Hyaluronic acid

Biofilms formation was developed in 96-well polystyrene microtiter plates (Sumitomo Bakelite, Tokyo, Japan), which were previously coated with human saliva, BSA, anti-PAc serum [38 (link)], anti-PAc monoclonal antibody [39 (link)] and anti-GbpC serum [40 (link)] for 1h at 4°C. After pre-coating, wells were washed with phosphate buffered saline (PBS) 2 times. Biofilm formation assays were performed using a previously described procedure [36 (link)]. Wells containing 180 μl of TSB with 0.25% sucrose were inoculated with 20 μl of S. mutans UA159 and gtfBC mutant, S. sanguinis, S. mitis and S. gordonii from a cell culture with an optical density (OD₆₀₀) of 0.4. The plates were then incubated with various concentrations of polypyrrole (Sigma-Aldrich, Co., St. Louis, MO) or hyaluronic acid (MW 5,000–150,000, Tokyo Chemical Industry, Co. LTD, Tokyo, Japan) at 37°C with 5% CO₂ for 16 h. To observe the effects of hyaluronic acid on polypyrrole-dependent biofilm formation, polypyrrole was pretreated with 7 mg/ml hyaluronic acid for 1 h at 37°C and applied to the biofilm formation assay. After incubation, the planktonic cells were removed by washing with distilled water (DW), and the adherent cells were stained with 0.25% safranin for 15 min. After washing with DW 2 times, safranin was extracted from biofilm with 70% (v/v) ethanol, and quantitatively measured by the absorbance at 492 nm.

Prednisolone, carbonyl-diimidazole (CDI), dimethyl-aminopyridine (DMAP), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC), and sodium alginate (AL-Na; low viscosity grade (viscosity of 1% aqueous solution at 25 °C = 32.7 ± 0.4 mPa·s (n = 3)), cat no. 154725, lot no. QR13046) were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). N-Trityl-glycine (Tr-G) was obtained from Sigma-Aldrich Co., LLC. (St. Louis, MO). Hyaluronic acid (viscosity of 1% aqueous solution at 25 °C = 1.2 ± 0.1 mPa·s (n = 3), product code H0595, lot no. 8FUKM-CI) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Non-viable and desiccated Mycobacterium tuberculosis H37 Ra was obtained from Becton, Dickinson and Company (Franklin Lakes, NJ) and used in the preparation of adjuvants. All other chemicals were of reagent grade.