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The inhibitory effects of silver diamine fluoride at different concentrations on matrix metalloproteinases
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903–908
Available online at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
The inhibitory effects of silver diamine fluoride at different concentrations on matrix metalloproteinases May L. Mei a , Q.L. Li b , C.H. Chu a,∗ , Cynthia K.Y. Yiu a , Edward C.M. Lo a a b
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:
Objective. To study the inhibitory effect of various commercially available concentrations of
Received 8 August 2011
silver diamine fluoride (SDF) solutions on matrix metalloproteinases (MMPs).
Received in revised form
Methods. Three SDF solutions with concentrations at 38%, 30% and 12% were studied. Two
4 January 2012
sodium fluoride (NaF) solutions at 10% and 3% were prepared, and they had the same fluoride
Accepted 16 April 2012
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
Silver diamine fluoride
MMP-9 were 82%, 65% and 60%, respectively (p < 0.001). The percentage inhibition on MMP-2,
Sodium fluoride
MMP-8 and MMP-9 by 38% SDF was significantly higher than the corresponding percentage inhibition by 10% NaF and 42% AgNO3 . Significance. 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 fluoride and silver ions, respectively. © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
A variety of chemical agents have been adopted in dentistry to control caries progression in dentin without surgical removal of the caries lesion. The first extensively reported agent in arresting dentin caries lesion is silver nitrate (AgNO3 ) solution, which is now considered as obsolete [1]. It was used because some researchers believed that silver nitrate could mechanically block dentin tubules; and silver could react with the organic material of the dentin to form silver albuminate [2].
Sodium fluoride (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 fluoride (SDF) was used in clinical trials to arrest dentin caries and the results were promising [5–7]. 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: [email protected] (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
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changes in mineral content such as the calcium and phosphate level, fluoride 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 cysteine–Zn2+ 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 fibronectin domain which has a strong affinity 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 fibrillar 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.
The null hypotheses tested are firstly, there is no difference in inhibitory effect on MMPs with solutions of SDF at 38%, 30% and 12%; and secondly, there is no difference in inhibitory effect on MMPs with solutions of 38% SDF, 42% AgNO3 and 10% NaF.
2.
Materials and methods
2.1.
Preparation of experimental solutions
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 fluoride (F− ) ions of the 38% and 12% SDF, respectively. The commercially available SDF solutions had high pH values (pH = 12–13) 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 1–10 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 purified recombinant human MMPs (MMP-2, MMP-8 and MMP-9) and Sensolyte MMP fluorimetric 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 fluorogenic 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 fluorometric 96-well microtiter plate (Fisher Scientific, 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 ) 903–908
plate also received 50 L of diluted MMP-substrate (dilution factor: 1:100) and 39 L assay buffer to give a total volume in each well of 100 L. There was a positive control containing recombinant MMPs only without the potential anti-MMP agent, it was used as a reference to calculate the percentage inhibition. A substrate control containing assay buffer only was used to check background fluorescence of the substrate. Another test control containing experimental solutions only without recombinant MMP was used to measure autofluorescence of the experimental solutions. Assay buffer was added to all controls wells to obtain the 100 L of total volume [17].
2.3.
Fluorescence reference standard
Briefly, 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 fluorescence resonance energy transfer (FRET) peptide.
2.4.
End-point reading
The reagents were then mixed gently in the dark for 10 s and fluorescence 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 fluorescence units, were converted to M (g/L) based on standard curves (R2 > 0.99) generated with EDANS fluorescence reference standard. A low concentration value reflected 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.
