**3. Discussion**

We considered cytotoxicity of any AM monomer to be a major determinant of whether the materials that incorporate the monomer in their resin phase are worthy of further study. We believe that direct contact cellular testing of the new agen<sup>t</sup> must be done at biologically relevant eluent concentrations. In this study, we employed conditions that reflect the accelerated leachability of UPH resins and included AMsil concentrations that significantly exceed the upper thresholds established experimentally in the previous work [45]. The direct toxicity of AMsils towards CCL1 cells and/or HGFs was demonstrated to be marginal or undetectable, except at the higher concentrations of monomers tested. These high AMsil levels correspond to unrealistically high levels of the unreacted monomers and are highly unlikely to ever be registered clinically. Our cytotoxicity results support the basic hypothesis of the study and sugges<sup>t</sup> that, from a biotoxicity viewpoint, AMsils can be safely utilized in design of AM new materials. Leachability studies of AM–UPE formulations employing high-performance liquid chromatography are currently underway in our laboratory, and are expected to confirm the conclusions derived from the tests based on the accelerated UPH leachability study.

Upon introduction of AMsil into the UPE resin, a shift towards lower CA values, consistent with the moderate increase in the overall hydrophilicity, was seen in all AMsil–UPE formulations. The detected range of CAs in AMsil–UPE resins (37.4–53.3◦) correlates very well with the range of CAs typical for the commercial resin composites (37.4–53.3◦) [46]. The lowest CAs detected in 10% AMsil2–UPE formulation make this resin a good candidate for the incorporation of the ACP filler in future design of AMRE composites. ACP-filled composite requires sufficient water absorption to initiate water-catalyzed transformation of ACP during which the remineralizing calcium and phosphate ions are released, by diffusion, into surrounding mineral-deficient tooth structures. There, they regenerate these mineral-depleted structures via redeposition of hydroxyapatite [47]. Taken together, the enhanced wettability should ease a diffusion of water into AMRE composite and result in the subsequent release of calcium and phosphate ions from the composite needed for demineralization prevention and/or active remineralization at the restoration site.

The range of DVC values attained in AMsil–UPE copolymers (60.7–86.7%), dependent on both the monomer type and its quantity in the resin, were higher or equal to the DVC reported for 2,2-bis[p-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bis-GMA)-based resins/composites with incorporated QA ionic dimethacrylate (67.9–70.7%) [25]. AMsil2 copolymers reached significantly higher DVC values (75.2–86.7%) compared to their AMsil1 counterparts (60.7–70.9%). In AMsil2–UPE

formulations, the inclusion of AM monomer apparently did not a ffect the high levels of DVC typically seen in UDMA-based resins [35,36]. This phenomenon has been attributed to the chain transfer reactions caused by UDMA's –NH– groups, resulting in increased mobility of the resin network's radical sites [48]. The DVCs attained in AMsil2–UPE copolymers sugges<sup>t</sup> limited mobility of cross-linked polymer matrix, thus reducing the likelihood of unreacted monomers leaching out to a minimum. The observed DVC decrease in AMsil1–UPE formulations compared to those of AMsil2–UPE is ye<sup>t</sup> to be explained.

The results of FS and E tests indicated a reduction of the copolymers' mechanical properties in going from the UPE control to 10% AMsil–UPE to 20% AMsil–UPE. The reduction was far more pronounced in AMsil1 series (50%) compared to the AMsil2 series (15%). This overall reduction in mechanical properties, particularly in AMsil2–UPE copolymers, should not disqualify this monomer from further exploration, as AM agen<sup>t</sup> in multifunctional AMRE composites. To compensate for the reduction in the mechanical properties, incorporation into composites of the reinforcing fillers in addition to ACP should be considered.

AMsils integrated into UPE resin were e ffective in reducing (*p* ≤ 0.002) *S. mutans* biofilms. Compared to the commercial control, *P. gingivalis* biofilm biomass was notably lower (71–85%) on AMsils–UPE copolymer disks, however, not statistically di fferent. As Gram-positive bacteria have peptidoglycan with long anionic polymers, called teichoic acids [49] (i.e., yielding a higher cell surface net negative charge than Gram-negative organisms), one could anticipate *S. mutans* to be more susceptible to AMsils. Notwithstanding, quaternary ammonium compound AM functionality can also be a ffected by the type of counter-ion [50], pendant active groups [51], molecular weight, and length of the alkyl chains [52].

AMsils show promise, as *S. mutans* planktonic and biofilm forms were reduced. Nonetheless, this reduction was only significant (*p* ≤ 0.002) for biofilms. When comparing planktonic and biofilm responses, similar trends were observed with an endodontic sealer. For example, bacteria tested with a resin-based root canal sealer did not statistically reduce the planktonic forms, while notably decreasing bacteria in monospecies biofilms [53]. These authors attributed this to the release of substances during the setting process. This attribute is unlikely applicable to our material, as DVC was high and aqueous extraction was conducted for 72 h. In another report, *Staphylococcus aureus* biofilms were demonstrated to be more susceptible to killing than the planktonic form of the same strain [54].

