Method: For in situ zymography etched dentin specimens were bonded with Optibond FL (Kerr) and Scotchbond 1XT (3M ESPE). Composite build-ups were made and sections of 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 in accordance with Mazzoni et al. 2012. In addition, to determine the type and nature of proteinases that cleaves the substrate a gelatin zymography was performed on demineralized dentin powder pulverized from extracted human molars and treated with the tested adhesives in accordance with Mazzoni et al., 2013.
Result: The in situ zymographic analyses revealed that HL created by the tested adhesives exhibited different degree of fluorescence that increased after prolonged time of fluorescein-conjugated gelatine exposure allowing localization of the enzymatic activity within the HL. The zymograms revealed that treatment with SB1XT and OFL produced an increase in MMP-2 and -9 expression.
Conclusion: In the present study the in situ zymography technique allowed the localization of gelatinolytic activity within the HL, while the nature and type of proteases were successfully determined using the zymographic analyses. The combination of these methodologies further supports the involvement of MMPs in the degradation of the HL.