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Results
The mean end-point values of the 10 test groups, positive and substrate controls were shown in Table 2. A low mean value (MMPs activities) indicated high inhibition of MMPs. The end-point values of substrate control were very low (Group 12), which indicated that background fluoresce of the substrate was very low. The end-point values of test controls also showed very low value which indicated only very slight autofluorescence of the 10 test solutions (close to assay control, data not shown). This demonstrated that the values of test groups were valid. The mean end-point values were significantly lower (hence, higher inhibitory effect) in SDF solutions with higher concentrations for all 3 MMPs studied. The mean end-point values of Saforide (38% SDF, pH = 13) for MMP-2, MMP-8 and MMP-9 were 2.38, 0.34 and 2.58, respectively. The mean endpoint value of 12% SDF (pH = 12) for MMP-2 was 9.17, which was not far from that of the reference (11.08). Saforide also had the lowest mean end-point values (or the greatest inhibition) for the 3 MMPs compared with 42% AgNO3 and 10% NaF solutions (Table 2). When comparing the commercial SDF product (pH value at 12–13) at 38%, 30% and 12% with the corresponding buffered group at pH 9, there was no statistically significant difference on the MMP-8 activities. Moreover, there was no statistically significant difference between 12% SDF at the two pH values for MMP-2, MMP-8 and MMP-9 activities. The percentage inhibitions of the experimental solutions for MMP-2, MMP-8 and MMP-9 were calculated and they are shown in Figs. 1–3, respectively. The percentage inhibition of 38% SDF solution for MMP-2, MMP-8 and MMP-9 were 79%, 94% and 82%, respectively. The percentage inhibitions of 30% SDF solution for MMP-2, MMP-8 and MMP-9 were lower than those of 38% SDF, but still were higher than 60%. The percentage inhibition of 12% SDF for MMP-2 was only 8%.
4.
Discussion
According to the results of this study, the two null hypotheses were rejected. Firstly, this study showed that 38% SDF had the greatest and 12% SDF had the lowest inhibition effects on MMP-2, MMP-8 and MMP-9. Secondly, 38% SDF showed a higher inhibitory effect than 42% AgNO3 and 10% NaF
Statistical analysis
All data were assessed for a normal distribution using Shapiro–Wilk test for normality. One-way ANOVA with LSD multiple comparison tests were used to detect differences between MMPs concentration values (g/L) of the relevant experimental groups. Student’s t-test was used to compare the mean end-point values of SDF solutions to the corresponding buffered SDF solutions. Analyses were performed with the computer software SPSS Statistics, V19.0 (IBM Corporation, Armonk, USA). The level of statistical significance for all tests was set at 0.05.
Fig. 1 – Percentage inhibition of MMP-2 of the experimental solutions.
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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 ) 903–908
Table 2 – Mean end-point values (MMPs activity) of the experimental solutions. End-point values (MMP activity) in g/L (mean ± SD)
Group (n = 10 each)
1. 38% SDF (pH 13) 2. 38% SDF (pH 9) 3. 30% SDF (pH 12) 4. 30% SDF (pH 9) 5. 12% SDF (pH 12) 6. 12% SDF (pH 9) 7. 10% NaF 8. 3% NaF 9. 42% AgNO3 10. 13% AgNO3 11. Positive control 12. Substrate control
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
Comparing SDF solution at different concentrations (38%, 30% and 12%) 1<3<5 Group 1, 3, 5 <0.001 p-Value
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
Comparing pH effect on SDF solution at different concentrations 1 < 2 (0.004) 38%-Group 1 vs 2 (p value) 3 < 4 (0.002) 30%-Group 3 vs 4 (p value) NS (0.468) 12%-Group 5 vs 6 (p value)
1 < 2 (0.007) NS (0.157) NS (0.264)
Fig. 2 – Percentage inhibition of MMP-8 of the experimental solutions.
Fig. 3 – Percentage inhibition of MMP-9 of the experimental solutions.
