The organic matrix in dentin is composed of type I collagen (90 %), within which endogenous enzymes such as matrix metalloproteinases (MMPs, e.g. gelatinases, collagenases and stromelysins) and cysteine cathepsins are entrapped [1]. These enzymes work synergistically to degrade the extracellular matrix (ECM), particularly collagen, thereby contributing to the deterioration of the dentin-resin interface. Both enzyme families are expressed in mature human odontoblasts and, although rarely co-expressed in other tissues, they appear to participate in physio-pathological processes in dentin, especially under acidic and inflammatory conditions [2]. MMPs are a group of zinc-dependent endopeptidases found in both sound and caries-affected dentin and are particularly relevant in hybrid layer degradation. Notably, MMPs such as MMP-2, MMP-8, and MMP-9, are activated in the acidic environment of dentin caries or during the bonding procedures, but their activity is most effective at near-neutral pH, as typically occurs after adhesive application [3], [4]. This shift in pH following bonding promotes collagen degradation within the hybrid layer. Given this mechanism, the inhibition of MMPs and cathepsins has emerged as a critical strategy to enhance resin-dentin bond durability and prolong the clinical longevity of adhesive restorations [5], [6].

Chlorhexidine (CHX) has been considered a standard for MMP inhibition in dentin [7]. Combined with its broad-spectrum antimicrobial properties, this makes it an effective agent for preserving the integrity of the collagen matrix [8]. CHX functions by chelating metal ions such as zinc and calcium that are crucial for the catalytic process of MMPs, thereby preventing the enzymatic breakdown of collagen [9], [10]. However, despite its efficacy, CHX has limitations, including its high water solubility, which facilitates passive leaching from the bonded interface, and therefore leads to burst-release rather than sustained inhibition over time [7], [11], [12]. Moreover, CHX’s ability to chelate calcium may interfere with calcium-dependent functional monomers such as 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP), potentially compromising chemical bonding to hydroxyapatite. This interaction has prompted the selection of experimental adhesive formulations devoid of 10-MDP, such as those based on 2-Hydroxyethyl Methacrylate (HEMA) and quaternary ammonium methacrylates (QAMs), which offer an alternative strategy for both adhesion and enzymatic inhibition without calcium-binding competition [13].

Even though the inhibitory effects of quaternary ammonium methacrylates (QAMs) on MMPs have been recognized for over a decade, with early studies demonstrating reduced enzymatic activity and improved dentin stability following QAM application, very few studies have tested QAMs as enzymatic inhibitors under clinically relevant conditions [14], [15], [16]. Most existing studies have been limited to models using purified enzymes, which do not fully replicate the structural complexity and enzymatic environment of bonded dentin. Moreover, the mechanism by which quaternary ammonium methacrylate (QAM)-based adhesives inhibit matrix metalloproteinases (MMPs) remains unclear and warrants further investigation. While QAMs are known to interact with the negatively charged collagen matrix and MMPs via their positive charge, it is not yet established whether this involves binding to the enzyme's active site, immobilization of the enzyme, or allosteric inhibition [17], [18]. Unlike CHX, QAMs can be copolymerized within adhesive formulations, potentially offering a more stable and prolonged inhibitory effect on MMPs while also providing antimicrobial benefits [14], [19], [20]. While both these effects have been explored separately, studies evaluating the combined effects of QAM on bacteria and enzyme inhibition are less explored. In addition, there are no studies evaluating these effects under physiologically-relevant testing conditions.

To evaluate the effectiveness of a QAM-based adhesive for MMP inhibition, this study employed a bioreactor system designed to simulate a physiologically relevant environment combining simultaneous mechanical and bacterial challenges [21]. The bioreactor allows for continuous fluid flow and dynamic loading conditions that closely mimic the oral cavity, providing a more accurate assessment of adhesive performance compared to static in vitro models. Therefore, this study aims to evaluate the potential inhibitory effect of a QAM-based dental adhesive on MMP activity in dentin and compare its efficacy to that of a control adhesive (without MMP inhibitors) and a 2 % CHX-based adhesive. The tested hypotheses are: 1) The QAM-based adhesive will improve the stability of the collagen matrix compared to the control adhesive or the 2 % CHX-based adhesive, and 2) The QAM-based adhesive will improve bond interface integrity under mechanical or bacterial challenges compared to the control adhesive or the 2 % CHX-based adhesive.