Use of crosslinkers to inactivate dentin MMPs
Introduction
Enzymatic degradation of resin-infiltrated dentin matrices by endogenous proteases results in the loss of stability of resin–dentin bonds created by contemporary adhesives [1]. Matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent endogenous proteases, are responsible for the turnover of collagen-based tissues [2]. Although MMPs are inactive in mineralized dentin, the application of etching acids in etch-and-rinse or acidic monomers in self-etch adhesives can uncover and activate these proteases [3], [4], [5], [6] resulting in progressive loss of collagen from the hybrid layers over time.
To maintain the durability of bonded restorations, use of synthetic MMP inhibitors has been demonstrated to be effective. Chlorhexidine (CHX), an inhibitor of MMP-2, -9 and -8 [7] has been shown to stabilize the resin–dentin interface [8], [9]. However, recent studies have demonstrated that CHX is only electrostatically bound to dentin [10], and the inhibitory effect of CHX on dentin MMPs may be lost in 1.5–2 years [11].
Biomodification of dentin matrices using collagen crosslinkers such as glutaraldehyde or proanthocyanidin improves the mechanical properties of dentin matrix by enhancing intra- and intermolecular crosslinks of collagen [12], [13] and inactivates matrix-bound MMPs [14], [15]. However, a wide range of crosslinkers are available, and their specific anti-MMP effects are still not clear. In our previous publication [16] we evaluated the effects of pretreatment of completely demineralized dentin matrix by synthetic or natural crosslinking agents on the loss of dry mass and the liberation of ICTP and CTX telopeptide fragments, as indirect measures of matrix degradation over 3, 7 and 14 days of incubation at 37 °C. In the current study we used the same model and same crosslinking agents to pretreat demineralized dentin for 1 or 5 min. The outcomes that were measured were total MMP activity, total extractable protein, gelatin zymographic activity of dentin extracts and the change in the extractability of MMP-2, -8 and -9 after pretreatment. The tested null hypotheses were that 1 or 5 min application of collagen crosslinkers does not reduce dentin protease activity, that the collagen crosslinkers do not change the release of proteins from dentin matrix, and that crosslinkers do not inhibit dentin matrix MMPs equally.
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Materials and methods
The source of synthetic and natural collagen crosslinkers and the concentrations used in this study are presented in Table 1. Reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise specified. Extracted non-carious human third molars were obtained with informed consent from 18 to 21-year-old patients under a protocol approved by the Ethical Committee of Oulu University, Finland. The teeth were stored at 4 °C in 0.9% NaCl containing 0.02% NaN3 to prevent microbial
Inhibition of MMP activity
The results of the generic total-MMP activity screening assay are shown in Fig. 1. The control group showed an 84.1 ± 15.8% increase in total MMP activity compared to the demineralized baseline level. All crosslinker pretreatment groups showed significant reductions in the total MMP activity compared to control (p < 0.05). After 1 min of pretreatment, the highest inhibition was 64% compared to the baseline activity observed at 5% grape seed group, which corresponds to 177% reduction compared to the
Discussion
Collagen crosslinking agents have been reported to increase the resistance of dentin collagen matrix to degradation [14], [18], [19]. To date, glutaraldehyde is the most commonly used synthetic cross-linker, while proanthocyanidin is frequently reported in dental research as a natural cross-linker [19], [20], [21], [22]. In the present study, we compared the effect of various plant-derived natural as well as synthetic cross-linkers on the MMP activity of collagen matrix.
Detection of MMPs
Acknowledgements
This study was supported by grant #8126472 from the Academy of Finland to AT-M (PI), EVO funding of Turku University Hospital to AT-M (PI) and by FINDOS Mobility Grant awarded to Dr. Seseogullari Dirihan as part of her PhD thesis. The authors wish to thank Mr. Aurelio Valmori for photographical assistance. The authors do not have a financial interest in products, equipment, and companies cited in the manuscript.
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