A PGA sheet measuring 2.3 cm in diameter (NEOVEIL sheet 0.15, GUNZE LIMITED, Kyoto Japan) was placed on the bottom of a glass vial and fixed using fibrin glue (BOLHEAL®, KM Biologics Co., Ltd., Kumamoto Japan). Fibrin glue comprises two solutions (i.e., fibrinogen and thrombin); 0.25 mL of each solution was used to form a 1-mm-thick layer of fibrin glue containing a PGA sheet. This was repeated three times to form a total of three layers per vial. Artificial gastric juice with pepsin was prepared under different conditions of pH1.2, 2.0, 4.0, 5.5, and 6.0; these five groups in total were set up with reference to the activity of pepsin, which peaks at pH 2.0 and drops sharply from pH 4.0 to pH 6.0 (Figure 1) . As a reference, the dissolution rate of fibrin glue by acid without pepsin was also measured at the same pH levels. Preparation of artificial gastric juice was prepared by adding 1,000 mL of water to 2.0 g of sodium chloride and 7.0 mL of 35% hydrochloric acid, according to the first solution of the JP Dissolution Test Method. In the pepsin-containing group, porcine gastric mucosa-derived pepsin (2,953 U per mg, BBI Solutions Ltd., Cardiff, United Kingdom) was added at 0.1%. The pH was adjusted using sodium hydroxide; hydrochloric acid was also used for fine adjustment. Artificial gastric juice (10 mL) was used for one sample (Figure 2).
A locking mixer with a shaking condition of 8 rpm (RM-80, AS One Co., Ltd., Osaka, Japan) was installed in an incubator (LST-300D, TOKYO RIKAKIKAI CO, LTD., Tokyo, Japan) and set at 37°C for the measurements. The mean number of remaining layers was measured every 30 minutes for the first 6 hours, every 6 hours until the third day, and every other day until the seventh day; the measurements were compared in three samples per group. As fibrin dissolved, its degradation products and additives contained in the fibrin glue leached into the artificial gastric juice and raised the pH; therefore, the pH was adjusted after each measurement. The timing of layer dissolution, which was defined as the release of the PGA sheet into the artificial gastric juice after shaking the vial gently, was recorded (Figure 3).