Juc 5000

Scanning electron microscopy was performed as described previously [17 (link)]. Briefly, the collagen gels were prefixed in 3% glutaraldehyde in phosphate buffer for 2 h, at room temperature, and postfixed in 1% osmium tetroxide in phosphate buffer for 1 h, at room temperature. After washing with phosphate buffer, the gels were incubated with 1.0% tannic acid in water for 30 min. The gels were again fixed in 1% osmium tetroxide for 1 h, at room temperature, and washed with phosphate buffer. The samples were then dried by the t-butyl alcohol freeze-drying method, mounted on metal stubs, coated with platina using an ion sputter (JUC-5000; JEOL), and observed under a scanning electron microscope (JSM-5200; JEOL).

A dentin block (5 mm × 5 mm × 2 mm) was prepared from extracted human teeth. The block was chemically cleaned with sodium hypochlorite solution and EDTA solution, sterilized in an autoclave, immersed in the E. faecalis bacterial solution, and cultured aerobically at 37 °C for 21 days. After the block was washed with 5 mL of sterile saline using a syringe, aPDT/PACT treatment was performed on the experimental group. Irradiation was performed under the same conditions described in Section 4.6. Control groups were also set up with the same conditions described in Section 4.6. As NaOCl treatment, dentin blocks were placed in 3% NaOCl solution (Dental Antiformin; Nishika, Yamaguchi, Japan) for 1 min.
The samples were washed with 5 mL of sterile saline and fixed with a mixture of 4% paraformaldehyde and 5% glutaraldehyde for 24 h at 4 °C. The samples were washed again in the same manner, dehydrated with an ascending ethanol series (50, 60, 70, 80, 90, 95, 100, 100%), replaced with t-butyl alcohol, and freeze-dried for 4 days using a freeze-dryer (EYE4 FDS-1000 type; Tokyo Rika Kikai Co. Ltd., Tokyo, Japan). Subsequently, platinum was vapor-deposited using a sputtering device (JUC-5000; JEOL, Tokyo, Japan), subjected to conductive treatment, and observed under SEM (JXA-8530FA; JEOL).