The inhibitory effects of silver diamine fluoride at different concentrations on matrix metalloproteinases
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], [6], [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 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.
Section snippets
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.
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).
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 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
Conclusion
For the first time, this study found an inhibitory effect of SDF solution at different concentrations on MMP activities. The 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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