*4.1. Materials*

Antibacterial resins (ABR-C and ABR-E) were prepared in house; the detailed preparation route can be found in a previous paper [50]. SDR Resin and adhesion-promoting resin (APR, Dentsply proprietary resin) were supplied by Dentsply Sirona's internal production (Milford, DE, USA). Other conventional dental resins and ingredients used in formulations were purchased from commercial sources, including 1,6-bis [methacryloyloxyethoxycarbonylamino]-2,4,4-trimethylhexane (UDMA), ethoxylated bisphenol A dimethacrylate (EBPADMA), trimethylolpropane trimethacrylate (TMPTMA), camphorquinone (CQ), dimethylaminobenzonitrile (DMABN), diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO), and butylated hydroxytoluene (BHT). Inorganic fillers were milled and silanated in house, including barium fluoroalumino borosilicate glass (BFBG), bariumalumino borosilicate glass (BABG), and AEROSIL fumed silica OX-50 (silanated OX-50). All fillers were surface treated by γ-methacryloxypropyl-trimethoxysilane.

Commercially available orthodontic cements studied include the following: Prime Master (Sun Medical Co., Shiga, Japan), Light Bond (Reliance Orthodontic Products, Alsip, IL, USA), Transbond XT (3M Unitek, Monrovia, CA, USA), NeoBond (Dentsply Sirona, Milford, DE, USA), and Grengloo (Ormco, Glendora, CA, USA).

Preparation of experimental orthodontic resin and cement. Experimental dental resin mixture with various concentrations of polymerizable antibacterial resin (0 (control), 4, 8, 12, 16 wt %) was mixed with other resins, namely UDMA (28~34 wt %), EBPADMA (20~26 wt %), APR (28~32 wt %), TMPTMA (6~8 wt %), CQ (~0.1 wt %), DMABN (~0.2 wt %), and BHT (~0.07 wt %), to a uniform resin blend at 55 ◦C using an overhead mechanical stirrer (Fisher Scientific, Pittsburgh, PA, USA). Experimental orthodontic cements were compounded with 75 wt % of inorganic filler mixture using a Ross double planetary mixer at 55 ◦C to a uniform paste (Ross Mixer, Charles Ross & Son Company, Hauppauge, NY, USA). The inorganic filler used in this study consists of three types of fillers in the filler blend: ~33 wt % BABG, ~65 wt % BFBG-2, and ~2 wt % silanated OX-50.

#### *4.2. Evaluation Methods*

MIC and MBC Testing. Three monomers were tested against controls, i.e., ABR-E, ABR-C, and SDR Resin (a proprietary dimethacrylate monomer, without imidazole moiety, synthesized by Dentsply Sirona). The monomers were diluted in UltraPure distilled water (Gibco, Gaithersburg, MD, USA) before use. Different concentrations of the three tested monomers, along with two controls, i.e., wide-spectrum antimicrobial agen<sup>t</sup> chlorhexidine (CHX) and conventional monomer TEGDMA, were incubated statically with the cariogenic organism *S. mutans* strain UA159 using mid-exponential cells for 48 h at 37 ◦C in the presence of 5% CO2. A fixed initial inoculum size of 9 × 10<sup>7</sup> CFU/mL of bacterial cells was inoculated per well and 2-fold dilutions of the experimental monomers, CHX, and TEGDMA were added. The following concentrations were tested: 0 to 125 μg/mL for CHX; 0 to 0.1% for the experimental monomers ABR-E, ABR-C, SDR Resin, and the control monomer TEGDMA. The MIC was determined as the lowest test concentration needed to ensure that culture did not grow over 10% of the relative cell, as determined by visual inspection of the growth inhibition of each well compared to that of the control well (without test compounds). For the MBC test, cells were serially diluted and

spot-plated on agar plates; MBC values were determined by using the actual reduction of the bacterial viable cell counts. The MBC was determined as the lowest concentration that allows less than 0.1% of the original inoculum to survive.