NS (0.849) NS (0.125) NS (0.758)
solution. A significant inhibition of MMP-2, MMP-8 and MMP-9 by 38% SDF could be a reason for the success of caries arrests in clinical trials. MMPs digest extracellular matrix components. In this study, EDANS and 4-(dimethylaminoazo) benzene-4carboxylic acid (DABCYL) fluorescence resonance energy transfer peptide was used as substrate for detection of MMP-2 activities. MMP-2 breaks down the peptide into EDANS and DABCYL. Upon this proteolytic cleavage, fluorescence initially was measured at excitation/emission at 340 nm/490 nm. The assay kits for detection of MMP-8 and MMP-9 share the same principle in the substrate digestion, except that the substrate used was EDANS/DABCYL-Plus. DABCYL-Plus is a quencher which is more water-soluble than DABCYL. The fluorescent MMPs assay kit used in this study was optimized to detect MMPs activities. It is noteworthy that the sensitivity of the assay kit depends on the MMP concentration, thus precaution in the dilution process during preparation of MMPs is critical. Moreover, the sensitivity also depends on endogenous activities of MMPs. In our pilot study, the kinetic reading (MMPs activities) of the reaction became fairly stable after incubation for 60 min. Therefore, the reaction in the assay was terminated by adding cessation solution after 1 h. Taking end point reading at other time points may give different reading and different percentage inhibition. An important limitation in this study is the use of recombinant MMPs to represent matrix-bound MMPs in dentin. MMP from different sources may vary in its endogenous activities. Proteolytic degradation of the dentinal proteins mediated by the host MMPs in caries lesion is intricate. The host MMPs may induce activation/inactivation effects on other MMPs, while the assay kits used in this assay may not be related to the combined action of MMPs. In addition, the recombinant MMPs
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903–908
act on EDANS/DABCYL, which is a commercial peptide and is not collagen. Nevertheless, the fluorescent MMPs assay kit is easier to use than the SDS-gel electrophoresis technique [19]. More importantly, the MMPs activities can be quantified to compare the inhibitory effect of different experimental solutions. Another advantage of this method is that the 96well-plate allows large numbers of samples to be evaluated at the same time [17]. Caries destruction in dentin is different from that in enamel because dentin contains about 30% by volume of organic matrix. In the past, the destruction of organic matrix was considered to be mainly caused by bacterial collagenases. Later, Kawasaki and Featherstone [20] showed that the bacterial collagenases were inactivated by the drop in pH during demineralization, and hence they might not be the main reason for the matrix destruction. Some researchers suggested that the host MMPs present in dentin matrix [14,15] or in saliva [6] have an important role in dentin caries process. Collagens can be degraded by MMP-8, resulting in (3/4) to (1/4) length peptides. These peptides can be further degraded by the gelatinases MMP-2 and MMP-9 [11]. In fact, MMPs are commonly involved in many processes and can contribute to both normal and pathological events. MMP-8 (neutrophil collagenases) cleaves interstitial collagen types I, II and III. They are capable of digesting other extra cellular matrix and non-extra cellular matrix molecules. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) not only degrade the denatured collagen molecules (gelatin), type IV collagen, but also other proteins to a lesser degree [21]. Recent studies suggest that the activation of MMP-2, MMP8 and MMP-9 have a crucial role in the destruction of dentin collagen in caries lesions [6]. One of the most commonly used concentrations in clinical trials of SDF solution is 38% [5,22]. A concentration at 12% was also used in a clinical study conducted in Nepal [7]. In addition, 30% SDF is available in the market and is a concentration commonly used in the commercial products sold in South America. These concentrations of SDF were therefore selected for this study. The design could have been easier if the study was performed in a controlled way at a defined amount of Ag, and a defined amount of F, and their combination having the same acidity (pH value). This study tested commercially available SDF solutions which are at high pH values. The high pH of the solutions may affect some MMPs which work around neutral pH [23]. Thus, in this study, reference