Mildfolm

Body weight (BW) measurement and blood sample collection were performed at the time of GCV administration, and weekly after PH transplantation. Sera were stored at - 20 °C until use, and they were subjected to the measurement of serum ALT levels (SRL Inc. Aichi, Japan). In all the models (Sham, Auto, Allo, FK, and ASC models), the mice were sacrificed at before (day 0) and 1 week after GCV administration (0 w), 1, 2, 4, and 8–11 weeks after PH transplantation (1, 2, 4, and 8 w, respectively). The resected livers were subjected to the histological analysis, flow cytometric analysis for hepatocyte polyploidy, and hepatic gene expression analysis (Fig. 1b and c). The part of liver tissues was fixed with 10 N Mildfolm (Wako) at room temperature for the histological examination. The unfixed liver tissues were embedded with FSC 22 Clear Frozen Section Compound (Leica Biosystems, Germany), and frozen in liquid nitrogen for the preparation of frozen specimens also for another histological examination. The remaining part of them were stored at -30 °C in RNAlater (QIAGEN, Hilden, Germany) for the analysis of mRNA expression. For the flow cytometric analysis, the livers were perfused as described above (2.2 PH isolation) to collect isolated hepatocytes.

We used female diabetic mice at 9 weeks of age (n = 120). Three days before starting this experiment, all mice were shaved and depilated. After anesthetizing the mice using isoflurane (Isoflurane inhalation solution; Pfizer Inc., Tokyo, Japan), all mice were cleansed, and full-thickness wounds measuring 12 mm in diameter were created on the middle of the back of each diabetic mouse as impaired wound healing models (Fig. 1). Aqueous silk-elastin solution [56 μl/animal, 20% (w/v) silk-elastin group: n = 30] or CMC gels (50 μl/animal, Granugel group, n = 30; 50 μl/animal, Intrasite gel system group, n = 30) were implanted into the resulting wounds, which were then covered with polyurethane film (Tegaderm; 3M Healthcare, Ltd., Tokyo, Japan). The control wounds were covered with polyurethane film alone. Thereafter, the wound area was covered with gauze, which was fixed to the skin around the wound areas with a nylon thread. At 14 and 21 days after application, all 60 mice were killed. The specimens were fixed with 20% formalin fluid (Mildfolm; Wako Pure Chemical Industries, Osaka, Japan). The specimens included the surrounding normal skin on the section through the center of the wound. For the histological examination, the sections were stained with hematoxylin and eosin.