Dentin Mass Loss. Completely demineralized dentin beams (2 mm × 1 mm × 6 mm) were prepared. Dentin beams were obtained from extracted molars after removing the enamel and superficial dentin. The beams were then submerged in 10% phosphoric acid for 18 h at 25 ◦C to completely demineralize the dentin. They were incubated in a control bu ffer (artificial saliva) to see how much of the insoluble collagen in demineralized dentin could be solubilized in 7 days of incubation at 37 ◦C. 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 for 30 s, 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.

Flexural Strength. Specimens were prepared based on ISO 4049:2019. Samples were carefully injected into 25 mm × 2 mm × 2 mm stainless steel molds, then covered by Mylar film and cured with a Spectrum 800 ® (Dentsply Sirona, Milford, DE, USA ) halogen lamp uniformly across the entire length of the specimen, using an irradiance of 850 mW/cm<sup>2</sup> for 5 × 20 s. After curing, specimens were immersed in DI water for 24 h at 37 ◦C prior to the 3-pt bending flexural test. Flexural test was performed on an Instron Universal Tester (Model 4400R, Norwood, MA, USA countr using a crosshead speed of 0.75 mm/min. Six specimens per sample set were prepared and tested.

Compressive Strength. Samples were carefully injected into ∅4 mm × 7 mm stainless steel molds, sandwiched between two Mylar films. Light curing of the specimen was carried out with a Spectrum 800 ® (Dentsply Sirona) halogen lamp at an irradiance of 850 mW/cm<sup>2</sup> for 20 s on both ends. After curing, specimens were immersed in DI water for 24 h at 37 ◦C, before being polished to ∅4 mm × 6 mm specimen size with 600 grit sand paper. The compression test was performed on an Instron Universal Tester (Model 4400R) using a crosshead speed of 5 mm/min. Six specimens per sample set were prepared and tested.

Ambient Light Sensitivity (ALS). ALS was tested on a Suntest unit (model CPS 56007014, Heraeus Noblelight, Buford, GA, USA ) with a UV filter that was calibrated to provide illuminance of 8000 ± 1000 lux tested by a Lux Meter HD400 (Extech, Nashua, NH, USA ). A spheroidal mass of approximately 0.03 gm of the material was weighed to be tested on a glass microscope slide, and the slide was positioned with a matte black cover on top of the cell with a UV filter in place. The Suntest light was turned on, and one should wait at least 10 s until the light output has stabilized. The matte black cover was removed and the timer was started simultaneously. After exposure, the slide with the sample was removed and the second microscope slide was immediately pressed against the material with a slight shearing action to produce a thin layer. The material was visually inspected to see whether it was physically homogeneous. If the material has begun to set, clefts and voids appear in the specimen when the thin layer is being produced. The time was recorded when the sample started to set and appeared physically non-homogeneous. In all, 3 runs were conducted for each sample.

Notched-Edge Shear Bond Strength. Caries-free and freshly extracted bovine incisors were carefully sectioned longitudinally through the distal, occlusal, and mesial surfaces using a precision low speed saw (TechCut 4 ™, Allied High Tech Products, Rancho Dominguez, CA, USA). The sectioned bovine incisors, with buccal surface exposed, were then mounted in a cylindrical block using cold-cure acrylics. The exposed surface was then coarse ground on a model trimmer until a flat enamel surface was exposed, followed by wet ground on a grinding wheel under running water using 120-grit and 320-grit SiC sanding papers until the surface was even and smooth when visually inspected. Enamel was acid etched using 34% phosphoric etching gel for 15 s, then thoroughly rinsed for 20 s. The notched-edge bonding jig contained a cylindrical plastic mold, resulting in samples with a defined bonding area (diameter of 2.38 mm). The antibacterial orthodontic cement restorative composite was then carefully placed into the center of the mold and good contact with the substrate was ensured. After light curing with a Spectrum 800 ® (Dentsply Sirona) halogen lamp at 850 mW/cm<sup>2</sup> for 20 s, the specimen was then carefully removed from the mold. Specimens were stored in DI water for 24 h at 37 ◦C prior to SBS testing. The SBS test was performed on an Instron Universal Tester (Model 4400R) using a crosshead speed of 1 mm/min. Seven specimens per sample set were prepared and tested.

