**4. Results**

A representative TEM image of calcium fluoride (nCaF2) nanoparticles is shown in Figure 1A. The nanoparticle size distribution ranged from 22 nm to 57 nm, with a mean particle size of 32 nm and is illustrated in Figure 1B.

Flexural strength and elastic modulus of the six composite groups (mean ± sd; *n* = 6) are shown in Figure 2A,B, respectively. The flexural strength was measured after one day of immersion in water at 37 ◦C. The flexural strength was significantly higher in EC and nCaF2 composites, when compared to the commercial Heliomolar control (*p* < 0.05). Flexural strength in groups with nCaF2+DMAHDM and nCaF2+MPC matched those of Heliomolar control composite (*p* > 0.05). However, flexural strength in nCaF2+DMAHDM+MPC was significantly lower than commercial Heliomolar control (*p* < 0.05).

The elastic modulus values of EC and nCaF2 were significantly greater than all other groups (*p* < 0.05). Other groups had comparable elastic modulus values to Heliomolar control (*p* < 0.05).

The accumulative F ion release is shown in Figure 3. At 70 days, the composite nCaF2+MPC had the highest F release of (0.40 ± 0.02) mmol/L (*p* < 0.05). Meanwhile, nCaF2+DMAHDM+MPC had F release of (0.25 ± 0.03) mmol/L, nCaF2+DMAHDM had (0.20 ± 0.03) mmol/L, and nCaF2 had (0.04 ± 0.01) mmol/L of fluoride ion release. Heliomolar control had the lowest F release of (0.004 ± 0.0003) mmol/L.

**Figure 1.** (**A**) TEM of nanoparticles of CaF2 (nCaF2) synthesized in this study. (**B**) Particle size distribution of nCaF2. The nCaF2 were synthesized via a spray-drying technique and collected using an electrostatic precipitator.

**Figure 2.** Mechanical properties of composites: (**A**) Flexural strength, and (**B**) Elastic modulus (mean ± sd; *n* = 6). The flexural strength was higher in experimental control (EC) and nCaF2 than commercial Heliomolar control (*p* < 0.05). Flexural strength in nCaF2+DMAHDM and nCaF2+MPC matched Heliomolar control (*p* > 0.05). However, the flexural strength in nCaF2+DMAHDM+MPC was reduced (*p* < 0.05). The elastic modulus of the EC and nCaF2 were higher than Heliomolar control (*p* < 0.05). All other groups had comparable elastic moduli to Heliomolar control after 1 day of immersion (*p* > 0.05). In each plot, different letters (a, b, c) indicate values that are significantly different from each other (*p* < 0.05).

**Figure 3.** Fluoride (F) ion release from composites (mean ± sd; *n* = 6) at pH 7.0. The incorporation of nCaF2, DMAHDM, and MPC increased the release of F ions with time (*p* < 0.05). Different letters (a, b, c, d, e) indicate significant differences between groups at day 70 (*p* < 0.05).

The calcium ion release is plotted in Figure 4. At 70 days, nCaF2+MPC had a Ca ion release of (0.32 ± 0.005) mmol/L, and nCaF2+DMAHDM+MPC had a similar ion release at (0.35 ± 0.006) mmol/L. Groups containing MPC had ion releases that were significantly higher when compared to other groups (*p* < 0.05). nCaF2 had a Ca ion release of (0.11 ± 0.004) mmol/L. nCaF2+DMAHDM had (0.18 ± 0.005) mmol/L, and Heliomolar control had close to zero Ca ion release.

**Figure 4.** Calcium (Ca) ion release from composite resins (mean ± sd; *n* = 6) at pH 7.0. The incorporation of nCaF2, DMAHDM, and MPC increased the release of Ca ions with time (*p* < 0.05). Different letters (a, b, c, d) indicate significant differences between groups at day 70 (*p* < 0.05).

Two-day biofilm colony forming units CFU on composites are shown in Figure 5: (A) Total microorganisms, (B) total streptococci and (C) mutans streptococci (mean ± SD; *n* = 9). CFU was reduced by 6 logs from a mean of 2.51 × 10<sup>8</sup> counts for Heliomolar control to 1.00 × 10<sup>2</sup> counts for the new nCaF2+DMAHDM+MPC composite (*p* < 0.05). nCaF2+DMAHDM reduced the CFU by 4 logs (*p* < 0.05). The combination of nCaF2+DMAHDM+MPC yielded the smallest CFU counts.

**Figure 5.** Colony-forming unit (CFU) counts of 2-day biofilm on composite discs (mean ± sd; *n* = 9): (**A**) Total microorganisms, (**B**) total streptococci, and (**C**) mutans streptococci (mean ± sd; *n* = 9).The incorporation of nCaF2+DMAHDM+MPC had the lowest CFU, followed by nCaF2+DMAHDM in the total microorganisms. The reduction of CFU in nCaF2+DMAHDM+MPC and nCaF2+DMAHDM was similar in total streptococci and mutans streptococci (*p* < 0.05). In each plot, different letters (a, b, c, d) indicate significant differences between groups (*p* < 0.05).

The bacterial metabolic activity of 2 days biofilm on composites is shown in Figure 6A. Metabolic activity was decreased from 0.18 (OD540/cm2) for commercial Heliomolar composite control, to 0.02 for the nCaF2+DMAHDM+MPC composite (*p* < 0.05). Biofilm lactic acid production results can be seen in Figure 6B. The lactic acid production was reduced from 0.72 mmol/L on commercial Heliomolar control composite to 0.29 mmol/L in the nCaF2+DMAHDM+MPC composite.

**Figure 6.** (**A**) The total metabolic activity, and (**B**) Lactic acid production (mean ± sd; *n* = 9) of composite specimens exposed to salivary biofilm. The incorporation of nCaF2+ DMAHDM had the best reduction in the metabolic activity and lactic acid production (*p* < 0.05). In each plot, different letters (a, b, c, d) indicate significant differences between groups (*p* < 0.05).

SEM results in Figure 7 indicate an extensive biofilm formation at 24 h, 48 h, and 96 h in all groups except those containing the antimicrobial DMAHDM. Heliomolar (CC), experimental control (EC), and nCaF2+MPC groups had the most biofilm formation at all time points, followed by the nCaF2 group. Biofilm formation increased with time. However, in DMAHDM-containing groups there was minimal attachment of biofilm observed. At 96 h, the DMAHDM-containing groups showed a reduction in biofilm attachment compared with earlier time points.

**Figure 7.** Scanning Electron Microscopy (SEM) images of biofilm formed on composite disc surfaces at 24 h, 48 h, and 96 h. All images are at 3000× magnification. Arrows indicate presence of minimal biofilm on DMAHDM-containing specimens. All other groups had full coverage of a substantial and mature biofilm.
