Abstract
Objective
To study the inhibitory effect of various commercially available concentrations of silver diamine fluoride (SDF) solutions on matrix metalloproteinases (MMPs).
Methods
Three SDF solutions with concentrations at 38%, 30% and 12% were studied. Two sodium fluoride (NaF) solutions at 10% and 3% were prepared, and they had the same fluoride ion concentrations as 38% and 12% SDF, respectively. Two silver nitrate (AgNO 3 ) 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 their inhibitory effect on three MMPs, which were MMP-2 (gelatinase A), MMP-8 (neutrophil collagenase) and MMP-9 (gelatinase B) using MMP assay kits. Positive control containing assay buffer at pH 9 and MMPs dilution was used to calculate the percentage inhibition.
Results
The percentage inhibition of 38%, 30% and 12% SDF on MMP-2 were 79%, 60% and 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, MMP-8 and MMP-9 by 38% SDF was significantly higher than the corresponding percentage inhibition by 10% NaF and 42% AgNO 3 .
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 AgNO 3 containing equivalent concentration of fluoride and silver ions, respectively.
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 (AgNO 3 ) solution, which is now considered as obsolete . 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 . Sodium fluoride (NaF) in various forms has also been used . Despite there is no clinical trial reported, a case report demonstrated using NaF varnish to arrest caries in a teenager . Furthermore, silver diamine fluoride (SDF) was used in clinical trials to arrest dentin caries and the results were promising .
Apart from clinical studies, laboratory studies have been performed to investigate the anti-caries effect of SDF on dentin caries . Most laboratory studies have focused on changes in mineral content such as the calcium and phosphate level, fluoride content and microhardness of dental hard tissues . 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 .
MMPs are metal-dependent endopeptidases commonly known as matrixins . 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 . MMPs can be activated by proteinases , chemical agents and, in caries lesion, the low pH of the environment. The activation is likely to be initiated through disturbance of the cysteine–Zn 2+ interaction of the cysteine switch. The prodomain links up with the catalytic domain by a hinge. The catalytic domain contains an active Zn 2+ -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 .
In the presence of zinc ion (Zn 2+ ) which acts as a co-factor, MMPs mediate the degradation of practically all extracellular matrix molecules, including native and denatured collagen . It is found that MMPs are present in dentin matrix or in saliva . They can be activated in an acidic environment or by lactate released by cariogenic bacteria . 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 . 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% AgNO 3 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 AgNO 3 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% HNO 3 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 .
| Group | Product | Chemicals | F − (ppm) | Ag + (ppm) | pH |
|---|---|---|---|---|---|
| 1 | Saforide 38% | 38% SDF | 44,800 | 255,000 | 13 |
| 2 | Buffered Saforide | 38% SDF + 10% HNO 3 | 44,800 | 255,000 | 9 |
| 3 | Cariestop 30% | 30% SDF | 35,400 | 200,000 | 12 |
| 4 | Buffered Cariestop 30% | 30% SDF + 10% HNO 3 | 35,400 | 200,000 | 9 |
| 5 | Cariestop 12% | 12% SDF | 14,150 | 80,000 | 12 |
| 6 | Buffered Cariestop 12% | 12% SDF + 10% HNO 3 | 14,150 | 80,000 | 9 |
| 7 | Fluoride solution A | 10% NaF | 44,800 | – | 9 |
| 8 | Fluoride solution B | 3% NaF | 14,150 | – | 9 |
| 9 | Ag solution A | 42% AgNO 3 | – | 255,000 | 10 |
| 10 | Ag solution B | 13% AgNO 3 | – | 80,000 | 10 |
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 . 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 μL activated MMP and 10 μL 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 plate also received 50 μL of diluted MMP-substrate (dilution factor: 1:100) and 39 μL assay buffer to give a total volume in each well of 100 μL.
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 μL of total volume .
2.3
Fluorescence reference standard
Briefly, 1 mM 5-((2-aminoethyl) amino) naphthalene-1-sulfonic acid (EDANS) was diluted to 5 μM 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 μM. Add 50 μL of the diluted EDANS of the above 8 concentrations from 5 to 0 μM to the well. A volume of 50 μL 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_Multilabel_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 μL 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 μM (μg/μL) based on standard curves ( R 2 > 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 .
2.5
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 (μg/μL) 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.
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 AgNO 3 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% HNO 3 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 .
| Group | Product | Chemicals | F − (ppm) | Ag + (ppm) | pH |
|---|---|---|---|---|---|
| 1 | Saforide 38% | 38% SDF | 44,800 | 255,000 | 13 |
| 2 | Buffered Saforide | 38% SDF + 10% HNO 3 | 44,800 | 255,000 | 9 |
| 3 | Cariestop 30% | 30% SDF | 35,400 | 200,000 | 12 |
| 4 | Buffered Cariestop 30% | 30% SDF + 10% HNO 3 | 35,400 | 200,000 | 9 |
| 5 | Cariestop 12% | 12% SDF | 14,150 | 80,000 | 12 |
| 6 | Buffered Cariestop 12% | 12% SDF + 10% HNO 3 | 14,150 | 80,000 | 9 |
| 7 | Fluoride solution A | 10% NaF | 44,800 | – | 9 |
| 8 | Fluoride solution B | 3% NaF | 14,150 | – | 9 |
| 9 | Ag solution A | 42% AgNO 3 | – | 255,000 | 10 |
| 10 | Ag solution B | 13% AgNO 3 | – | 80,000 | 10 |
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