groups of corresponding SDF concentrations buffered with 10% nitric acid to produce a lower pH value were added to investigate whether the acidicity would influence the inhibition effect of SDF. In addition, freshly prepared AgNO3 and NaF solutions containing the same concentrations of Ag+ and F− ions as the 12% and 38% SDF solutions were also selected for comparison purpose. To prevent photo-chemical reaction of Ag+ containing solutions, the solutions were kept in opaque glass bottles and the process was performed in a dark room. Despite clinical studies showing that SDF is effective in arresting dentin caries, the mechanism is not clearly known. SDF has been shown to remineralize carious dentin and increase its microhardness [9]. Recent study reported the antimicrobial properties of SDF to cariogenic bacteria such as Streptococcus mutans and Actinomyces naeslundii [10,24]. An
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in vivo study with rat molars demonstrated a significant reduction in progression of dentinal caries lesion with MMP inhibitors, along with reduced salivary MMP activities [25]. This study showed that 38% SDF had a significant inhibition of MMP-2, MMP-8 and MMP-9, and the inhibition can be important in halting destruction of organic substance in dentin caries lesion. The results also showed SDF has a concentration-dependent inhibitory effect on the 3 MMPs which are present in dentin. SDF solution at 38% can inhibit up to 80% of the activities of the 3 MMPs. This may be one of the major reasons for the success of 38% SDF in arresting caries in clinical trials [5,22]. This study found that 12% SDF solution could hardly inhibit MMP-2 and only slightly inhibit MMP-8 and MMP-9. This may explain why 12% SDF solution was not effective in arresting dentin caries [7]. Typical MMPs have a pro-peptide domain to maintain the enzymes as inactive zymogen [21]. MMPs can be activated by proteinases [11], chemical agents and, in caries lesion, the low pH of the environment. The activated MMPs work around neutral pH [23] but not in acidic environment [11]. In caries process, the bacterial acids cause a drop in pH but salivary buffer systems provide transient pH neutralization [6]. This allows the pH-activated MMPs to degrade the organic matrix [25]. In this study, the MMPs were pre-activated by APMA before mixing with the test solutions. Yan et al. [23] reported MMPs work around neutral pH and their activities can be adversely affected by a higher pH environment. The commercial SDF products used in this study have very high pH values (pH = 12 or 13). We found the high pH of 8% SDF had a significant inhibition effect on MMP-2 and MMP-9. However, the difference in the MMP activities (end point values) between commercial 12% SDF solutions and buffered SDF solutions (pH = 9) did not demonstrate that higher pH had a significantly greater inhibition effect on MMPs. We also found high pH of SDF had no additional inhibition effect on MMP-8. In this study, the percentage inhibitions on MMP-2, MMP-8 and MMP-9 by 38% SDF solution were significantly higher than the corresponding percentage inhibitions by 10% NaF and 42% AgNO3 . Comparing the results of equivalent F− and Ag+ solutions, F− seems to contribute more to this inhibitory effect than Ag+ . Theoretically, the reactive series of metals shows the reactivities of metals. Silver is a metal which is less reactive than hydrogen and can hardly replace H+ in a reaction. Thus Ag+ is averse to bind to specific site of MMPs to inactivate their catalytic functions. However, a dose dependent inhibition of both MMP-2 and MMP-9 with silver particles was shown previously, but the mechanism is not clear [18]. Fluoride has been shown to be able to alter MMP-2 activities in enamel [26], while information on the effect of fluoride on MMP-2, MMP-8 and MMP-9 is lacking. This study found that F− ion in NaF solution had inhibitory effect on MMPs, especially on MMP-8 and MMP9. The percentage inhibition of 38% SDF for MMP-2, MMP-8 and MMP-9 were significantly higher than the corresponding percentage inhibition on equivalent F− in NaF solution.
5.
Conclusion
For the first 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 ) 903–908
inhibitory effect on MMP-2, MMP-8 and MMP-9 is related to the concentration of SDF solutions, 38% has significantly greater inhibition on MMPs than 30% and 12%. In addition, it has significantly greater inhibition on MMPs than 10% NaF and 42% AgNO3 solutions that have equivalent concentrations of fluoride and silver ions, respectively.
