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Dental pulp was harvested from the extracted incisors of 6-week-old male green fluorescent protein (GFP)-transgenic SD rats (SD-Tg(CAG-EGFP)Cz-0040sb) (Japan SLC, Inc., Hamamatsu, Japan) and suspended in phosphate-buffered saline containing 0.1% collagenase and 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA). DPSCs were cultured in an alpha modification of Eagle’s medium (α-MEM) (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) with 20% fetal bovine serum (GIBCO) on plastic dishes at 37 °C in 5% humidified CO₂. Non-adherent cells were washed off, and adherent cells were continuously expanded until the third passage. DPSCs expressed the cell surface markers of mesenchymal stem cells, such as CD29 and CD90, and their pluripotency to chondrocytes, adipocytes, and osteoblasts was confirmed [13 (link)].

Dental pulp was harvested from the extracted incisors of 6-week-old male Sprague–Dawley rats (Chubu Kagakushizai, Nagoya, Japan) or green fluorescent protein (GFP)-transgenic SD rats (SD-Tg(CAG-EGFP)Cz-0040sb; Japan SLC, Inc., Hamamatsu, Japan). DPSCs were isolated from the dental pulp by digestion with phosphate-buffered saline containing 0.1 % collagenase and 0.25 % trypsin. DPSCs were cultured with alpha modification of the Eagle’s medium (α-MEM; GIBCO, Billings, MT, USA) supplemented with 20 % fetal bovine serum embryonic stem cell-qualified (GIBCO) [21 (link)]. DPSCs from passage 3 to 6 were used for all experiments. All experimental protocols were conducted according to the Regulations for Animal Experiments in Aichi Gakuin University, and were approved by the Institutional Animal Care and Use Committees of Aichi Gakuin University.

DPSCs were isolated from dental pulp tissue by collecting the incisors of 6-week-old male Sprague-Dawley rats (Chubu Kagakushizai, Nagoya, Japan) and Green fluorescent protein (GFP)-transgenic SD rats (SD-Tg[CAG-EGFP] Cz-0040sb; Japan SLC Inc., Hamamatsu, Japan) using 0.1% collagenase and 0.25% trypsin. DPSCs were cultured in minimum essential medium Eagle, alpha modification (MEM-α; Gibco Laboratories Inc., Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; GIBCO) and 1% penicillin streptomycin at 37 °C in a humidified 5% CO₂ incubator. Nonadherent cells were washed away, and adherent cells were grown continuously until passage 3. DPSCs were used at passages 3–6 for all experiments.
This study was approved by the Institutional Animal Care and Use Committee of Aichi Gakuin University (AGUD 437-2), and all animal experiments were carried out following national guidelines and the relevant national laws on the protection of animals.