Healthy premolars were collected from 10 adults (15–20 years of age; five men and five women) for orthodontic purposes at the Department of Oral and Maxillofacial Surgery, the Affiliated Stomatological Hospital of Sun Yat-sen University. All participants provided informed consent for the collection and use of their tissues. The protocols were approved by the University Ethics Committee.
PDLSCs were isolated and cultured as previously reported [6 (link)]. Briefly, periodontal tissue was gently separated from the surface of the middle third of the root and then digested with 3 mg/mL collagenase type I and 4 mg/mL dispase (Gibco-BRL, Gaithersburg, MD, USA) at 37°C for 1 h. Colony-forming cells were collected and then cultured in alpha modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL), 100 μg/mL streptomycin, 100 U/mL penicillin (Hyclone, Logan, UT, USA), 200 μM l -ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and 5 mM l -glutamine (Gibco-BRL) at 37°C in an atmosphere containing 5% CO2. The medium was changed every 3 days. Cells were used at passages 3–5. For intermittent PTH treatment, cells were treated with 10−12 M PTH (1-34) (Sigma-Aldrich) for 6 h, followed by another 6 h treatment after 32 h. Cells were harvested at various times after treatment with 10−12 M PTH (1-34) for RNA and protein isolation.
PDLSCs were isolated and cultured as previously reported [6 (link)]. Briefly, periodontal tissue was gently separated from the surface of the middle third of the root and then digested with 3 mg/mL collagenase type I and 4 mg/mL dispase (Gibco-BRL, Gaithersburg, MD, USA) at 37°C for 1 h. Colony-forming cells were collected and then cultured in alpha modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL), 100 μg/mL streptomycin, 100 U/mL penicillin (Hyclone, Logan, UT, USA), 200 μM