Although AM functionality is observed, we believe that the full potential of the AMsil monomers has not ye<sup>t</sup> been realized. As reported for other quaternary ammonium compounds [47], the current AMsil–UPE copolymer formulations are likely to have N<sup>+</sup> charges randomly distributed throughout the material. As the mechanism of AM action is contingent on contact, it would be advantageous to develop fabrication methods that would favor charge density at the materials surface. Further, others have demonstrated that proteins can diminish the AM capability of quaternary ammonium methacrylates (reviewed by [6]). Currently, there is insu fficient information concerning the interaction of proteins with quaternary ammonium methacrylates. Elucidation of the protein–material interactions would yield valuable information to develop strategies to maximize AM e fficacy of materials with a charge-based AM mechanism of action.

In conclusion, our novel AM dental monomers (AMsil1 and AMsil2) exhibited minimal or no toxicity upon direct contact with biologically relevant concentrations, while reducing *S. mutans* and *P. gingivalis* biofilm forms. AMsils made the UDMA/PEG-U/EHMA resin more hydrophilic. This would be an advantageous feature in AMRE composites that require water to induce their remineralizing e ffects. At 10 mass % level of AMsil monomer, DVC of the ensuing AMsil–UPE copolymers was only marginally lower than in UPE control and still exceeded DVCs typically seen in the commercial composites based on bis-GMA/triethyleneglycol dimethacryalate (TEGDMA) resins. The mechanical properties of AMsil–UPE copolymers were reduced (11–57%) compared with the UPE control. The extent of reduction depended on both the type and the concentration of AMsil monomer in the resin. This finding should

not disqualify the AMsil–UPE resins from use in AMRE composites intended for Class V restorations where the mechanical stability is not a critical factor.

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

## *4.1. Monomer Synthesis*

The synthesis and validation protocols for the AMsils are described in detail by Okeke et al. (2019) [40]. In brief, AMsil1 and AMsil2 were synthesized at 50–55 ◦C by reacting equimolar amounts of tertiary amine, DMAEMA, with IPTMS and BrUDTMS, respectively, in the presence of chloroform and butylated hydroxytoluene. DMAEMA, IPTMS, and butylated hydroxytoluene were purchased from Sigma, St. Louis, MO, USA. BrUDTMS was purchased from Gelest Inc., Morrisville, PA, USA. Reactants and solvents (chloroform, diethyl ether, hexane; Sigma, St. Louis, MO, USA) used during synthesis and the subsequent purification were used as received, without further purification. The reaction yields were 94.8% and 36.0% for AMsil1 and AMsil2, respectively. Due to the generally hygroscopic nature of QA monomers, the AMsils were stored under vacuum (25 mm Hg) before being used for resin formulation and/or copolymer disk specimen preparation.

## *4.2. Structural Verification*

Purified monomers were characterized by 1H and 13C NMR spectroscopy as described [40]. Briefly, spectra were obtained using a Bruker Advance II (600 MHz) spectrometer equipped with a Broadband Observe room temperature probe (Bruker, Corp., Billerica, MA, USA). Monomers were dissolved in deuterated dimethyl sulfoxide containing tetramethylsilane.

#### *4.3. Experimental Resin Formulation*

UPE resin was formulated from the commercially available monomers UDMA, PEG-U, and EHMA at 2.8:1.0:1.7 mass ratio (corresponds to the average mass ratio of the ternary UDMA-based formulations explored by our group so far). A conventional visible light initiator system comprised of camphorquinone and ethyl-4- *<sup>N</sup>*,*<sup>N</sup>*-dimethylamino benzoate (4EDMAB) was introduced to the resin at concentrations of 0.2 mass % camphorquinone and 0.8 mass % 4EDMAB. AMsils were blended into UPE resin to yield (AMsil1 or AMsil2)–UPE resin with 10 or 20 mass % of AM component. Addition of AMsil1 or AMsil2 to the light-activated UPE resin took place in the absence of blue light. The rationale for the chosen levels of AM monomers is based on the previously reported AM activities of similar QA methacrylates in camphorquinone /4EDMAB-activated bis-GMA/TEGDMA resins [25]. Once all components were introduced, the mixture was stirred magnetically (38 rad/s) at 22 ◦C until a uniform consistency was achieved. CLEARFIL SE Protect BOND (Kurary America, Inc., New York, NY, USA) was used as a comparative commercial AM material. This resin was prepared using equal quantities of primer (containing MDPB) and bonding (containing bis-GMA-HEMA) agent.

