TEM/FEI-SEM immunohistochemical identification of MMP-2 within the hybrid layer

Objective: Degradation of hybrid layers (HL) within resin-infiltrated dentin has shown to result from multiple degradation factors, including the collagenolytic activity of specific extracellular proteinases (matrix metalloproteinases-MMPs). Inhibition of host-derived MMPs may, therefore, slow the degradation of HL. This study aimed to determine the contribution and effectiveness of chlorhexidine (CHX), as a MMP inhibitor, on the preservation of the mechanical properties of HL and on the distribution of MMP-2 within HL.

Methods: Mechanical integrity of HL was assessed by microtensile bond testing. Etched dentin substrates were assigned to two treatment groups: 1) 2% CHX+Adper Scotchbond 1XT (CHX); 2) Adper Scotchbond 1XT (Control). Composite build-ups were made and beams were prepared for microtensile bond testing and either immediately pulled to failure or stored in artificial saliva for 12 months and then tested. In additional adhesive interfaces the effect of CHX on distribution of MMP-2 within the HL was assessed with immunohistochemical labeling (Breschi et al., Oper Dent, 2004) using monoclonal antibody anti-MMP2 and then investigated under high resolution SEM/ TEM.

Results: Microtensile test showed similar immediate bond strength (MPa) between groups [CHX=39.3±9.1; Control=42.6±8.7]. After 12 months, bond strength remained stable in CHX-pretreated specimens [CHX=32.3±7.2], while significantly decreased in control specimens [20.5±8.2; p<0.05]. The FEI-SEM/TEM analyses revealed MMP-2 distribution within the HL: after 12 months of artificial aging the MMP-2 patterns were increased only in the control group, while minor labeling was observed in the CHX-pretreated specimens.

Conclusions: The immunohistochemical and microtensile bond strength data of this study confirmed that CHX inhibits MMP-2 expression within the HL and effectively contributes to preserve its mechanical integrity over time. Further in vivo studies should be done to confirm that the dentinal MMPs activity can be inhibited by synthetic non-toxic MMP-inhibitors that may stabilize the organic matrix of resin-infiltrated dentin interfaces.