Effect of pretreatment with collagen crosslinkers on dentin protease activity

Graphical abstract


  • Dentin contains proteases which are uncovered and activated during the adhesive procedures.

  • We evaluated the inhibitory effect of crosslinkers on MMP- and cathepsin K-mediated degradation.

  • Collagen crosslinkers hold much promise for increasing the durability of resin-dentin bonds.



This study evaluated the effect of dentin pretreatment with collagen crosslinkers on matrix metalloproteinase (MMP) and cathepsin K mediated collagen degradation.


Dentin beams (1 mm × 2 mm × 6 mm) were demineralized in 10% H 3 PO 4 for 24 h. After baseline measurements of dry mass, beams were divided into 11 groups ( n = 10/group) and, were pretreated for 5 min with 1% glutaraldehyde (GA); 5% GA; 1% grape-seed extract (GS); 5% GS; 10% sumac (S); 20 μM curcumin (CR); 200 μM CR; 0.l% riboflavin/UV (R); 0.5% R; 0.1% riboflavin-5-phosphate/UV (RP); and control (no pretreatment). After pretreatment, the beams were blot-dried and incubated in 1 mL calcium and zinc-containing medium (CM, pH 7.2) at 37 °C for 3, 7 or 14 days. After incubation, dry mass was reassessed and aliquots of the incubation media were analyzed for collagen C-telopeptides, ICTP and CTX using specific ELISA kits. Data were analyzed by repeated-measures ANOVA.


The rate of dry mass loss was significantly different among test groups ( p < 0.05). The lowest 14 day mean dry mass loss was 6.98% ± 1.99 in the 200 μM curcumin group compared to control loss of dry mass at 32.59% ± 5.62, p < 0.05, at 14 days. The ICTP release over the incubation period (ng/mg dry dentin) ranged between 1.8 ± 0.51 and 31.8 ± 1.8. CTX release from demineralized beams pretreated with crosslinkers was significantly lower than CM (5.7 ± 0.2 ng/mg dry dentin).


The results of this study indicate that collagen crosslinkers tested in this study are good inhibitors of cathepsin K activity in dentin. However, their inhibitory effect on MMP activity was highly variable.


Current dental adhesive techniques utilize acid-etching procedures or acidic monomers to create micromechanical retention for adhesive resin attachment to dentin. Dentin matrices contain proteolytic enzymes , the most recognized group being matrix metalloproteinases (MMPs) that were developmentally secreted as inactive proenzymes. These proteases are uncovered and activated during the acid etching step in adhesive procedures . In addition to MMPs, cysteine cathepsins were recently discovered in normal and carious dentin , increasing the list of potential endogenous proteases in dentin matrices.

Crosslinking agents have been reported to increase the stiffness of collagen by promoting additional hydrogen bonding and/or the formation of covalent inter- and -intra-molecular cross-links that prevents the long rod-like helical collagen molecules from sliding past each other under mechanical stress, and also reduce biodegradation by endogenous proteases . Several synthetic (glutaraldehyde, carbodiimides etc.) and natural crosslinking (grape seed, cocoa, berries etc.) agents have been used for these purposes . Gluteraldehyde has been used as a well-known crosslinking agent for the immobilization of proteins via the formation of a three-dimensional network as a result of intermolecular covalent crosslinking . Concerns about cytotoxicity limit their use in many applications . Natural polyphenols are capable of stabilizing collagen structure via the formation of multiple hydrogen bonds between collagen polypeptides . Polyphenolic compounds of vegetable tannin present in fruits, nuts, vegetables, seeds, leaves and flowers, such as grape seed, sumac berries, or curcumin are crosslinking reagents that may decrease biodegradation of collagen, and do not have high cytotoxicity, compared to gluteraldehyde .

The use of collagen crosslinkers to improve the mechanical properties of demineralized dentin and to increase collagen crosslinks has been investigated extensively and have been shown to increase the durability of resin–dentin bonding interfaces . Long pretreatment times limit the clinical relevancy of these agents. However, Liu et al. using demineralized dentin obtained excellent cross-linking with 15% proanthacyanidins in 60 s. Additionally, a synthetic crosslinking agent, carbodiimide, was recently shown to inhibit protease activity after an application time of 1 min.

Since there are a large variety of synthetic and natural crosslinkers available, a better understanding of the ability of these agents to interact with the endogenous protease activity in dentin matrix is of great interest. Thus, the aim of this study was to evaluate the effect of different collagen crosslinkers on their ability to inactivate dentin proteases. The null hypotheses tested were that pretreatment of demineralized dentin matrix with collagen crosslinkers does not inhibit dentinal MMPs or/dentinal cathepsin-K activity, and does not reduce the loss of dry mass of demineralized dentin matrix.

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Effect of pretreatment with collagen crosslinkers on dentin protease activity
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