EDC inactivates endogenous MMPs within the hybrid layer

Purpose: In situ zymography is a valuable technique to investigate gelatinolytic/collagenolytic activity within the hybrid layers created by dentin bonding agents. Recent studies reported that the use of 1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide Hydrochloride (EDC) as collagen cross-linker in bonding procedures could prevent dentin collagen degradation, thus improving the bond durability. The aim of this study was to investigate the gelatinolytic activity within the hybrid layer created with an EDC-containing conditioner by means of in situ zymography. Methods and materials: Middle/deep dentin exposed from 12 human sound molars was etched with 35% phosphoric acid for 15 s. Specimens were then assigned to: Group (1) dentin was pretreated with 0.3 M EDC water solution for 1 min then bonded with Adper Scotchbond 1XT (SB1XT-3M ESPE); Group (2) SB1XT was applied on untreated etched dentin (control). Composite build-ups were made and sections of the adhesive/dentin interface were obtained using a low-speed diamond saw under water irrigation. Interfaces were glued on microscope slide and in situ zymography reactions were performed using fluorescein-conjugated gelatin (E-12055, Molecular Probes, Eugene, OR, USA). Gelatin solution was applied on the adhesive interface, protected with cover slips and incubated in dark humid condition. The gelatinolytic activity was observed as green fluorescence (absortion: 488 nm and emission lp530 nm) with a TCS SP2 AOBS confocal laser scanning microscope (Leica Microsystems). Negative control sections were incubated as previously described, with standard non-fluorescent gelatin. Results: Hybrid layers of SB1XT (Group 2) exposed to fluorescein-conjugated gelatine exhibited intense collagenolytic activity, while almost no fluorescence signal was recorded in specimens pre-treated with 0.3 M EDC-containing conditioner (Group 1). Negative controls showed only a faint signal mainly caused by auto-fluorescence of the dentin. Conclusion: The in situ zymography technique employed in this study clarified that EDC can contribute to inactivate endogenous dentin MMPs within the hybrid layer created by an etch-and-rinse adhesive. This may be responsible for preventing loss of bond strength over time. Supported, in part, by grants: FIRB RBAP1095CR and PRIN 2009SAN9K5 and 2009FXT3WL from MIUR, Italy, and R01DE015306 from NIH/NIDCR to DHP (PI).