Nanofilled Antibacterial Adhesives Downregulate the Metabolism of Streptococcus Mutans Biofilms

Objectives: Determine the lactate dehydrogenase (LDH) activity and bioluminescence (BIO) of non-disrupted Streptococcus mutans biofilms grown on aged dental adhesive resins containing nitrogen-doped titanium dioxide nanoparticles (N_TiO₂).
Methods: Experimental adhesives were formulated using varying ratios of N_TiO₂ (20%, 25%, 30%, v/v). Two commercial adhesives served as control groups (OSP and PUB). Disk-shaped specimens for LDH (diameter=8.0mm, thickness=0.5mm, n=6/group) and for BIO testing (diameter=6.0mm, thickness=0.5mm, n=12/group) were fabricated and photopolymerized (40 sec/each; 385-515nm, 1,000mW/cm²; VALO). Specimens were then aged in sterile DPBS (0, 1, 3 and 6 months; 37°C, dark conditions [control]). LDH activity was measured by growing S. mutans biofilms (1, 2, and 15 days; UA159-ldh, 37°C, microaerophilic) on the surfaces of specimens. Bacterial cells were then lysed before measuring the LDH activity with a commercially available kit (Sigma Aldrich). BIO was measured by growing S. mutans biofilms for 24-hours on the surfaces of specimens in the presence of continuous light irradiation (dark, 410nm, 600nm, or 780nm) and then measuring the bioluminescence values (RLUs) of intact biofilms with a Biotek Synergy HT multi-well plate reader. Experimental data for LDH and BIO were analyzed using two-factor General Linear Models and post hoc Student-Newman-Keuls tests (∝=0.05, SAS Software).
Results: LDH values ranged from 0.68±0.32 milliunits/mL (30% N_TiO_2, 2-day biofilms, 0 months aged) to 10.99±2.40 milliunits/mL (30% N_TiO₂, 15 days biofilm growth, 6 months aged) and BIO values ranged from 2,203.33±501.57 RLU (30% N_TiO_2, 410nm, 0 months aged) to 94,737±41,757 (0% N_TiO_2, 600 nm, 1 month aged) RLU.
Conclusions: Significant differences (p < 0.0001) were observed from the interaction between parameters of interest (nanoparticles x control x time) for LDH and BIO. Nanofilled experimental materials were demonstrated to exhibit superior antibacterial properties and support biofilms with lower LDH activity.