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).
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Available online at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
The inhibitory effects of silver diamine fluoride at different concentrations on matrix metalloproteinases May L. Mei a , Q.L. Li b , C.H. Chu a,∗ , Cynthia K.Y. Yiu a , Edward C.M. Lo a a b
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:
Objective. To study the inhibitory effect of various commercially available concentrations of
Received 8 August 2011
silver diamine fluoride (SDF) solutions on matrix metalloproteinases (MMPs).
Received in revised form
Methods. Three SDF solutions with concentrations at 38%, 30% and 12% were studied. Two
4 January 2012
sodium fluoride (NaF) solutions at 10% and 3% were prepared, and they had the same fluoride
Accepted 16 April 2012
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
Silver diamine fluoride
MMP-9 were 82%, 65% and 60%, respectively (p < 0.001). The percentage inhibition on MMP-2,
Sodium fluoride
MMP-8 and MMP-9 by 38% SDF was significantly higher than the corresponding percentage inhibition by 10% NaF and 42% AgNO3 . Significance. 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 fluoride and silver ions, respectively. © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
A variety of chemical agents have been adopted in dentistry to control caries progression in dentin without surgical removal of the caries lesion. The first extensively reported agent in arresting dentin caries lesion is silver nitrate (AgNO3 ) solution, which is now considered as obsolete [1]. It was used because some researchers believed that silver nitrate could mechanically block dentin tubules; and silver could react with the organic material of the dentin to form silver albuminate [2].
Sodium fluoride (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 fluoride (SDF) was used in clinical trials to arrest dentin caries and the results were promising [5–7]. 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: [email protected] (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
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changes in mineral content such as the calcium and phosphate level, fluoride 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 cysteine–Zn2+ 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 fibronectin domain which has a strong affinity 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 fibrillar 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.
The null hypotheses tested are firstly, there is no difference in inhibitory effect on MMPs with solutions of SDF at 38%, 30% and 12%; and secondly, there is no difference in inhibitory effect on MMPs with solutions of 38% SDF, 42% AgNO3 and 10% NaF.
2.
Materials and methods
2.1.
Preparation of experimental solutions
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 fluoride (F− ) ions of the 38% and 12% SDF, respectively. The commercially available SDF solutions had high pH values (pH = 12–13) 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 1–10 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 purified recombinant human MMPs (MMP-2, MMP-8 and MMP-9) and Sensolyte MMP fluorimetric 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 fluorogenic 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 fluorometric 96-well microtiter plate (Fisher Scientific, 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 ) 903–908
plate also received 50 L of diluted MMP-substrate (dilution factor: 1:100) and 39 L assay buffer to give a total volume in each well of 100 L. There was a positive control containing recombinant MMPs only without the potential anti-MMP agent, it was used as a reference to calculate the percentage inhibition. A substrate control containing assay buffer only was used to check background fluorescence of the substrate. Another test control containing experimental solutions only without recombinant MMP was used to measure autofluorescence of the experimental solutions. Assay buffer was added to all controls wells to obtain the 100 L of total volume [17].
2.3.
Fluorescence reference standard
Briefly, 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 fluorescence resonance energy transfer (FRET) peptide.
2.4.
End-point reading
The reagents were then mixed gently in the dark for 10 s and fluorescence 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 fluorescence units, were converted to M (g/L) based on standard curves (R2 > 0.99) generated with EDANS fluorescence reference standard. A low concentration value reflected 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.
