2.2.2. AMsil2

Regardless of time, AMsil2 concentration exerted a main effect (*p* ≤ 0.001) on the number of viable CCL1s (Figure 4). Paired comparisons indicated that at 24 h exposure, CCL1 cells exposed to ≥3.64 mmol/L were lower (*p* ≤ 0.05) than the number of live cells exposed to all lower concentrations. At 72 h exposure, the percentage of live CCL1 cells exposed to ≥1.82 mmol/L AMsil2 was at least 3- to 4-fold lower (*p* ≤ 0.001) compared to all concentrations ≤ 0.91 mmol/L.

Like cell viability, AMsil2 concentration exerted an effect (*p* ≤ 0.001) on CCL1 metabolic activity. Viability and metabolic activity of CCL1 cells exposed to AMsil2 showed a strong positive linear correlation at 24 h (R<sup>2</sup> = 0.91, *p* ≤ 0.0005) and 72 h (R<sup>2</sup> = 0.93, *p* ≤ 0.0005) (data not shown).

AMsil2 exhibited a concentration effect (*p* ≤ 0.001) on HGF cell viability (Figure 5). Paired comparisons indicated that exposure to ≥1.82 mmol/L AMsil2 reduced the number of live HGFs by more than 3-fold (*p* ≤ 0.05) compared to the control group. When considering the effect of time regardless of AMsil2 concentration, the number of live cells was consistently lower (~23% difference of means). Like viability, AMsil2 concentration exhibited an effect (*p* ≤ 0.001) on HGF metabolic activity. At 24 and 72 h, viability and metabolic activity of HGF cells exposed to AMsil2 showed a positive linear correlation (R<sup>2</sup> = 0.94; (*p* ≤ 0.0005) and R<sup>2</sup> = 0.77; (*p* ≤ 0.001), respectively) (data not shown).

**Figure 4.** Percent control value of viability of CCL1 cells exposed to 2-fold serial dilutions of AMsil2 (≤7.28 mmol/L) for 24 or 72 h. Data represent mean ± SEM for five independent replicates tested in triplicate. **\*** indicates *p* ≤ 0.05 when compared to concentrations ≤ 0.91 mmol/L within the same time period. + indicates *p* ≤ 0.05 when compared to 0.455, 0.228, or 0.114 mmol/L concentrations within same time period.

**Figure 5.** Percent control value of viability of HGF cells exposed to 2-fold serial dilutions of AMsil2 (≤7.28 mmol/L) for 24 h or 72 h. Data represent mean ± SEM for five independent replicates tested in triplicate. + indicates *p* ≤ 0.05 when compared to concentrations ≤ 0.455 mmol/L within same time period. \* indicates *p* ≤ 0.05 when compared to concentrations ≤ 0.91 mmol/L within same time period. ˆ indicates *p* ≤ 0.05 when compared to concentrations ≤ 0.114 mmol/L within same time period. \$ indicates *p* ≤ 0.05 when compared to concentrations ≤ 0.228 mmol/L within same time period.

For both AMsils, control wells (with or without cells) in which the tetrazolium salt reagen<sup>t</sup> was omitted resulted in negligible optical density values. Positive control wells containing unexposed cells (i.e., no AMsils) that were given an equal volume of culture medium were not significantly different from cells that were previously treated with the viability stain (data not shown).

#### *2.3. Hydrophobicity*/*Hydrophilicity of the Resins*

Copolymers comprised of UPE resin with added AMsils generally exhibited lower contact angles (CAs) (Figure 6), suggesting change in their hydrophilic/hydrophobic balance toward more hydrophilic surfaces. At 10 mass % monomer in the resin, CAs of both AMsil1–UPE and AMsil2–UPE copolymers (46.9 ± 5.9◦ and 37.4 ± 9.2◦, respectively) were significantly lower (23% and 38% reduction, respectively; *p* ≤ 0.01) than the CA of the UPE control 60.8 ± 5.1◦. The apparent increase in the CA in going from 10% to 20% AMsil in the resin was significant only for AMsil2 (*p* ≤ 0.035). The overall order of the decreasing relative hydrophilicity (evidenced by the increasing CA values) of the examined UPE-based copolymers was as follows: (10% AMsil2–UPE ≥ 10% AMsil1–UPE) > (20% AMsil2–UPE = 20% AMsil1–UPE) > UPE control.

**Figure 6.** The contact angle (CA) values of AMsil–UPE and UPE control indicative of the changes in resin's overall hydrophilicity/hydrophobicity upon introduction of AMsil monomers at 10 and 20 mass % relative to UPE. Shown are mean values + standard deviation of four repetitive measurements in each experimental group.

#### *2.4. E*ff*ect of AMsils on Degree of Vinyl Conversion (DVC)*

Introduction of 10% and 20% AMsil1 into UPE reduced (*p* ≤ 0.05) the mean vinyl moiety conversion upon photopolymerization by 31% and 20%, respectively (Figure 7). No significant effect was observed with the increasing levels of AMsil1 in the resin. Although reduced, the DVC observed amongs<sup>t</sup> the AMsil2 groups was not statistically different from one another or the UPE control group.

**Figure 7.** The values for degree of vinyl conversion (DVC) attained 24 h post-cure in AMsil–UPE copolymers compared to no-AM UPE control. Shown are mean values + standard deviation of three repetitive measurements.

#### *2.5. Mechanical Properties of AMsil–UPE Copolymers*

The FS and E of AMsil–UPE copolymers were, generally, diminished compared to the UPE resin control (Figure 8). The extent of reduction in FS and E varied with the type and the concentration of AMsil. In all AMsil–UPE formulations, the FS values were significantly (*p* ≤ 0.05) lower than the UPE control counterparts. In both 10 mass % AMsil formulations, the E was reduced, although not statistically significant. At 20 mass %, the E of both AMsil formulations were notably lower (*p* ≤ 0.0008) than the UPE resin control. Both FS and E reductions ranged from moderate (11–13%) for 10 mass % AMsil formulations to substantial (25–57%) for 20 mass % AMsil formulations.

**Figure 8.** (**a**) Flexural strength and (**b**) tensile elasticity of AMsil–UPE copolymers in comparison with the UPE control. Indicated are mean values + standard deviation of three specimens.

#### *2.6. Bacterial Testing*

For planktonic bacterial testing, the number of *S. mutans* colony-forming units/mL observed amongs<sup>t</sup> the AMsil groups were not statistically different from one another or the control groups (UPE only and commercial control). However, compared to UPE resin, AMsil1–UPE and AMsil2–UPE (10% mass) copolymers reduced the colonization of *S. mutans* biofilm 4.7- and 1.7-fold, respectively (*p* ≤ 0.002) (Figure 9). *S. mutans* biofilms exposed to AMsil1–UPE were at least 2.8-fold lower (*p* ≤ 0.005) than that observed with AMsil2–UPE.

**Figure 9.** *Streptococcus mutans* biofilm growth inhibition by the experimental AMsils–UPE (10 mass %) copolymers compared to UPE control resin. Bar height indicates mean + standard deviation of 5 specimens/group.

*P. gingivalis* biofilm biomass on copolymer disks exposed to AMsil1–UPE and AMsil2–UPE were lower (71% and 85%, respectively) than that observed with the commercial control, albeit not statistically different (*p* ≤ 0.07) (Figure 10).

**Figure 10.** *Porphyromonas gingivalis* biofilm growth inhibition by the experimental AMsils–UPE (10 mass %) copolymers compared to UPE control resin. Bar height indicates mean + standard deviation of 5 specimens/group.