JIS Z 2801. This test was conducted at Antimicrobial Test Laboratories (ATL, Round Rock, TX, USA), an independent and GLP complied testing institution. JIS Z 2801 was adopted as ISO standard (ISO-22196). This test is designed to quantitatively test the ability of hard surfaces to inhibit the growth of microorganisms over a 24-h period of contact. The test microorganism selected for this test was *Streptococcus mutans* (*S mutans*, ATCC ® 25175). The following protocol was used by ATL: (1) test microorganism *S mutans*, ATCC ® 25175 was prepared by growth in tryptic soy broth for 24 h; (2) the suspension of the test microorganism was standardized by dilution in a Dey-Engley neutralizing broth (D/E Broth) at 0.9 mL blanks; (3) control and test surfaces were inoculated with microorganisms, and then the microbial inoculum was covered with a thin sterile film; (4) microbial concentrations were determined at "time zero" by elution followed by dilution and plating to tryptic soy agar (Difco); (5) a control was run to verify that the neutralization/elution method e ffectively neutralized the antimicrobial agen<sup>t</sup> in the antimicrobial surface being tested; (6) the inoculated covered control and antimicrobial test surfaces were allowed to incubate undisturbed in a humid environment for 24 h at 36 ± 1 ◦C; and (7) after incubation, microbial concentrations were determined and the reduction of microorganisms relative to the control surface was calculated as follows:

$$\text{R (Average Log reduction)} = \text{Log (B/C)},\tag{1}$$

where

B = Average number of viable cells on the control pieces after 24 h.

C = Average number of viable cells on the test pieces after 24 h.

ISO-22196 Antimicrobial Test. This test was conducted at Antimicrobial Test Laboratories (ATL, Round Rock, TX, USA), an independent and GLP complied testing institution. ISO method 22196 is a quantitative test designed to assess the performance of materials' antimicrobial capabilities on hard non-porous surfaces. The method can be conducted using contact times ranging from 10 minutes up to 24 h. For the ISO 22196 test, non-antimicrobial control surfaces are used as the baseline for calculations of microbial reduction. The test microorganism selected for this test was *Staphylococcus aureus* 6538 (*S. aureus* 6538). This bacterium is a Gram-positive, spherical-shaped, facultative anaerobe. *Staphylococcus* species are known to demonstrate resistance to antibiotics such as methicillin and is commonly used in standard test methods as a model for Gram-positive bacteria. *S. aureus* pathogenicity can range from commensal skin colonization to more severe diseases such as pneumonia and toxic shock syndrome (TSS). The following protocol was used by ATL: (1) *S. aureus* 6538 prepared by growth in tryptic soy broth for 18 h and the suspension of the test microorganism was standardized by dilution in broth at 1:500 by volume; (2) control and test surfaces were inoculated with microorganisms, and then the microbial inoculum was covered with a sterile thin film; (3) microbial concentrations were determined at "time zero" by elution, followed by dilution and plating to tryptic soy agar; (4) a control was run to verify that the neutralization/elution method e ffectively neutralized the antimicrobial agen<sup>t</sup> in the antimicrobial surface being tested; (5) the inoculated covered control and test surfaces were allowed to incubate undisturbed in a humid environment at 36 ± 1 ◦C for 24 h; and (6) after incubation, microbial concentrations were determined and the reduction of microorganisms relative to the control surface was calculated as follows:

$$Percent\text{ }Reduction = \left(\frac{B-A}{B}\right) \times 100$$

$$Log\_{10}\text{ }Reduction = Log\left(\frac{B}{A}\right)$$

where B = Number of viable test microorganisms on the control carriers after the contact time.

A = Number of viable test microorganisms on the test carriers after the contact time.

Statistical Analysis. Statistical analysis was conducted with Minitab 17 Statistical Software (Minitab ®, State College, PA, USA). Normality of data was checked using the Anderson–Darling test. Mechanical properties and shear bond strength data were analyzed with one-way analysis of variance (ANOVA) and Tukey's test. Significance levels of 0.05 were used for ANOVA and Tukey's tests.