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Results
The mean end-point values of the 10 test groups, positive and substrate controls were shown in Table 2. A low mean value (MMPs activities) indicated high inhibition of MMPs. The end-point values of substrate control were very low (Group 12), which indicated that background fluoresce of the substrate was very low. The end-point values of test controls also showed very low value which indicated only very slight autofluorescence of the 10 test solutions (close to assay control, data not shown). This demonstrated that the values of test groups were valid. The mean end-point values were significantly lower (hence, higher inhibitory effect) in SDF solutions with higher concentrations for all 3 MMPs studied. The mean end-point values of Saforide (38% SDF, pH = 13) for MMP-2, MMP-8 and MMP-9 were 2.38, 0.34 and 2.58, respectively. The mean endpoint value of 12% SDF (pH = 12) for MMP-2 was 9.17, which was not far from that of the reference (11.08). Saforide also had the lowest mean end-point values (or the greatest inhibition) for the 3 MMPs compared with 42% AgNO3 and 10% NaF solutions (Table 2). When comparing the commercial SDF product (pH value at 12–13) at 38%, 30% and 12% with the corresponding buffered group at pH 9, there was no statistically significant difference on the MMP-8 activities. Moreover, there was no statistically significant difference between 12% SDF at the two pH values for MMP-2, MMP-8 and MMP-9 activities. The percentage inhibitions of the experimental solutions for MMP-2, MMP-8 and MMP-9 were calculated and they are shown in Figs. 1–3, respectively. The percentage inhibition of 38% SDF solution for MMP-2, MMP-8 and MMP-9 were 79%, 94% and 82%, respectively. The percentage inhibitions of 30% SDF solution for MMP-2, MMP-8 and MMP-9 were lower than those of 38% SDF, but still were higher than 60%. The percentage inhibition of 12% SDF for MMP-2 was only 8%.
4.
Discussion
According to the results of this study, the two null hypotheses were rejected. Firstly, this study showed that 38% SDF had the greatest and 12% SDF had the lowest inhibition effects on MMP-2, MMP-8 and MMP-9. Secondly, 38% SDF showed a higher inhibitory effect than 42% AgNO3 and 10% NaF
Statistical analysis
All data were assessed for a normal distribution using Shapiro–Wilk test for normality. One-way ANOVA with LSD multiple comparison tests were used to detect differences between MMPs concentration values (g/L) of the relevant experimental groups. Student’s t-test was used to compare the mean end-point values of SDF solutions to the corresponding buffered SDF solutions. Analyses were performed with the computer software SPSS Statistics, V19.0 (IBM Corporation, Armonk, USA). The level of statistical significance for all tests was set at 0.05.
Fig. 1 – Percentage inhibition of MMP-2 of the experimental solutions.
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Table 2 – Mean end-point values (MMPs activity) of the experimental solutions. End-point values (MMP activity) in g/L (mean ± SD)
Group (n = 10 each)
1. 38% SDF (pH 13) 2. 38% SDF (pH 9) 3. 30% SDF (pH 12) 4. 30% SDF (pH 9) 5. 12% SDF (pH 12) 6. 12% SDF (pH 9) 7. 10% NaF 8. 3% NaF 9. 42% AgNO3 10. 13% AgNO3 11. Positive control 12. Substrate control
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
Comparing SDF solution at different concentrations (38%, 30% and 12%) 1<3<5 Group 1, 3, 5 <0.001 p-Value
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
Comparing pH effect on SDF solution at different concentrations 1 < 2 (0.004) 38%-Group 1 vs 2 (p value) 3 < 4 (0.002) 30%-Group 3 vs 4 (p value) NS (0.468) 12%-Group 5 vs 6 (p value)
1 < 2 (0.007) NS (0.157) NS (0.264)
Fig. 2 – Percentage inhibition of MMP-8 of the experimental solutions.
Fig. 3 – Percentage inhibition of MMP-9 of the experimental solutions.
