This in vitro study evaluated at baseline (T0) and over time (T12 months), the effect of a multi-mode universal adhesive compared with two experimental formulations blended with different concentrations of benzalkonium chloride (BAC), on bond strength and endogenous enzymatic activity. Specimens were assigned to the following groups according to the adhesive protocol: G1) All-Bond Universal (ABU) self-etch (SE); G2) ABU + 0.5% BAC SE; G3) ABU + 1% methacrylate BAC SE; G4) ABU etch-and-rinse (E&R); G5) ABU + 0.5% BAC E&R; G6) ABU + 1% methacrylate BAC E&R. Gelatin zymography was performed on dentin powder obtained from eight human third molars. Endogenous enzymatic activity within the hybrid layer was examined using in situ zymography after 24 h (T0) or 1-year storage in artificial saliva (T12). Forty intact molars were prepared for microtensile bond strength test at T0 and T12. Results were statistically analyzed with three-way ANOVA (α = 0.05). Gelatin zymography assay and in situ zymography quantification analyses indicated that all the BAC-containing formulations decreased matrix metalloproteinase expression. However, in situ zymography showed a general trend of enzymatic activity increase after aging. Microtensile bond-strength testing showed decrease in bond strength over time in all the tested groups; performances of the 1% methacrylate BAC experimental groups were worse than the control. BAC-containing adhesives reduce endogenous enzymatic activity both immediately and over time. However, independently from the adhesive employed, increase in the gelatinolytic activity over time and decrease in bond strength was found (especially in the BAC + 1% methacrylate groups), probably due to impaired polymerization properties. Adhesives containing protease inhibitors are practical and efficient tools in clinical practice for enhancement of the longevity of dental restorations. However, extensive investigation of the mechanical and adhesive properties of the material is necessary prior to their clinical use.

The aim of the present study was to evaluate the effect of 0.1% chitosan (Ch) solution as an additional primer on the mechanical durability and enzymatic activity on dentine using an etch-and-rinse (E&R) adhesive and a universal self-etch (SE) adhesive. Microtensile bond strength and interfacial nanoleakage expression of the bonded interfaces for all adhesives (with or without pretreatment with 0.1% Ch solution for 1 min and air-dried for 5 s) were analyzed immediately and after 10,000 thermocycles. Zymograms of protein extracts from human dentine powder incubated with Optibond FL and Scotchbond Universal on untreated or Ch-treated dentine were obtained to examine dentine matrix metalloproteinase (MMP) activities. The use of 0.1% Ch solution as an additional primer in conjunction with the E&R or SE adhesive did not appear to have influenced the immediate bond strength (T0) or bond strength after thermocycling (T1). Zymography showed a reduction in MMP activities only for mineralized and demineralized dentine powder after the application of Ch. Application of 0.1% Ch solution does not increase the longevity of resin–dentine bonds. Nonetheless, the procedure appears to be proficient in reducing dentine MMP activities within groups without adhesive treatments. Further studies are required to comprehend the cross-linking of Ch with dentine collagen.