A novel dry-bonding approach to reduce collagen degradation and optimize resin-dentin interfaces

Abstract

In dentistry, the wet-bonding approach relies on water to maintain demineralized collagen expanded for proper resin infiltration; nevertheless, hydrolytic instability of the resin-dentin interface is inevitable with current bonding techniques. Considering dimethyl sulfoxide's (DMSO) ability to "biomodify" collagen and precipitate enzymes, the aim was to test whether the use of DMSO would permit adequate resin bonding to H₃PO₄-etched dehydrated dentin and assess its impact on collagen degradation by host-derived enzymes. Etched dentin surfaces from extracted sound human molars were randomly bonded in wet or dry conditions using aqueous or ethanolic DMSO solutions as pretreatments and bonding resins with or without DMSO. Bonded teeth were sectioned into resin-dentin slabs for confocal in situ zymography and beams for microtensile bond strength test. Demineralized powdered dentin was incubated in the tested DMSO -media and a hydroxyproline assay evaluated dissolution of collagen peptides. Zymography was performed on protein extracts obtained from dry and wet H₃PO₄-ecthed dentin powder treated with the DMSO- media. The correlative biochemical analysis demonstrated that reduction of water content during dentin hybridization by the innovative dry-bonding approaches with DMSO is effective to inactivate host-derived MMP-2 and MMP-9 and thus reduce collagen degradation while simultaneously optimizing resin-dentin bonding.

Figure 1

(A) Microtensile bond strength of wet- and extensively air-dried demineralized dentin bonded with SBMP using DMSO solutions as dentin pre-treatments or incorporated in the bonding agent (DMSO/SBMP). Pretreatment solutions consisted of DMSO dissolved in either water (DMSO/H2O) or ethanol (DMSO/EtOH). Heights of bars indicate the mean values (MPa) of 8 teeth per group (n = 8) and standard deviations. Columns identified by different capital letters represent significant differences according to Tukey Test (p < 0.05) for wet-dentin groups. Columns identified by different lowercase letters represent significant differences for dry-dentin groups. * represent significant differences between wet- and dry-dentin for each pre-treatment. (B) Graphical presentation of proportional prevalence of fracture modes for all experimental groups.

Figure 2

Representative CLSM scans (63x/1.4NA oil immersion objectives) for in situ zymography of wet- and extensively air-dried demineralized dentin bonded with SBMP. DMSO was solvated in water (DMSO/Water) or in ethanol (DMSO/Ethanol) and used as dentin pre-treatments or incorporated in the bonding agent (DMSO in Primer). Isolated red fluorescence signals, originated from Rhodamine B in adhesive, delineate the morphology of adhesive interface (A–H). Green fluorescence signals designate collagenolytic activity originated from quenched FITC-conjugated collagen breakdown by endogenous enzymes. (A1-H1) depict the localization of collagenolytic activity on the hybrid layer and surrounding areas. (A2-H2) show isolated FITC fluorescence revealing different levels of collagenolytic activity according to the different pretreatments and dentin conditions. While untreated control groups (A2 and E2) exhibited higher FTIC fluorescence signals, pretreatments with DMSO/H2O (C2 and G2) and DMSO/EtOH (D2 and H2) indicate reduced endogenous enzymatic activity especially on dry dentin (F2 and G2). Incorporation of DMSO in the bonding resin produced similar FITC fluorescence signals (D2) to control group in wet condition; however, a slight reduction of enzymatic activity in the hybrid layer was observed for the dry-bonding protocol (H2).

Figure 3

Hydroxyproline content derived from wet- and dry- demineralized dentin powder (n = 5) treated with DMSO after incubation for 7 days at 37 ºC. Control groups were incubated in distilled water (WET Control and DRY Control). SBMP also served as a control incubation solution (Wet SBMP and Dry SBMP). Experimental treatments consisted of DMSO solvated in water (DMSO/H2O), ethanol (DMSO/EtOH) or incorporated in the SBMP primer (DMSO/SBMP). Dissolved collagen from the demineralized dentin was expressed as μg hydroxyproline per mg dry mass of the baseline demineralized dentin powder. Groups with different upper ase letters were significantly different (p < 0.05) according to Dunn’s multiple comparison test.

Figure 4

Gelatin zymograms of wet (A) and dry (B) demineralized dentin powder treated with DMSO solvated in water (DMSO/H2O), ethanol (DMSO/EtOH) or incorporated in SBMP. Control groups consisted of untreated dentin powder (Control H2O), SBMP and ethanol. Pure MMP-2 and MMP-9 extracts from odontoblasts were used as specific enzyme molecular mass standards. Molecular masses, expressed in kDa, are reported in the standard lane (Std). The graph (C) shows band intensities for proMMP-9, actMMP-9 and actMMP-2 calculated according to the peak area method. Complete inhibition of actMMP-2 and MMP-9 activity was not observed for neither of the DMSO treatments. Nevertheless, fainted bands indicate partial inactivation of MMP-2 and -9.