NS (0.849) NS (0.125) NS (0.758)
solution. A significant inhibition of MMP-2, MMP-8 and MMP-9 by 38% SDF could be a reason for the success of caries arrests in clinical trials. MMPs digest extracellular matrix components. In this study, EDANS and 4-(dimethylaminoazo) benzene-4carboxylic acid (DABCYL) fluorescence resonance energy transfer peptide was used as substrate for detection of MMP-2 activities. MMP-2 breaks down the peptide into EDANS and DABCYL. Upon this proteolytic cleavage, fluorescence initially was measured at excitation/emission at 340 nm/490 nm. The assay kits for detection of MMP-8 and MMP-9 share the same principle in the substrate digestion, except that the substrate used was EDANS/DABCYL-Plus. DABCYL-Plus is a quencher which is more water-soluble than DABCYL. The fluorescent MMPs assay kit used in this study was optimized to detect MMPs activities. It is noteworthy that the sensitivity of the assay kit depends on the MMP concentration, thus precaution in the dilution process during preparation of MMPs is critical. Moreover, the sensitivity also depends on endogenous activities of MMPs. In our pilot study, the kinetic reading (MMPs activities) of the reaction became fairly stable after incubation for 60 min. Therefore, the reaction in the assay was terminated by adding cessation solution after 1 h. Taking end point reading at other time points may give different reading and different percentage inhibition. An important limitation in this study is the use of recombinant MMPs to represent matrix-bound MMPs in dentin. MMP from different sources may vary in its endogenous activities. Proteolytic degradation of the dentinal proteins mediated by the host MMPs in caries lesion is intricate. The host MMPs may induce activation/inactivation effects on other MMPs, while the assay kits used in this assay may not be related to the combined action of MMPs. In addition, the recombinant MMPs
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 903–908
act on EDANS/DABCYL, which is a commercial peptide and is not collagen. Nevertheless, the fluorescent MMPs assay kit is easier to use than the SDS-gel electrophoresis technique [19]. More importantly, the MMPs activities can be quantified to compare the inhibitory effect of different experimental solutions. Another advantage of this method is that the 96well-plate allows large numbers of samples to be evaluated at the same time [17]. Caries destruction in dentin is different from that in enamel because dentin contains about 30% by volume of organic matrix. In the past, the destruction of organic matrix was considered to be mainly caused by bacterial collagenases. Later, Kawasaki and Featherstone [20] showed that the bacterial collagenases were inactivated by the drop in pH during demineralization, and hence they might not be the main reason for the matrix destruction. Some researchers suggested that the host MMPs present in dentin matrix [14,15] or in saliva [6] have an important role in dentin caries process. Collagens can be degraded by MMP-8, resulting in (3/4) to (1/4) length peptides. These peptides can be further degraded by the gelatinases MMP-2 and MMP-9 [11]. In fact, MMPs are commonly involved in many processes and can contribute to both normal and pathological events. MMP-8 (neutrophil collagenases) cleaves interstitial collagen types I, II and III. They are capable of digesting other extra cellular matrix and non-extra cellular matrix molecules. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) not only degrade the denatured collagen molecules (gelatin), type IV collagen, but also other proteins to a lesser degree [21]. Recent studies suggest that the activation of MMP-2, MMP8 and MMP-9 have a crucial role in the destruction of dentin collagen in caries lesions [6]. One of the most commonly used concentrations in clinical trials of SDF solution is 38% [5,22]. A concentration at 12% was also used in a clinical study conducted in Nepal [7]. In addition, 30% SDF is available in the market and is a concentration commonly used in the commercial products sold in South America. These concentrations of SDF were therefore selected for this study. The design could have been easier if the study was performed in a controlled way at a defined amount of Ag, and a defined amount of F, and their combination having the same acidity (pH value). This study tested commercially available SDF solutions which are at high pH values. The high pH of the solutions may affect some MMPs which work around neutral pH [23]. Thus, in this study, reference groups of corresponding SDF concentrations buffered with 10% nitric acid to produce a lower pH value were