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

Abstract

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.

Keywords: Acrylamide; Adhesive interface; Dental restorations; In-situ zymography; MMP-2 MMP-9; Methacrylate.

Figure 1:

Molecular structure, molecular weight and log P information for the different monomers (HEMA – methacrylate control, DMAM – monoacrylamide control, TMAAEA – triacrylamide, DEBAAP – diacrylamide) and inhibitors (NNGH, phenanthroline) used in this study.

Figure 2:

(A) Degradation of monomers incubated in an enzyme solution (2units CE + 2 units PCE) at 37 °C (n=3). The percentage of monomer remaining was calculated from NMR peak integration of the monomer and degradation product. DMAM and DEBAAP showed no degradation product, even after 30 days (curves are superimposed). (B) Comparison between degradation in enzyme solution and in PBS.

Figure 3:

Hydroxyproline concentration obtained from dentin extracts incubated with different monomers (HEMA, DMAM, TMAAEA, DEBAAP) and inhibitors (NNGH, phenanthroline). Ethanol and DMSO were used as the negative controls. Similar letters indicate statistical similarity (α=0.05).

Figure 4.

(A) Zymographic analysis of recombinant enzymes (MMP-2 and -9) and dentin powder treated with monomers, showing a faint activity of MMP-2 active-form (66-kDa) regardless of the group. A higher molecular weight band of approximately 130KDa was detected, except for the TMAAEA group, suggesting a complex of enzymes had formed. (B) Gelatin zymography of recombinant enzymes (MMP-2 and -9) treated with Ethanol (control) and TMAAEA for 1h, 37°C, before electrophoresis. The monomer TMAAEA was not able to reduce enzymatic activity. (C). Zymographic analysis of recombinant enzymes (MMP-2 and -9) and extracted enzymes (min). TMAAEA and NNGH were added to the activity buffer of their respective groups, after electrophoresis. Only NNGH showed complete inhibition of the activity of the recombinant and extracted enzymes. Original gel images (uncropped) shown in Figure S10.

Figure 5:

In situ zymography. (A) Dentin slice treated with water (control) or (B) NNGH, and incubated for 24h with quenched fluorescein-labeled gelatin. (C) Confocal images 40x in the green channel showing the adhesive-dentin interface of SB (Single bond), HEMA, DMAM, DEBAAP and TMAAEA with standard non-fluorescent gelatin (NC- negative control) and quenched fluorescein-labeled gelatin; bar = 10 μ; In red: D = Dentin; HL = Hybrid layer; A = Adhesive. Images processed with Imaris ^™ software (Zeiss).

Figure 6:

Enzyme activity for recombinant MMP-2/9 incubated with different monomers (HEMA, DMAM, TMAAEA, DEBAAP). Ethanol was used as the negative controls. All results were statistically similar (α=0.05).

Figure 7:

Shear storage modulus (G’) for demineralized dentin discs incubated with different monomers (HEMA, DMAM, TMAAEA, DEBAAP). Ethanol was used as the negative controls. Similar lower-case letters indicate no statistical difference (α=0.05).

Figure 8:

Absorption spectra in the mid-IR region obtained with ATR for dentin discs at baseline and after being incubated with different monomers (HEMA, DMAM, TMAAEA, DEBAAP). Ethanol was used as the negative control. Notable changes in peak position and area are shown in Table 1.