**3. Discussion**

As demonstrated by MIC and MBC test results reported in Table 1, ABR-C and ABR-E displayed distinct antibacterial activities against cariogenic *S. mutans* cells. Not surprisingly, the MIC values of ABR-C and ABR-E were higher than that of benchmark wide-spectrum bactericidal chlorhexidine, which is also highly leachable and does not offer sustained antibacterial activity once leached out. Also as expected, the 8 μg/mL MBC of ABR-C and ABR-E is higher than CHX's MBC of 4 μg/mL, whereas no bactericidal activities were observed at all for SDR Resin and TEGDMA. It has been shown that, for an organic QAS compound, 10~14 carbon atoms for the N-alkyl group are associated with higher antimicrobial activity [43,44]. Another study also showed that imidazolium's N-alkyl's chain length can play a critical role in antitumor activity and cytotoxicity; C-12 was found exhibiting high antitumor activity against ~60 tumor cell lines as well as low cytotoxicity in most cases. Longer chain length could improve antitumor potency but also increase imidazolium's cytotoxicity [45]. As demonstrated in their chemical structures in Schemes 3 and 4, respectively, both ABR-C and ABR-E have a 12-carbon atom chain length on their N-alkyl group, which helps in explaining their identical MIC and MBC values.

The dentin mass loss results (Figure 1) indicate that the ABR resins, even at low concentrations, are effective against matrix metalloproteinases (MMPs) for minimizing the collagen degradation. MMPs belong to a larger group of proteases known as the metzincin superfamily. These collagenases are capable of degrading triple-helical fibrillary collagens, such as those in dentin, into distinctive fragments. More specifically, certain MMPs are considered to contribute to the gradual degradation of collagen fibrils in hybrid layers established during dentin bonding. A wide range of MMPs, including MMP-2, MMP-8, MMP-9, and MMP-20, have been detected in the human dentin matrix [46]. It is well reported that CHX has broad anti-MMP activity in addition to antimicrobial capability. However, the long-term *in vivo* anti-MMP activity of CHX may not be as effective, which has been attributed to possible leaching out of the CHX [47,48]. It has also been reported that cationic quaternary ammonium methacrylates may exhibit dentin MMP inhibition comparable with that of CHX but that higher concentrations were required [49].

The synthetic strategy in this investigation focused on covalently linking antibacterial compounds that contain functional groups with strong antibacterial activities to a variety of backbones and polymerizable groups compatible with conventional dental resin systems. The antibacterial capability of the active compound could be either enhanced or reduced by polymerization. This depends on how the compound eradicates bacteria, either by disrupting bacterial membrane or via depleting the bacterial food supply. In order for an antibacterial resin to be a viable option for industrial-scale production and application, there are several essential requirements that need to be fulfilled: (1) synthesis of ABR should be straightforward and not cost-prohibitive; ideally, well-established synthetic routes are preferred; (2) ABR should have good storage stability (sufficient shelf life); (3) end products that contain ABR should be able to maintain their antibacterial activities through recurrent bacterial challenges; (4) ideally, the antibacterial effect can be achieved with relatively small dosing and minimum bacterial resistance in the target pathogenic microorganisms; (5) no drastic compromises to the other vital properties should be introduced by ABR. As shown in Table 2, even at up to 12 wt % loading level, there are no significant decreases in flexural strength or flexural modulus with the incorporation of the

imidazole-based polymerizable antibacterial monomer ABR-C, as compared to control. The flexural strength of the resin mixture with 16 wt % antibacterial monomer showed lower flexural strength, but flexural modulus still retained 84% value as compared to control.

During this investigation, it was found that a group of polymethacrylate resins containing at least one imidazole moiety could be readily prepared via appropriate hybrid methacrylate–acrylate resins or polyacrylate resins with proper control of the conversion of the imidazole addition. This is an e ffective approach to incorporating an imidazole moiety into a polymerizable resin as novel acid-free functional resins. Moreover, such imidazole-containing polymerizable resins may be further chemically modified by reacting with a variety of halogenated alkyls to form polymerizable resins with ionic moiety of imidazolium, which results in a unique class of polymerizable ionic liquid resins.

