4.9.2. Biofilm

A bioluminescent *S. mutans* strain JM 10 (derivative of wild type UA159 [59] was used to assess the AM properties of AMsils. Methods of the real-time bioluminescence assay were as described [41,58].

*Porphyromonas gingivalis*, strain FDC 381 (ATCC ® BAA-1703) was propagated in Becton Dickinson BBL chopped meat carbohydrate, pre-reduced II broth, using a shaking incubator (37 ◦C, anaerobic conditions). Three-day cultures were diluted in broth to approximate 5 × 10<sup>6</sup> CFU/mL. Copolymer disks, vertically supported in a 24-well plate, were immersed in 1.6 mL of the bacterial suspension. In anaerobic conditions, the plate was incubated at 37 ◦C for 4 days. The copolymer disks were washed thrice in sterile 0.89% NaCl solution. Thereafter, the biofilm was displaced from the copolymer disks by transferring them to a sterile glass tube containing 1 mL of saline, vortexed (1 min), sonicated (10 min), and vortexed (1 min). Each disk was visually examined to ensure that the biomass was removed. The resulting suspensions were used to make 10-fold serial dilutions and subsequently spread onto the surface of Brucella agar with hemin and vitamin K1 (Sigma-Aldrich, St. Louis, MO, USA) plates. After incubation (3 days at 37 ◦C, anaerobic conditions), colony-forming units were enumerated.

#### *4.10. Statistical Analyses*

Analysis of variance and multiple paired comparisons (two-sided, 95% confidence interval) were used to analyze the experimental data as a function of material makeup and/or exposure/incubation time and establish a statistical significance of di fferences between the experimental groups. Correlation coe fficient (*r*) was calculated to determine the functional dependence between cellular metabolic activity and viability. (SigmaPlot ™, Systat Software, San Jose, CA, USA and/or Microsoft O ffice Excel 2016; Microsoft, Redmond, WA, USA). Graphics were created using Microsoft O ffice Excel 2016 and/or DeltaGraph6 for Windows ® (Red Rock Software, Inc., Salt Lake City, UT, USA).

**Author Contributions:** D.R.B. conceptualized and conducted biotesting, fabricated copolymer specimens, and conducted statistical analyses. A.A.G. conducted biotesting, physicochemical/mechanical testing of copolymers. S.A.F. conducted monomer synthesis/validation, fabrication of copolymer resins, and measured their physicochemical/mechanical properties. S.S.K., F.L.E.F., and R.D.H. conducted S. mutans biofilm analyses. D.S. conceptualized the AM monomers, performed literature compilation, and statistical data interpretation. All authors contributed to the preparation/review of this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Institute for Dental and Craniofacial Research (grant R01 DE26122), American Dental Association (ADA), and ADA Foundation.

**Acknowledgments:** Assistance of U.C. Okeke (formerly ADA Foundation) in monomer synthesis/characterization are gratefully acknowledged. Technical expertise of H. Kim (Integrated Pharma Services) to assess AM properties of *P. gingivalis* is appreciated. Authors gratefully acknowledge donation of UDMA and PEG-U from Esstech, Essington, PA, USA.

**Conflicts of Interest:** D.R.B., A.A.G., S.A.F., and D.S. are employees of the non-profit ADA Foundation, which has applied for a patent describing synthesis and uses of polymerizable multifunctional antimicrobial quaternary ammonium monomers.

**Disclaimer:** The sole purpose of identifying certain commercial materials and equipment in this article was to adequately define the experimental protocols. Such identification, in no instance, implies recommendation or endorsement by the ADA or ADA Foundation, or means that the material/equipment specified is the best available for the purpose.
