Nanostructural analysis of the adhesive interface in dentistry

The subject of this thesis is the stability of the adhesive interface in dentistry. Success in adhesive dentistry means long lasting restorations. However, there is substantial evidence that this ideal objective is not achieved. Current research in this field aims at increasing the resin-dentin bond durability. This doctoral research examines the fundamental processes responsible for the aging mechanisms involved in the degradation of resin-bonded interfaces, as well as some potential approaches to prevent and counteract this degradation. Resin-dentin bond degradation is a complex process that is not completely understood, involving the hydrolysis of both the resin and the collagen component of the hybrid layer. The hydrophilic and acidic characteristics of current dentin adhesives have made hybrid layers highly prone to water sorption, which causes polymer degradation and results in decreased resin-dentin bond strength over time. These unstable polymers inside the hybrid layer may result in an incomplete encapsulation of collagen fibers, which become vulnerable to mechanical and hydrolytical fatigue, as well as degradation by host-derived proteases with collagenolytic activity. These enzymes, such as matrix metalloproteinases (MMPs) and cysteine cathepsins, have a crucial role in the degradation of type I collagen, the organic component of the hybrid layer. The first part of this thesis aims to review the current knowledge regarding adhesion to the tooth substrate (Chapter 1), focusing on the fundamental processes that are responsible for the degradation of the adhesive interface (Chapter 2). Since the permeability of adhesives to water is particularly evident in simplified adhesive formulations, the research activity was focused on self-etch and universal adhesive systems' behavior. Thus, the research study reported in Chapter 3 showed that the bond strength and nanoleakage expression of two-step and one-step self-etch tested bonding systems were affected by storage for 6 month and 1 year in artificial saliva. Although it is generally accepted that the permeability of adhesives to water is particularly evident in simplified adhesive formulations, the stability over time was not related to the number of steps of bonding systems, but to their chemical formulations. The performance of a new universal (or multi-mode) adhesive system through storage in artificial saliva was also investigated. The original results presented in Chapter 4 found that improved bonding effectiveness of the tested universal adhesive system on dentin was obtained when the adhesive was applied with the self-etch approach. Indeed, the etch-and-rinse approaches tested (both on wet and dry dentin) resulted in immediate bond strength comparable to the self-etch mode but expedited long-term aging resulted in reduced bond strength and increased nanoleakage expression, irrespective of dentin wetness. Moreover, the results of the zymographic analysis showed evident changes in dentinal MMP-2 and -9 enzyme activities after the application of the tested adhesives, revealing differences in the extent of enzyme activation. These findings exhibit that the activation of endogenous MMPs is not related to the adhesive system or the strategy employed. Thus, regardless of the approach and the material used in bonding procedures, a stable and durable bond is not achieved. Therefore, experimental strategies that aim to enhance the adhesive interface, particularly improving the durability of the resin-dentin bond strength by inhibiting intrinsic collagenolytic activity and increasing the resistance of dentin collagen matrix to enzymatic degradation are needed. The last part of the thesis is focused on both the strategies to inhibit the proteolytic and collagenolytic activity of the endogenous proteases and the methods to increase the mechanical strength of collagen network and its resistance to enzymatic degradation (Chapter 5). Chlorhexidine (CHX) has been used as a non-specific MMP inhibitor to prevent degradation of hybrid layers. However, CHX is water-soluble and may leach out of hybrid layers, compromising its long-term anti-MMP effectiveness. An entirely different approach is to treat the acid-etched dentin containing activated matrix-bound MMPs with cross-linking agents that inactivate the catalytic site of proteases. In particular, the ability of a cross-linker agent, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), to prevent collagen degradation was evaluated under occlusal cycle loading. Previous research successfully utilized EDC to increase the durability of resin-dentin bonds by increasing the mechanical properties of the collagen matrix; however, the 1 to 4 hrs required for that procedure was clinically unacceptable. For this reason, the purpose of the last part of the research, presented in Chapter 6, was to evaluate the ability of 0.5 M EDC short-time (1 min) pre-treatment to improve the stability of demineralized dentin collagen matrices by quantifying the release of telopeptide fragments over time. The results showed that EDC application for 1 min may be a clinically relevant and effective means for stabilizing the collagen network not only by strengthening the fibrils, but also by reducing the enzymatic degradation rate. Thus, dentin collagen reinforcement and strengthening through EDC cross-linking might be of importance to improve the bond strength and structural integrity of the resin-dentin interface over time against the enzymatic and hydrolytic degradation.