**2. Results**

By coupling with different mono-ols, diols, polyols (such as triols), monoamines, diamines, and/or polyamines, a range of methacrylate resins with (poly)imidazole moieties were prepared. During the processes of synthesizing imidazolium-based polymerizable resins from imidazole-containing monomers, a facile process based on imidazole and (meth)acrylated resins was developed. As demonstrated in Schemes 1 and 2, a variety of imidazole-containing polymerizable monomers were able to be prepared accordingly.

**Scheme 1.** Isosorbide-based bisimidazole–dimethacrylate resins.

The polymerizable imidazole-based resins were further converted into polymerizable imidazolium-based monomers by reacting them with a variety of halogenated alkyls. A variety of polymerizable imidazolium-based antibacterial resins (ABRs) were successfully synthesized as exhibited in Schemes 3 and 4. The primary evaluations of these novel ABRs' antibacterial activity include the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against cariogenic *S. mutans* bacterial strain UA159.

**Scheme 2.** Various polymerizable mono-imidazole monomers synthesized.

**Scheme 3.** Polymerizable alkylimidazoliumbromide–dimethacrylate monomer, antibacterial resin C (ABR-C).

**Scheme 4.** Polymerizable alkylimidazoliumbromide–methacrylate monomer ABR-E.

Table 1 presents the results of MIC and MBC for representative ABR resins and SDR Resin (a proprietary dimethacrylate monomer, without imidazole moiety, synthesized by Dentsply Sirona) in comparison to frequently used controls, namely chlorhexidine (CHX) as wide-spectrum antimicrobial agen<sup>t</sup> and triethylene glycol dimethacrylate (TEGDMA). TEGDMA was chosen as control monomer because it is widely used in the composition of resins for dental-related applications, including but not limited to restorative, adhesive, and orthodontic compositions. In addition, it was not known to have antibacterial potencies against common cariogenic oral bacteria strains. Furthermore, by using a modified JIS Z 2801 test method, a preliminary antibacterial test against *S. mutans* strain UA159 was used to evaluate the antibacterial e ffectiveness for such formulated compositions. After 24 h contact, a bacterial reduction of 99.88% was exhibited by test sample containing ABR-E (Scheme 4), as compared to control. This indicated that imidazolium-based polymerizable resin could be highly effective in suppressing the growth of bacteria such as *S. mutans*.

**Table 1.** Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of novel antibacterial resins ABR-E and ABR-C against *Streptococcus mutans* strain UA159, as compared to SDR Resin, triethylene glycol dimethacrylate (TEGDMA), and chlorhexidine.


The loss of dry mass from completely demineralized dentin beams (2 mm × 1 mm × 6 mm) in bu ffer solutions pre-dipped into ABR-C and ABR-E resins, versus in control bu ffer (artificial saliva) without pre-dip, was studied. The amount of soluble collagen in these demineralized dentin beams was measured after 7 days of incubation at 37 ◦C. As Figure 1 indicates, dentin beams without pre-dip lost about 15.5% of their dry mass due to degradations of endogenous matrix metalloproteinases (MMPs) and cathepsins. When the dentin beams were pre-dipped in 1–4% of imidazolium-based ABR resins (ABR-C, ABR-E) for 30 s and then dropped in incubation medium, the dentin beams only lost about 3.5–5% of their dry mass. Furthermore, the hydroxyproline (HYP), an amino acid unique to collagen, content in the medium was also measured. The collagen peptide fragments that were solubilized by the endogenous proteases of the dentin matrix were hydrolyzed to amino acids in 6N HCl and then analyzed for hydroxyproline. In the control group, the proteases released about 10.5 μg HYP/mg dry dentin after 7 days; beams pre-dipped in 1% or 4% of ABR-C released less than half as much hydroxyproline and the beam treated with ABR-E yielded a similar result.

As the designed imidazolium-based resin still comprises a methacrylate functional group, no compatibility or copolymerization issues were found when mixed with typical dimethacrylate-type dental resins, such as 1,6-bis[methacryloyloxyethoxycarbonylamino]-2,4,4-trimethylhexane (UDMA), ethoxylated bisphenol A dimethacrylate (EBPADMA), and TEGDMA. Even with 16 wt % loading of ABR-C into the control orthodontic resin, no mixability issue was observed and the resulting resin mixture was found uniform. The 3-point bending flexural strength and modulus of unfilled orthodontic

resins containing 4~16 wt% ABR-C, along with control, were measured following ISO-4049 method and the results are presented in Table 2.

**Figure 1.** Effect of pre-dipping in antibacterial resins ABR-C and ABR-E on loss of dry mass of demineralized dentin beams after incubation for 7 days at 37 ◦C.



Within the same row, means that do not share the same superscript(s) are significantly different (α = 0.05).

The 75 wt % filled experimental orthodontic cements incorporating various concentrations of antibacterial monomer ABR-C were also studied. The current investigation examined flexural and compressive strengths, flexural modulus, ambient light sensitivity, and notched-edge shear bond strength (NE-SBS) to bovine enamel. Results along with statistical analysis are presented in Table 3. For reference, the shear bond strength to bovine enamel, using the same NE-SBS method, and ambient light sensitivity of five commercially available orthodontic bonding cements were also evaluated, as presented in Figures 2 and 3, respectively.

Antibacterial testing was also conducted at an independent and good laboratory practice (GLP) complied testing institution. As shown in Table 4, for orthodontic cement paste formulations that incorporated imidazolium-based dimethacrylate antibacterial monomer (ABR-C), the ISO-22196 antimicrobial testing results against ATCC 6538 showed significant levels of antibacterial effects—up to over 5 logs of microorganism reduction—when compared with control. Such highly effective bactericidal effects for the imidazolium-based polymerizable resins were promising due to a relatively low-level loading and significantly reduced probability of leaching out owing to the dimethacrylate functional groups.



Within the same row, means that do not share the same superscript(s) are significantly different (α = 0.05).

**Figure 2.** Notched-edge shear bond strength to bovine enamel of commercial orthodontic cements. Values that do not share the same superscript(s) are significantly different (α = 0.05).

**Figure 3.** Ambient light sensitivity of commercial orthodontic cements. Values that do not share the same superscript are significantly different (α = 0.05).


**Table 4.** ISO-22196 antimicrobial test of orthodontic cements (filled with 75 wt % inorganic filler) that contain various concentrations of imidazolium-based polymerizable antibacterial monomer using *S. aureus* 6538. The limit of detection for this assay is 5 CFU/carrier.