## *4.4. Biocompatibility Tests*

Direct contact cytotoxicity of AMsils was determined following described protocols [55,56]. Briefly, immortalized mouse subcutaneous connective tissue fibroblasts (NCTC clone 929 [L-cell, L-929, Strain L derivative]; American Type Culture Collection (ATCC), Manassas, VA, USA) (CCL1) or HGF (Applied Biological Materials, Inc., Richmond, BC, Canada) were exposed to 2-fold serial dilutions (AMsil1: ≤8.34 mmol/L; AMsil2: ≤7.28 mmol/L). Chosen concentrations corresponded to approx. 7% mass fraction of AMsil1 or AMsil2 in the copolymer resin and a maximum of 2% leaching. To allow for the possibility of restoration multiplicity and variable size, a 2-fold greater dilution was also included in the testing. These calculations are based on the accelerated leachability study of UDMA/PEG-U/2-hydroxyethyl methacrylate (HEMA) resin (abbreviated UPH; a close analog to UPE resin used in this study) and ACP-UPH composites [57]. After 24 and 72 h incubation, cells were assessed for cell viability (LIVE/DEAD ® Viability/Cytotoxicity kit, Life Technologies, Corp., Grand

Island, NY, USA) and metabolic activity (CellTiter ® AQueous One Solution Reagent; Promega, Corp., Madison, WI, USA). Controls were without the AMsils and/or cells. The CCL1 cells and HGFs were maintained, at 37 ◦C and 5% CO2, in 10% serum-supplemented Eagle's minimum essential medium (ATCC) and PriGrow III medium (Applied Biological Materials, Inc.), respectively. For experiments, cells were obtained from a subconfluent stock culture. Means were obtained from 5 independent replicates tested in duplicate.

#### *4.5. Contact Angle (CA)*

Changes in hydrophilicity/hydrophobicity of UPE resins due to the introduction of AMsils were assessed by CA measurements (drop shape analyzer DSA100, Krüss GmbH, Hamburg, Germany). Following the deposition of the sessile droplets of the resin on the substrate, they were imaged after 1 min resting time with a charge-coupled device camera at the points of intersection (three-phase contact points) between the drop contour and the projection of the surface (baseline). The CA water values were calculated employing the Krüss Advance software. Four repetitive measurements were performed in each group.

#### *4.6. Copolymer Specimen Preparation*

For biotesting, UPE and AMsil1–UPE and AMsil2–UPE copolymer specimens were fabricated by filling circular openings of a flat stainless-steel molds (6 mm diameter, 0.5 mm thickness) with the resins. Each side of the mold was covered with Mylar film and a glass slide, firmly clamped, and then cured (2 min/side: Triad 2000; Dentsply International, York, PA, USA).

Specimens were subjected to extraction in Dulbecco's phosphate-bu ffered saline lacking both calcium and magnesium (Life Technologies, Grand Island, NY, USA) for 72 h, at 37 ◦C and 6.3 rad/s using an orbital shaker. After extraction, the disks were dried under vacuum (desiccator; ~22 ◦C) for 7 days. Specimens were sterilized for 12 h using an Anprolene gas sterilization chamber (Andersen Products, Inc., Haw River, NC, USA). Prior to bacterial testing, specimens were degassed for ≥5 days under vacuum (desiccator; ~22 ◦C).

#### *4.7. Degree of Vinyl Conversion (DVC)*

DVC of UPE and (AMsil1 or AMsil2)–UPE resins was determined by collecting the near-IR (NIR) spectra (Nexus; ThermoFisher, Madison, WI, USA) before and 24 h after the light cure and calculating the reduction in =C–H absorption band at 6165 cm<sup>−</sup><sup>1</sup> in the overtone region in going from monomers to polymers. By maintaining a constant specimen thickness, a need for an invariant internal standard was eliminated. The DVC was calculated as

$$\text{DVC (\%)} = \text{[(area}\_{\text{mononner}} - \text{area}\_{\text{polymer}}) / \text{area}\_{\text{mononner}}] \times 100 \tag{1}$$

where areapolymer and areamonomer correspond to the areas under 6165−<sup>1</sup> absorption peak after and before the polymerization, respectively.

#### *4.8. Mechanical Properties of Copolymers*

Test specimens (2 mm × 2 mm × 25 mm) for flexural strength (FS) and elastic modulus (E) determinations were photopolymerized in the same manner as the copolymer disks for biological testing. Polymerized specimens did not undergo any additional treatment. The FS and E of UPE and (AMsil1 or AMsil2) UPE copolymer specimens was tested employing the Universal Testing Machine (Instron 5500R, Instron Corp., Canton, MA, USA). The load was applied (crosshead speed of 1 mm/min) to the center of a specimen positioned on a test device with supports 20 mm apart. The FS and E of the specimens (three replicates/experimental group) were calculated as instructed in the ISO4049:2009 document.

#### *4.9. Bacterial Testing*
