Mechanistic study of the stabilization of dentin-bonded restorative interfaces via collagen reinforcement by multi-acrylamides.

Hydrolytically and enzymatically-stable multi-acrylamides have been proposed to increase the long-term durability of dental adhesive interfaces as alternatives to methacrylates. The aim of this study was to investigate the mechanical and biochemical properties of experimental adhesives containing multi-functional acrylamides concerning collagen reinforcement and metalloproteinases (MMP) activity. Multi-functional acrylamides, TMAAEA (Tris[(2-methylaminoacryl) ethylamine) and DEBAAP (N,N-Diethyl-1,3-bis(acrylamido) propane), along with the commercially available DMAM (N,N-dimethylacrylamide) (monofunctional acrylamide) and HEMA (2-Hydroxyethyl methacrylate) (monofunctional methacrylate - control) were tested for stability against enzymatic hydrolysis by cholesterol esterase/pseudocholinesterase (PC/PCE) solutions for up to 30 days. Collagen-derived substrate and gelatin zymography were performed to examine the effect of the compounds on the biological activity of human recombinant and dentin-extracted gelatinases MMP-2 and MMP-9. In situ zymography was carried out by fluorescent collagen degradation combined with confocal microscopy analysis. Hydroxyproline content was measured in collagen derived from dentin extracts though reaction with Ehrlich's reagent p-dimethylaminobenzaldehyde (DMAB), generating a stable chromophore measured at 550 nm. Storage shear modulus of demineralized dentin discs treated with the tested compounds was measured by oscillatory rheometry, in order to investigate potential collagen reinforcement. FT-IR was performed to determine qualitative differences in collagen based on observed changes in amide bands. The results were analyzed by ANOVA/Tukey's test (α = 0.05). Multi-acrylamides survived 30 days of incubation in cholinesterase/pseudo-cholinesterase (PC/PCE) solutions, while HEMA showed approximately 70 % overall degradation. Incubation with multi-acrylamides reduced collagen degradation as evidenced by the reduced hydroxyproline levels and by the 30 % increase inshear storage modulus. Biochemical and zymography assays showed no noticeable inhibition of recombinant and extracted MMPs enzymatic activity. The infra-red spectroscopy results for multi-functional acrylamides treated samples demonstrated shifts of the amide II bonds and marked increase in intensity of the bands 1200 cm-1, which may indicate partial collagen denaturation and some degree of cross-linking of the compounds with collagen, respectively. The multi-acrylamides exhibited not only comparable mechanical properties but also demonstrated significantly enhanced biochemical stability when compared to the widely used methacrylate control. Clinical relevance: These findings highlight the potential of multi-acrylamides to increase the bonding stability to tissues and, ultimately, contribute to the longevity of dental restorations.

Role of Proteolytic Enzyme Inhibitors on Carious and Eroded Dentin Associated With a Universal Bonding System.

OBJECTIVES: The aim of this study was to evaluate the effect of proteolytic inhibitors on the bond strength of a universal adhesive system (etch-and-rinse mode) applied to artificial carious and eroded dentin. METHODS: Ninety molars were prepared and randomly divided into three groups according to the substrate: N, no challenges; ACD, artificial carious dentin simulation and ERO, artificial erosion simulation with orange juice. All groups were redivided into three subgroups according to the dentin pretreatment: W, water; CHX, 2% digluconate chlorhexidine; and E-64 (trans-epoxysuccinyl-L-leucylamido-[4-guanidino] butane), 5 µM E-64 inhibitor. They constituted a total of nine groups (n=10): N-W, N-CHX, N-E64, ACD-W, ACD-CHX, ACD-E64, ERO-W, ERO-CHX, and ERO-E64. All specimens were restored with Adper Single Bond Universal/Filtek Z250. Beams (0.64 mm2) were obtained and subjected to the microtensile test (µTBS) in a universal testing machine at 0.5 mm/min. The failure mode of the interfaces was determined by optical microscopy (40× magnification). Data were statistically analyzed by three-way analysis of variance and Tukey tests (p<0.05). RESULTS: All individual factors (p<0.0001) and the interaction between substrate and treatment (p=0.0011) and between substrate and time (p=0.0003) were statistically significant. The caries substrate contributed negatively to bond strength. Chlorhexidine reduced bond strength for normal and eroded conditions. Only the normal substrate was negatively affected by time despite the pretreatment. CONCLUSIONS: The universal bonding system appears to be a promising bonding strategy for the maintenance of bond strength to affected dentin. E-64 did not affect bonding to the dentin in contrast to the use of chlorhexidine, which, when associated with the universal system, did affect the microtensile bond strength for artificial carious dentin.