The approval for the present study was provided by the Committee on Ethics of the Stomatology Hospital of Shandong University (protocol no. GR201806). From April, 2019 to September, 2019, freshly extracted teeth were collected from orthodontic volunteers aged 16-22 years at the Stomatology Hospital of Shandong University. All participants provided signed informed consent in accordance with the Helsinki Declaration. The method of hPDLSC isolation from teeth was similar to a previously reported one (31). Briefly, the extracted teeth were placed in a 15 ml centrifuge tube with α-MEM (Biological Industries) amended with 5% antibiotics (100 U/ml penicillin, 10 mg/ml streptomycin) on ice. They were transferred to an ultra-clean workbench as soon as possible for use in further experiments. Each tooth was washed thrice in phosphate-buffered saline (PBS; Biological Industries) containing 5% antibiotics. Periodontal ligament tissue from the middle third of the root surface was gently scraped with a sterile surgical blade and cut into tiny fragments, which were then seeded into culture dishes (25 cm). hPDLSCs were grown at 37°C in medium supplemented with α-MEM, 20% fetal bovine serum (FBS; Biological Industries) and 1% antibiotics in a 5% CO2 incubator. Following inoculation 3-4 h, the culture dish (25 cm) was turned over to ensure that the hPDLSCs touched the culture medium. The medium was refreshed after every 3 days until the cells grew out from the tissue sections. The cells were passaged at 80-90% confluence and the cells at passages 3 to 5 were used for further experiments.

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