Research Regarding Inhibiting MMPs
Inside Dentistry (ID): What are matrix metalloproteinases (MMPs), and what effect do they have on dentin?
Annalisa Mazzoni, DDS, PhD (AM): MMPs are enzymes that are everywhere in the body, and their activity regulates several processes. However, there was little research conducted about them in relation to dentistry and their effect on dentin until my colleagues and I began studying them around 2005. We started our research more on the biochemical side in order to gain a better understanding. To determine which types of MMPs were in the dentin substrate and what effect they had on adhesive dentistry, we put together laboratory techniques and developed protocols to analyze MMPs on tooth structure. We were primarily interested in the clinical side of the research regarding MMPs and adhesives, but we needed to build a knowledge base about the enzymes on the substrate first.
ID: What are the differences between the various types of MMPs?
AM: Their differences primarily just involve the substrates on which they act. Because dentin is the major substrate of interest in adhesive dentistry, and it contains a lot of collagen, we focused our attention on the enzymes that are able to degrade collagen: MMP-2, MMP-8, MMP-9, and, in an indirect way, MMP-3.1-6 We wanted to understand the localization and activity associated with those specific enzymes.
ID: The actual behavior of each enzyme is fairly similar though, right?
AM: Yes. The behavior of each was similar among those that we studied. What changes from one MMP to another is the 3-dimensional structure that they possess. The way that they act on collagen in terms of degradation, however, is the same.
ID: What impact can effectively inhibiting MMPs have on long-term oral health?
AM: Hybrid layer degradation is perhaps one of the most important topics in adhesive dentistry. We know that restorations made with the latest adhesives and composites can last for a long time, but they do not last forever. At some point, leakage and secondary caries are likely to occur. The hybrid layer's effect on the substrate and its interaction with the adhesive play significant parts in how long the restoration can last before needing to be replaced. Of course, incorporating an MMP inhibitor will not allow a restoration to last forever, but it is one way to prolong and enhance a restoration's stability and mechanical properties. Creating a stable, long-lasting hybrid layer is an important aspect of preserving and improving the longevity of restorations.
ID: What were the challenges involved in researching MMPs and their inhibitors?
AM: The main challenge, as I mentioned previously, was that little was known about this topic in dentistry, even though it was very well understood in other sectors of medicine. Therefore, the first difficulty involved adapting all of the biochemical, histochemical, and morphological research protocols to the dentin substrate. Any time that you work on a different substrate, you need to adapt and revise the protocols. We also needed to develop reliable testing methods that we could use to compare the materials.
ID: Once you accomplished that, you were able to study alcohols,7 chlorhexidine,8 Galardin,9 chitosan primer,10 and more. What have you learned?
AM: Yes, we have studied a number of different types of MMP inhibitors. We started with compounds that were already being used in clinical dentistry, such as chlorhexidine, which has been used as an antibacterial compound for many years. The end goal was to identify compounds that could be blended with adhesives to add an MMP-inhibition effect. Of course, each compound has a different chemical nature, so they all behave differently. Although chlorhexidine exhibits antibacterial properties, which is why it is often used for periodontal treatments, other compounds, such as carbodiimide, are cross-linkers, which are known to stiffen collagen. We wanted to test these compounds to see if we could make collagen more robust in order to resist MMP activity. Again, our intent was to blend these compounds into an adhesive formulation. That would be the perfect result: an adhesive that could function like a bioactive material with antimicrobial and remineralizing properties—or, in our case, MMP-inhibiting and collagen-strengthening properties. Realizing this goal remains a challenge, however. Although these compounds act effectively when used as part of a separate step in the application of adhesives, when incorporated into an adhesive formulation, their efficacy very often declines.
ID: Do any of these materials present any disadvantages or result in any side effects?
AM: We only tested materials that we knew would have no side effects at the concentrations that we were using. We knew that we had to select compounds that were nontoxic; otherwise, the research would become self-limiting. There's no point to finding the best compound to inhibit MMPs if it will not have any realistic potential clinical application.
ID: After conducting all of this research, if you were to place a restoration on yourself today, what MMP inhibitor would you use?
AM: Our latest work has concentrated on carbodiimide,11 which is a cross-linker. We have found that it can be used at a very low concentration, produces no side effects, and seems to remain stable for a long period of time. The other advantage of this type of compound is that we can blend it with water, acetone, or alcohol, which is important for adhesive formulations because the primers in adhesive formulations contain approximately 40% solvent. If we can identify an effective MMP-inhibiting compound that can be dissolved in a solvent such as acetone or alcohol, it could potentially be combined into the formulation of an adhesive. We have 7 years of in vitro research demonstrating positive results with carbodiimide, so we are optimistic about its potential.
Annalisa Mazzoni, DDS, PhD, is an associate professor in the Department of Biomedical and Neuromotor Sciences at the University of Bologna, Italy, and one of the foremost researchers in the field of adhesive dentistry with respect to MMPs and the hybrid layer.
1. Mazzoni A, Pashley DH, Tay FR, et al. Immunohistochemical identification of MMP-2 and MMP-9 in human dentin: correlative FEI-SEM/TEM analysis. J Biomed Mater Res A. 2009;88(3):697-703.
2. Mazzoni A, Mannello F, Tay FR, et al. Zymographic analysis and characterization of MMP-2 and -9 forms in human sound dentin. J Dent Res. 2007;86(5):436-440.
3. Mazzoni A, Carrilho M, Papa V, et al. MMP-2 assay within the hybrid layer created by a two-step etch-and-rinse adhesive: biochemical and immunohistochemical analysis. J Dent. 2011;39(7):470-477.
4. Santos J, Carrilho M, Tervahartiala T, et al. Determination of matrix metalloproteinases in human radicular dentin. J Endod. 2009;35(5):686-689.
5. Mazzoni A, Papa V, Gobbi P, et al. Identification of MMP-8 in human sound dentin. Ital J Anat Embryol. 2010;115(Suppl 1/2):111.
6. Mazzoni A, Papa V, Nato F, et al. Immunohistochemical and biochemical assay of MMP-3 in human dentine. J Dent. 2011;39(3):231-237.
7. Tezvergil-Mutluay A, Agee KA, Hoshika T, et al. Inhibition of MMPs by alcohols. Dent Mater. 2011;27(9):926-933.
8. Breschi L, Mazzoni A, Nato F, et al. Chlorhexidine stabilizes the adhesive interface: a 2-year in vitro study. Dent Mater. 2010;26(4):320-325.
9. Breschi L, Martin P, Mazzoni A, et al. Use of a specific MMP-inhibitor (galardin) for preservation of hybrid layer. Dent Mater.2010;26(6):571-578.
10. Baena E, Cunha SR, Maravić T, et al. Effect of chitosan as a cross-linker on matrix metalloproteinase activity and bond stability with different adhesive systems. Mar Drugs. 2020;18(5):263.
11. Comba A, Scotti N, Mazzoni A, et al. Carbodiimide inactivation of matrix metalloproteinases in radicular dentine. J Dent. 2019;82:56-62.