added to investigate whether the acidicity would influence the inhibition effect of SDF. In addition, freshly prepared AgNO3 and NaF solutions containing the same concentrations of Ag+ and F− ions as the 12% and 38% SDF solutions were also selected for comparison purpose. To prevent photo-chemical reaction of Ag+ containing solutions, the solutions were kept in opaque glass bottles and the process was performed in a dark room. Despite clinical studies showing that SDF is effective in arresting dentin caries, the mechanism is not clearly known. SDF has been shown to remineralize carious dentin and increase its microhardness [9]. Recent study reported the antimicrobial properties of SDF to cariogenic bacteria such as Streptococcus mutans and Actinomyces naeslundii [10,24]. An
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in vivo study with rat molars demonstrated a significant reduction in progression of dentinal caries lesion with MMP inhibitors, along with reduced salivary MMP activities [25]. This study showed that 38% SDF had a significant inhibition of MMP-2, MMP-8 and MMP-9, and the inhibition can be important in halting destruction of organic substance in dentin caries lesion. The results also showed SDF has a concentration-dependent inhibitory effect on the 3 MMPs which are present in dentin. SDF solution at 38% can inhibit up to 80% of the activities of the 3 MMPs. This may be one of the major reasons for the success of 38% SDF in arresting caries in clinical trials [5,22]. This study found that 12% SDF solution could hardly inhibit MMP-2 and only slightly inhibit MMP-8 and MMP-9. This may explain why 12% SDF solution was not effective in arresting dentin caries [7]. Typical MMPs have a pro-peptide domain to maintain the enzymes as inactive zymogen [21]. MMPs can be activated by proteinases [11], chemical agents and, in caries lesion, the low pH of the environment. The activated MMPs work around neutral pH [23] but not in acidic environment [11]. In caries process, the bacterial acids cause a drop in pH but salivary buffer systems provide transient pH neutralization [6]. This allows the pH-activated MMPs to degrade the organic matrix [25]. In this study, the MMPs were pre-activated by APMA before mixing with the test solutions. Yan et al. [23] reported MMPs work around neutral pH and their activities can be adversely affected by a higher pH environment. The commercial SDF products used in this study have very high pH values (pH = 12 or 13). We found the high pH of 8% SDF had a significant inhibition effect on MMP-2 and MMP-9. However, the difference in the MMP activities (end point values) between commercial 12% SDF solutions and buffered SDF solutions (pH = 9) did not demonstrate that higher pH had a significantly greater inhibition effect on MMPs. We also found high pH of SDF had no additional inhibition effect on MMP-8. In this study, the percentage inhibitions on MMP-2, MMP-8 and MMP-9 by 38% SDF solution were significantly higher than the corresponding percentage inhibitions by 10% NaF and 42% AgNO3 . Comparing the results of equivalent F− and Ag+ solutions, F− seems to contribute more to this inhibitory effect than Ag+ . Theoretically, the reactive series of metals shows the reactivities of metals. Silver is a metal which is less reactive than hydrogen and can hardly replace H+ in a reaction. Thus Ag+ is averse to bind to specific site of MMPs to inactivate their catalytic functions. However, a dose dependent inhibition of both MMP-2 and MMP-9 with silver particles was shown previously, but the mechanism is not clear [18]. Fluoride has been shown to be able to alter MMP-2 activities in enamel [26], while information on the effect of fluoride on MMP-2, MMP-8 and MMP-9 is lacking. This study found that F− ion in NaF solution had inhibitory effect on MMPs, especially on MMP-8 and MMP9. The percentage inhibition of 38% SDF for MMP-2, MMP-8 and MMP-9 were significantly higher than the corresponding percentage inhibition on equivalent F− in NaF solution.
5.
Conclusion
For the first time, this study found an inhibitory effect of SDF solution at different concentrations on MMP activities. The
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inhibitory effect on MMP-2, MMP-8 and MMP-9 is related to the concentration of SDF solutions, 38% has significantly greater inhibition on MMPs than 30% and 12%. In addition, it has significantly greater inhibition on MMPs than 10% NaF and 42% AgNO3 solutions that have equivalent concentrations of fluoride and silver ions, respectively.
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).
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