Carbodiimide has previously been investigated as a cross-linking agent to increase the mechanical properties of the dentin matrix (Bedran-Russo et al., 2010
). However, a 1- to 4-hour application time was required for the mechanical properties of the collagen matrix to increase, making it impossible for use in clinical applications (Bedran-Russo et al., 2010
). The results of the present study indicate that even a short pre-treatment (i.e
., 1 min) of acid-etched dentin matrix is sufficient to inactivate endogenous protease activity of dentin without significantly stiffening the collagen.
Since the rhMMP-9 inactivation potential of non-specific EDC was similar to or higher than that of the specific MMP inhibitor GM6001, the first null hypothesis—that EDC does not inhibit soluble rhMMP-9—was rejected. The results of the soluble rhMMP-9 assay showed that even at 0.01 M, EDC successfully inhibited rhMMP-9 by over 99%. This is in line with previous work which showed a large decrease in MMP-9 activity following treatment with 0.1 M EDC for 4 hrs (Calero et al., 2002
Based on the rhMMP-9 results, a 1-minute pre-treatment time was selected for inactivation of matrix-bound protease activity in completely demineralized dentin. Previous work has shown that endogenous proteases in dentin matrices slowly lower the modulus of elasticity of incubation time as collagen peptides are slowly solubilized (Carrilho et al., 2009
; Tezvergil-Mutluay et al., 2010
). When the decreases in stiffness of various groups were compared, the highest residual stiffness was obtained in the group treated with 0.3 M EDC, whereas the lowest stiffness was observed with the control (46% drop), where no pre-treatment was used. This suggests that the endogenous protease activities in the EDC pre-treated dentin beams were much lower than those in the control beams. The same trend was observed with the evaluation of dry mass and the hydroxyproline assay, which are indirect measures of solubilization of collagen from the dentin matrix. The loss of dry mass in the control group was around 5 times more than in the EDC pre-treated groups. Likewise, the control group released 5 to 6 times more hydroxyproline compared with the EDC pre-treated groups. This requires rejection of the second null hypothesis, that EDC does not inactivate matrix-bound proteases. We speculate that the EDC cross-links peptide chains in MMPs and cysteine cathepsins (Tersariol et al., 2010
; Nascimento et al., 2011
) of the dentin matrix. Cross-linking decreases the molecular mobility of the catalytic sites in these enzymes, which are critical for their function (Liu et al., 2011
). This cross-linking is done very rapidly in acid-etched dentin that is 30 vol% collagen and 70 vol% water (Pashley et al., 2011
). The carboxyl and amino groups in collagen may not be as accessible as those in MMPs (Orgel et al., 2006
; Perumal et al., 2008
), thereby permitting more rapid cross-linking of MMPs than of collagen.
There are potential advantages of inactivating the proteolytic enzymes of the dentin matrix (i.e
., MMPs and cathepsins) by using EDC instead of using non-specific MMP inhibitors such as chlorhexidine, or specific MMP inhibitors like Galardin (Breschi et al., 2010
). To be effective, enzyme inhibitors must remain firmly bound to the enzyme. While specific MMP inhibitors such as Galardin can inhibit MMPs at micromolar concentrations (Galardy et al., 1994
), chlorhexidine requires millimolar concentrations, because collagen is not saturated by chlorhexidine until it reaches 30 mM (Kim et al., 2010
). High concentrations of chlorhexidine (e.g.
, 2 wt%) can interfere with conversion of monomers to polymers (Hiraishi et al., 2010
). Water-soluble inhibitors may slowly leach from resin-dentin interfaces over time. Recent studies showed that chlorhexidine inhibition of resin-dentin bonds was effective for 9 to 12 mos, after which it begins to lose its effectiveness (Ricci et al., 2010
; Sadek et al., 2010
). We speculate that functional stresses on hybrid layers may cause them to compress 0.6% under function and then rebound (Wood et al., 2008
), which may allow unbound water within the hybrid layers to elute residual chlorhexidine from the acid-etched collagen matrix.
Within the limits of this in vitro
study, it may be concluded that EDC can inactivate matrix-bound dentin proteinases in demineralized dentin matrices with a 1-minute application time. This result requires rejection of the third null hypothesis. Once cross-linked, these covalent bonds may be stable and therefore contribute to improved durability of resin-dentin bonds (Bedran-Russo et al., 2010
) if newly synthesized odontoblast MMPs (Lehmann et al., 2009
) in dentinal fluid cannot diffuse around resin tags in dentinal tubules bonded to dentin to reach the hybrid layer. This speculation needs to be tested by longitudinal in vivo
studies and in vitro
bond strength studies on the use of EDC to stabilize resin-bonded dentin, if EDC can be shown to be non-toxic to pulpal soft tissues. The use of EDC on acid-etched dentin would not prevent hydrolysis of the resin polymer component of the hybrid layer.