Icps 8000

S-PRG eluate was prepared as described previously^{38 (link)}, and was provided by Shofu Inc. (Kyoto, Japan). Briefly, S-PRG filler was mixed with an equal amount of distilled water and mixed gently at room temperature for 24 h, followed by centrifugation at 3,000 × g, 23 °C for 6 h to separate the filler and the liquid. The supernatant was then filtered (pore size: 0.45μm) to remove any residual insoluble material and the resulting filtrate was used as the S-PRG eluate. The concentrations of ions released from S-PRG filler except for F⁻ (i.e., Al³⁺, BO₃³⁻, Na⁺, SiO₃²⁻, and Sr²⁺) were measured by using an emission spectrophotometer (ICPS-8000, Shimadzu Co., Kyoto, Japan). In addition, the concentration of F⁻ was confirmed with a F⁻ electrode (Model 9609BNWP, Orion Research Inc., Beverly, MA, USA) using an ion selective electrode meter (Model 720 A, Orion Research Inc.). The ion concentrations of the S-PRG eluate were as follows: Al³⁺ = 19.6 ppm, BO₃³⁻ = 1,656.5 ppm, Na⁺ = 618.5 ppm, SiO₃²⁻ = 13.9 ppm, Sr²⁺ = 126.8 ppm, and F⁻ = 141.0 ppm. The S-PRG eluate was diluted with brain heart infusion (BHI) broth (Difco Laboratories) at concentration of 25%, 12.5%, 6.3%, and 0%, respectively, and was used in subsequent studies.

S-PRG eluates were formulated by mixing DW with S-PRG filler (Shofu Inc., Kyoto, Japan) in a 1:1 ratio (1 L: 1,000 g), followed by stirring at 23 °C for 24 h. And the mixture was centrifuged to precipitate the S-PRG filler. The supernatant solution was then filtered using a Chromato Disk (25A Hydrophilic Type, Diameter: 25 mm, pore size: 0.2 µm; GL Sciences Inc., Tokyo, Japan) to obtain the various solution concentrations (25%, 50%, and 100%)^{27 (link)}. Furthermore, the elemental analyses of the BO₃^3–, Na⁺, Sr²⁺, Al³⁺, SiO₃^2–, and F^– ions released from the S-PRG fillers were performed using inductively coupled plasma atomic emission spectroscopy (ICPS-8000, Shimadzu Corp., Kyoto, Japan) and fluoride ion electrodes (Model 9609BNWP, Orion Research, Boston, MA, USA) (Table 2).Table 2

Concentrations of ions released from S-PRG filler.

	BO33−	Na+	Sr2+	F−	Al3+	SiO32−
25% S-PRG	348.3	97.9	34.2	28.6	2.4	2.7
50% S-PRG	696.6	195.8	68.3	57.3	4.9	5.4
100% S-PRG	1,393.2	391.6	136.7	114.5	9.7	10.8

Concentration values are in parts per million.

An endodontic sealer containing S-PRG filler (prototype; Shofu, Kyoto, Japan; PRG+), an experimental endodontic sealer containing S-PRG-free silica filler (Shofu, Kyoto, Japan; PRG−), AH Plus (an epoxy resin-based sealer; Dentsply Sirona, Charlotte, NC, USA; AH), and Canals N (a zinc oxide-noneugenol sealer; Showa Yakuhin Kako, Tokyo, Japan; CN) were used in this study (Table 1). Each was mixed according to the manufacturers’ instructions, poured into a polypropylene container (8 mm in diameter, 3 mm high), and either placed immediately in culture medium (alpha MEM, 10 mL, FUJIFILM Wako Pure Chemical, Osaka, Japan; fresh group) or incubated at 37 °C under 100% humidity for five days to induce complete setting before immersion in the culture medium (set group). The culture medium containing the sealer was shaken at room temperature for 24 h and sterilized by passing it through a membrane filter (0.45 µm pore size, Sartorius, Göttingen, Germany).
Elemental analysis of each sealer extract was conducted with inductively coupled plasma atomic emission spectroscopy (ICP-AES: ICPS-8000, Shimadzu, Kyoto, Japan). The concentration of fluoride ions released from each set sealer sample was measured using a fluoride ion sensor (Horiba, Kyoto, Japan).

PRG gel paste was provided by SHOFU Inc. (Kyoto, Japan). The main component of PRG gel paste is S-PRG filler, which is produced by reacting multifunctional glass (porous SiO₂ glass coated fluoroboroaluminosilicate glass) with polyacrylic acid solution, with the result that various ions are existed in the stable glass ionomer phase in the glass particles^{8 (link)}. Other components of the prophylaxis paste are glycerin and sorbitol solution as humectants, silicic anhydride as an abrasive compound, carboxymethyl cellulose as a binding material, and sodium lauryl sulfate as a foaming agent. The concentrations of Al³⁺, BO₃³⁻, Na⁺, SiO₃²⁻, and Sr²⁺ ions released from the PRG gel paste were measured using an emission spectrophotometer (ICPS-8000, Shimadzu Co., Kyoto, Japan). The concentration of F⁻ was confirmed with a F⁻ electrode (Model 9609BNWP, Orion Research Inc., Beverly, MA, USA) using an ion selective electrode meter (Model 720A, Orion Research Inc.). S-PRG filler-free gel paste was also provided by SHOFU Inc., and contained the same amounts of components as the PRG gel paste, but with no S-PRG filler.