Cell culture and characterization

The present study was approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University (Qingdao, China; no. QYFYWZLL26135). Written informed consent was obtained from each patient or from the parents/guardians of those participants who were minors. To obtain HPDLCs, premolars (healthy orthodontic tooth extractions) without caries, periodontal disease nor periapical periodontitis freshly extracted between December 2017 and December 2018 were selected. The patients (20 males and 20 females) were between 12 and 25 years old. All the cells from 60 teeth were pooled for different assays. The primary culture of HPDLCs was established according to a method described previously (15). First, the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; HyClone; Cytiva) containing 20% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) in an atmosphere under 5% CO₂ at 37˚C. The morphology and growth of the cells were observed. After the cells reached 80% confluency, they were passaged by trypsinization (Beijing Solarbio Science & Technology Co., Ltd.). After passaging, the medium was replaced with medium containing 10% FBS. The third-generation cells were identified by vimentin (cat. no. PB9359; Boster Biological Technology) and keratin (cat. no. BM0031; Boster Biological Technology) staining with the streptavidin-biotin complex (cat. no. SA1021, Boster Biological Technology) immunohistochemical method. Cells in the third to fifth generation with good viability were used for the subsequent experiments.

Primary HUASMCs (cat. no. 8030) were purchased from ScienCell Research Laboratories, Inc. and subcultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂ after recovery. The third generation HUASMCs were prepared on cell slides at 1x10⁴ cells/ml for α-smooth muscle actin (α-SMA; cat. no. BS70000; Bioworld Technology, Inc.) immunofluorescence staining. The cells in the exponential growth phase from the third generation to the eighth generation were used for the subsequent experiments.