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Polymicrobial Colonization of Athletic Mouth Guards Following Mechanical Chew Simulation.Mouth guard material is used for athletic mouth guards, temporary dental splints, whitening trays, medicinal depositories and catheter tubing. This study evaluated biofilm formation on mouth guard materials after simulated use to ascertain the propensity of mouth guards to harbor bacteria. Three different mouth guards were evaluated (n=18/group): two boil-and-bite guards, Shock Doctor® (SD) Nano 3D and The Wrightguard™ (TWG) and one custom 100% EVA Buffalo guard (EVA). In vitro wear of the fabricated guards were simulated via a chewing simulator for 120,000 cycles per guard. The most visibly worn 8x10mm section of each guard was determined and cut to standardize specimen size. All guard specimens were incubated with equal cell densities of both S. aureus (SA) and Candida albicans (CA) strains and colony-forming units (cells/ml) were measured as a quantification of biofilm growth. A one-way ANOVA with Tukey's-HSD test was used to analyze biofilm retention (CFU/ml) on non-simulated and simulated EVA, SD, and TWG guards. Neither EVA (2.89±1.09 - 3.28±0.74; p=.486) nor TWG (10.22±3.31 - 11.17±5.46; p=.725) groups exhibited a significant increase in CFUs after 6 months of simulated use. In contrast, SD guards (3.33±1.55 - 9.72±5.37; p=.019) exhibited a significant increase in mean CFUs of CA when non-simulated guards were compared to simulated guards. Like with CA, neither EVA (3.11±1.90 - 3.50±1.63; p=.712) nor TWG (9.83±4.09 - 12.56±5.58; p=.358) groups exhibited significant increases in adherent SA following 6 months of simulated use. However, Shock Doctor (5.11±2.20 - 9.67±4.77; p=.06) guards had a significant increase in adherent SA CFUs when non-simulated guards were compared to simulated guards. With chewing simulation parameters remaining constant for all guard types, significantly greater adherence could indicate a greater change in surface topography/roughness from simulated use. While EVA guards exhibited the lowest measured adherence of CA and SA, and TWG exhibited the highest CA and SA levels of adherence, neither of their respective material compositions undergo surface changes as readily as SD guards.The increased propensity of both fungal and bacterial species to adhere to mouth guards after continuous use indicates a potential concern for regular mouth guard users.