F344 rats

A total of 56 female, 6-week-old F344 rats were obtained from CLEA JAPAN (Tokyo, Japan). The animals were fed a diet, CE-2, purchased from CLEA JAPAN, and water was given ad libitum. The rat cages were placed in a conventional room, where the temperature was maintained at approximately 25 °C and the humidity was maintained at 55% to 70%. All experiments were performed according to the guidelines of The Laboratory Animal Care Committee of Azabu University (#170412-2: http://kitei.azabu-u.ac.jp/reiki/Re05_Hon_Main_Frame.exe?UTDIR=D:\EFServ2\ss000001DD\reiki&TID=2&SYSID=1331). Changes in body weight were recorded every week to assess the general health of the rats.

F344 rats (five-week-old, male) (CLEA Japan Inc., Tokyo, Japan) were sacrificed, and the femur and tibia are harvested after removing the muscles. Then, the bones were broken into small pieces using bone scissors. After washing with phosphate buffered saline (PBS), the bone fragments were enzymatically dissociated in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich) supplementing with 0.1% type II collagenase (Sigma Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS, Sigma Aldrich) and 2% antibiotics/antimycotics for 1 h at 37°C. The digests were filtered by a 70 µm cell strainer (Corning Falcon, NY, USA) and the bone fragments remaining on the cell strainer were washed three times with PBS. The bone fragments were cultured with DMEM containing 10% FBS and 2% antibiotic–antimycotic solution in 10 cm plates at 37°C under 5% CO₂. After 3 days of culture, the bone fragments were removed, and the medium was changed. When cultured cells reached 80% confluence, they were sub-cultured as mesenchymal stem/progenitor cells (MSCs) until passage 3 (P3) for subsequent experiments.