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Updated: Mar 27, 2026
Detection of Functional Matrix Metalloproteinases by Zymography
Published on: November 8, 2010
Use of crosslinkers to inactivate dentin MMPs
R Seseogullari-Dirihan1, F Apollonio2, A Mazzoni3
1Finnish Doctoral Program in Oral Sciences (FINDOS) University of Turku, Institute of Dentistry, Turku, Finland.
Objectives:
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.
Methods:
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.
Results:
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.
Significance:
The result of this work suggests that the effect of the crosslinkers is source-dependent. The use of crosslinkers for as little as 1min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices.
Insights
Short-term collagen crosslinker pretreatment effectively inactivates endogenous matrix metalloproteinase (MMP) activity in demineralized dentin. Even one minute of application significantly reduces protease activity, crucial for dental biomaterials.
Area of Science:
- Biomaterials Science
- Dental Research
- Biochemistry
Background:
- Demineralized dentin matrix harbors endogenous matrix metalloproteinases (MMPs) that can degrade collagen.
- Uncontrolled MMP activity compromises the integrity and longevity of dental biomaterials and restorations.
- Effective inhibition of MMPs is essential for improving the durability of dentin-based treatments.
Purpose of the Study:
- To evaluate the efficacy of various collagen crosslinkers in reducing endogenous MMP activity in demineralized dentin.
- To determine the optimal pretreatment time (1 or 5 minutes) for crosslinker application.
- To assess the impact of crosslinkers on MMP levels and activity using multiple analytical techniques.
Main Methods:
- Six collagen crosslinkers (glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, curcumin) were tested.
- Demineralized dentin beams were pretreated for 1 or 5 minutes, with untreated beams serving as controls.
- Matrix-bound MMP activity was assessed using generic MMP assays, in situ zymography, gelatin zymography, and multiplex bead technology for MMP-8, MMP-2, and MMP-9.
Main Results:
- Crosslinker pretreatment significantly reduced MMP activity by 21-70%, while untreated samples showed increased activity (up to 84%).
- Zymography confirmed reduced gelatinolytic activity and lower extractable total protein content in treated samples.
- Multiplex analysis revealed decreased release of MMP-8, MMP-2, and MMP-9 following crosslinker application.
Conclusions:
- Collagen crosslinkers, even with short pretreatment times (1 minute), effectively inactivate endogenous protease activity in demineralized dentin.
- The efficacy of crosslinkers is source-dependent, highlighting the need for tailored approaches.
- These findings support the use of crosslinkers to enhance the stability of demineralized dentin in dental applications.
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