Su fficient mechanical properties of the orthodontic bonding cement are important as the bending force and torque during teeth straightening and aligning could be quite significant. There is a slight reduction of flexural strength with a higher loading amount of ABR-C; however, flexural modulus can be maintained with up to 3 wt % of ABR-C loading (Table 3). A similar trend was also observed for compressive strength. Ambient light sensitivity (ALS) is another important property for light curable orthodontic cement or adhesive, as adequate ALS is needed for the bracket to be carefully placed and aligned at the correct position and angle, before it is light cured. As also exhibited in Table 3, the ALS values of ABR-C-containing orthodontic cements are all at 2 min or higher, which compared well with commercially available orthodontic cements, as shown in Figure 3.

Orthodontic cement with the capability to prevent the development of WSLs without compromising enamel bond strength would be desirable. Orthodontic treatments last an average of two to three years, during which time orthodontic cement must be capable of bonding to the enamel with high bond strength in order to resist masticatory loads. As demonstrated in Table 3, for experimental orthodontic cements incorporating 1–4 wt % ABR-C, there is no drastic compromise to the SBS to enamel except for 3 wt % ABR-C. More importantly, their SBS values are all comparable to those of the commercially available orthodontic cements products (Figure 2), with mean SBS ranging from 20.1 MPa to 36.0 MPa. However, this bond strength to enamel should not be too high either, to prevent damages to the enamel while removing the bracket.

The strong e ffectiveness in eradicating bacteria for the imidazolium-based polymerizable resins was further demonstrated by the formulated experimental orthodontic cements. As exhibited in Table 4, even at a low-level loading (1 wt %) of such imidazolium-based polymerizable monomer, a reduction of over 2 logs of microorganisms can be achieved by ABR-C. When the loading level of ABR-C was increased from 2 wt % to 4 wt %, reductions of around 5 logs or higher were observed. Moreover, with optimized compositions, not only can a highly e ffective antibacterial capability be achieved, but balanced mechanical properties can also be maintained (Table 3). The potent antibacterial property of this novel polymerizable imidazolium resin o ffers another crucial benefit—no severe cytotoxicity was introduced [50]. Conventional QAS-based polymerizable resins, on the other hand, could be less effective and a high dose loading (up to 30 wt %) is needed, which frequently leads to significant drops in mechanical properties and elevated cytotoxicity [51,52].

E ffective antimicrobial agents are expected to regulate the oral biofilm at levels compatible with good oral health while not sacrificing the beneficial properties of the resident oral microflora. Compounds comprising imidazolium moieties have exhibited various antibacterial, antioxidant, and antifungal properties; imidazolium salts' overall mechanism of antibacterial function is similar to that of QAS—mainly by disturbing the planktonic cell membrane [52]. Nonetheless, comprehensive studies by Koo et al. have shown that ABR-MC (ABR-modified composite) impaired biofilm initiation by disrupting bacterial accumulation, cell colonization, and subsequent biofilm development, if left unintervened [50]. The anti-biofilm properties of ABR-MC were assessed using an EPS-matrix producing *S. mutans* in an experimental biofilm model. Using high-resolution confocal fluorescence imaging and biophysical methods, they observed severely reduced biomass, remarkable disruption of bacterial accumulation, and defective 3D matrix structure on the surface of ABR-MC. Mechanism-wise, it was speculated that the distinct difference in the basicity between N-substitute imidazole (pKaH of 7.20 for 1-methylimidazole) and aliphatic tertiary amine (pKaH of 10.7 for triethylamine) might be the major contributor to the increased polarity and higher potential in its charged conjugated moiety (imidazolium). However, the exact mechanism contributing to the much more pronounced bactericidal effects as compared to conventional QAS compounds will require further investigation. Other areas of relevant investigation include the further study of the hydrolytical stability of these antibacterial monomers.

#### **4. Materials and Methods**
