This study evaluated the endogenous matrix metalloproteinase (MMP) activity of demineralized dentin matrix following 1 or 5 min pretreatment by various collagen crosslinkers. Generic MMP activity assay, total protein analysis, in situ zymography, gelatin zymography and multiplex bead technology were used to evaluate matrix-bound MMP activity.
Six different crosslinkers; glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, and curcumin were used. Demineralized dentin beams were pretreated with respective crosslinkers for 1 or 5 min. Demineralized dentin beams with no crosslinker pretreatment served as control. The reduction in the total activity of dentin matrices were measured using generic MMP activity assay. Dentin slabs were used for in situ zymography and evaluated by using hydrolysis of self-quenched fluorescein-conjugated gelatin under confocal microscopy. Dentin beam extracts were used for total protein assay and multiplex analysis and powder extracts were used for gelatin zymography.
MMP activity in crosslinker pretreated samples decreased significantly between 21% and 70%, whereas untreated control samples’ activity increased up to 84%. Zymograms confirmed a decrease in the gelatinolytic activity and in the amount of extractable total protein content. Multiplex analysis of extracts of crosslinker-treated dentin showed a reduction in the MMP-8, MMP-2 and MMP-9 release.
The result of this work suggests that the effect of the crosslinkers is source-dependent. The use of crosslinkers for as little as 1 min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices.
Enzymatic degradation of resin-infiltrated dentin matrices by endogenous proteases results in the loss of stability of resin–dentin bonds created by contemporary adhesives . Matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent endogenous proteases, are responsible for the turnover of collagen-based tissues . 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 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 has been shown to stabilize the resin–dentin interface . However, recent studies have demonstrated that CHX is only electrostatically bound to dentin , and the inhibitory effect of CHX on dentin MMPs may be lost in 1.5–2 years .
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 and inactivates matrix-bound MMPs . However, a wide range of crosslinkers are available, and their specific anti-MMP effects are still not clear. In our previous publication 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.
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% NaN 3 to prevent microbial growth, and were used within 1 month after extraction.
|1% Glutaraldehyde (v/v)
5% Glutaraldehyde (v/v)
|Distilled water||Protein crosslinker OHC(CH 2 ) 3 CHO||Merck, Finland||S5334503-928|
|1% Grape seed extract (w/v)
5% Grape seed extract (w/v)
|Hot water||Vitis vinifera , Natural proanthocyanidin source||Mega-natural gold grape seed extract CA, USA||13682503-01|
|0.1% (−) Riboflavin (w/v)
0.5% (−) Riboflavin (w/v) with UVA light 365 nm, 7 mW/cm 2
|Distilled water||Enzyme cofactor C 17 H 20 N 4 O 6||Sigma–Aldrich, Finland||OSOM1704Y|
|S||10% Sumac (w/v)||Hot water||Rhus coriaria , Natural proanthocyanidin source||Collected natural seed|
|RP1||0.1% Riboflavin-5-phosphate (w/v) with UVA light 370 nm, 3 mW/cm 2||Distilled water||C 17 H 20 N 4 P||Sigma–Aldrich, Finland||24887210|
|20 μM Curcumin (w/v)
200 μM Curcumin (w/v)
|0.2% Ethanol in distilled water||(1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione||LKT LAB||458-37-7|