Porphyromonas gingivalis

The strain of Porphyromonas gingivalis (BCRC14417) was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Fresh P. gingivalis cells from blood agar plates supplemented with hemin (5 μg/mL; Sigma-Aldrich, St Louis, MO, USA) and menadione (1 μg/mL; Sigma-Aldrich) were inoculated into 5 mL of trypticase soy broth (TSB, Difco, Detroit, MI, USA) supplemented with 2.5% yeast extract, hemin, and menadione. The cultures were incubated anaerobically in an N₂-H₂-CO₂ (80:10:10) atmosphere at 37 °C by using a Concept 400 Anaerobic Workstations (Ruskinn, Sanford, ME, USA). Organisms were harvested by centrifugation, washed with phosphate-buffered saline (PBS), and re-suspended in PBS. The numbers of bacteria were determined with a spectrophotometer (at an optical density at 600 nm) based on a standard curve established by colony formation on bacterial plates. To prepare heat-killed P. gingivalis cells, bacterial suspensions in PBS were heated at 80 °C for 30 min, washed with PBS, and re-suspended in RPMI 1640 medium (Gibco, Carlsbad, CA, USA).

Chitosan (CTS) with 85% deacetylation (molecular weight < 5 kDa), type B gelatin (GEL), methacrylic anhydride (MA), fluorescamine (98%), dimethyl sulfoxide (DMSO), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; 95%) were purchased from Sigma-Aldrich (Aldrich, St. Louis, MO, USA). Gallic acid (GA; 3,4,5-trihydroxybenzoic acid monohydrate, 98%), pyrogallol (PG; 1,2,3-trihydroxybenzene, 99%), and tannic acid (TA, 95%) were obtained from Alfa Aesar (Heysham, UK), and their chemical structures are shown in Figure 1a.
Escherichia coli (E. coli; ATCC 8739), Staphylococcus aureus (S. aureus; ATCC 25923), Porphyromonas gingivalis (P. gingivalis; A7436), Streptococcus mutans (S. mutans; ATCC 700610) strains, and L929 fibroblast cell line (RM60091) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Gibco (Langley, OK, USA). The alamarBlue reagent was from Invitrogen (Grand Island, NY, USA). Bacto tryptic soy broth and Wilkins-Chalgren anaerobe broth were purchased from Becton Dickinson (Sparks, MD, USA) and Oxoid (Hampshire, UK), respectively. All reagents were of analytical grade and used as received without further purification.