Crf 1

All surgeries and handling were performed based on the guidelines of the Animal Ethics Committee of Kitasato University (Permission number: 2018-087). A total of 32 six-month-old male C57BL/6J mice (Charles River Laboratories Japan, Inc., Yokohama, Japan) were used for this study. The mice were fed a standard laboratory diet, CRF-1 (Oriental Yeast, Tokyo, Japan), and housed under controlled temperature (23 ± 2°C) and humidity (55 ± 10%) conditions and a 12-hour light/dark cycle. Mice were randomly divided into four treatment groups of eight mice each, namely, Group CONT (immobile control), Group 0.2, Group 1.0, and Group 1.0/BMP-2. A 2.0-mm critical sized bone defect was created in the right femur according to previous studies (see below for details) [19 (link), 20 (link)]. In Group CONT, the defect was fixed with the Segmental MouseDis without transportation of the bone segment. Mice in Groups 0.2 and 1.0 underwent fixation with the device and the bone segment was moved 0.2 mm/day for 10 days and 1.0 mm/day for 2 days, respectively. Mice in Group 1.0/BMP-2 underwent fixation with the device and received an injection of 2.0 μg of rhBMP-2 into the bone defect site immediately after the defect-creating surgery and the bone segment was moved 1.0 mm/day for 2 days.

Male and female C57BL/KsJ-m+/+Lepr^db(+/db) mice were obtained from Japan SLC (Shizuoka, Japan) and were housed in plastic cages (2–3 mice per cage) with free access to drinking filtered tap water and a pelleted basal diet CRF-1 (Oriental Yeast Co., Ltd., Tokyo, Japan) under controlled conditions of humidity (50±10%), light (12/12 h light/dark cycle) and temperature (23±2°C). Mice were maintained in the specific pathogen free (SPF) facility at Gifu University Life Science Research Center in accordance with Institutional Animal Care Guidelines. The protocol was approved by the Committee on the Ethics of Animal Experiments of Gifu University (the authorization number is 25–8). DEN was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in phosphate buffered saline to make 1% (w/v) solution, according to manufacturer’s instruction. Metformin was supplied by Dainippon Sumitomo Pharma Co. (Tokyo, Japan). The maximum solubility of Metformin is 346 mg/ml in water.

Experimentally naive male C57BL/6J mice (CLEA Japan Inc., Tokyo, Japan) were used. Mice were housed in groups of four to five per cage [model# GM500; Tecniplast, Buguggiate (VA), Italy] with paper chips as bedding (PaperClean; Japan SLC, Inc., Hamamatsu, Japan). The mice were maintained in a specific pathogen-free (SPF) vivarium at 22 ± 1°C and 55 ± 5% humidity under a 12-h light-dark cycle (light on at 7:00 A.M.). Periodic microbial examinations were made to verify SPF throughout the entire housing period. Mice had free access to standard mouse feed (CRF-1, Oriental Yeast, Ltd., Tokyo, Japan) and chlorinated water (free chlorine, 2 ppm) throughout the entire experimental period. The mice health states were monitored daily by animal technicians. Mice were euthanized humanely once they show rapid and continuous weight loss or emaciation. All experiments were approved by the Animal Experiment Committee of the Tokyo Metropolitan Institute of Gerontology and were carried out according to its guidelines (Animal Protocol Approval Numbers 17012 and 20018).

Seven‐week‐old naïve male BALB/cAJcl mice were purchased from CLEA Japan, Inc. The mice were transported from the Fuji Breeding Center of CLEA Japan, Inc. to our animal facility. Three independent cohorts of 70‐73 mice were used. After arrival, mice were group‐housed (two to four per cage) in a plastic cage (250 × 182 × 139 mm), with paper chips for bedding (Paper Clean; Japan SLC, Inc.), covered with a stainless steel grid lid. The mice were acclimated for at least 1 week before the experiments started (8‐14 weeks of age at the start). Rooms were maintained on a 12‐hour light/dark cycle (lights on at 7:00 am) and at 23 ± 2°C. The mice were provided with filtered tap water and food pellets (CRF‐1; Oriental Yeast Co., Ltd.) ad libitum, although mice assigned to the stress conditions described below did not have access to water or food for the duration of stress exposure. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee of Fujita Health University.