
-ultilabel Plate Reader (1420 Victor PerkinElmer, Boston, USA)
with an associated computer program (Wallac 1420 manager
PerkinElmer, Boston, USA) at room temperature. The reaction
in the assay was terminated by adding 50 L cessation solution to each well 1 h after the reaction. The data (end-point
reading of MMP activities) obtained, initially expressed as
relative uorescence units, were converted to M (g/L)
based on standard curves (R2 > 0.99) generated with EDANS
uorescence reference standard. A low concentration value
reected a high inhibitory effect on MMPs. The percentage
inhibition was calculated from the difference between the
mean values of the test group and the reference (positive
control) group divided by that of the reference group [18].
2.5.
3.
905
Results
4.
Discussion
Statistical analysis
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d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903908
Group (n = 10 each)
MMP-2
MMP-8
2.38 0.16
4.65 1.34
4.41 0.32
5.72 0.81
9.17 1.51
9.65 0.77
7.89 0.55
8.51 1.09
11.08 1.74
10.64 1.67
11.08 0.68
0.02 0.01
0.34 0.08
0.32 0.16
0.77 0.09
0.98 0.32
1.21 0.18
1.18 0.16
1.66 0.43
2.30 0.54
1.49 0.20
2.87 0.31
5.28 0.55
0.02 0.02
2.58 0.48
3.48 0.54
4.91 0.52
5.57 1.03
5.60 0.71
6.01 0.61
5.01 0.76
5.65 1.39
6.77 1.27
12.35 1.89
13.97 0.27
0.01 0.02
1<3<5
<0.001
1<3<5
<0.001
MMP-9
Comparing 38%SDF (Ag-255,000 ppm and F-44,800 ppm), 10%NaF (F-44,800 ppm), and 42%AgNO3 (Ag-255,000 ppm)
1<7<9
1<9<7
Group 1, 7, 9
<0.001
<0.001
p-Value
1<7<9
<0.001
1 < 2 (0.007)
NS (0.157)
NS (0.264)
NS (0.849)
NS (0.125)
NS (0.758)
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903908
907
5.
Conclusion
For the rst time, this study found an inhibitory effect of SDF
solution at different concentrations on MMP activities. The
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d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903908
Acknowledgements
The authors acknowledge Dr. Epasinghe Don Jeevanie for her
support in this research. This study was supported by the
NSFC/RGC Joint Research Scheme (Grant No. N HKU 776/10
and NSFC No.81061160511).
references