Normal human impacted third molars (n = 18) were collected from sixteen adults (18–20 yr of age) at the Dental Clinic of the National Institute of Dental & Craniofacial Research (NIDCR) under approved guidelines set by NIH Office of Human Subjects Research and University of Southern California IRB. Root apical papilla was gently separated from the surface of the root, minced and digested in a solution of 3 mg/ml collagenase type I (Worthington Biochemicals Corp., Freehold, NJ) and 4 mg/ml dispase (Roche Diagnostic/Boehringer Mannheim Corp., Indianapolis, IN) for 30 minutes at 37°C. Single cell suspensions of SCAP were obtained by passing through a 70 µm strainer (Falcon, BD Labware, Franklin Lakes, NJ), seeded at 1×104 into 10 cm culture dishes (Costar, Cambridge, MA), and cultured with alpha-Modification of Eagle's Medium (GIBCO/Invitrogen, Carlsbad, CA) supplemented with 15% FBS (Equitech-Bio Inc., Kerrville, TX), 100 µM L-ascorbic acid 2-phosphate (WAKO, Tokyo, Japan), 2 mM L-glutamine (Biosource/Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO2. To assess colony-forming efficiency, day 10 cultures were fixed with 4% formalin, and then stained with 0.1% toluidine blue. Aggregates of ≥50 cells were scored as colonies. The proliferation rate of sub-confluent cultures (first passage) of SCAP was assessed by BrdU incorporation for 6 hours, using BrdU staining Kit (Zymed/Invitrogen). Conditions for the induction of calcium accumulation were as reported previously [18] (link). Calcium accumulation was detected by 2% Alizarin Red S (pH 4.2) staining. The induction of adipogenesis was as previously reported [8] (link). DPSCs and PDLSCs were isolated and cultured as previously described [8] (link), [9] (link). In some experiments, SCAP, DPSCs, and PDLSCs were obtained from the same donor or donors. All primary cells used in this study were at 1–3 passages. For each experiment, same passage of SCAP, DPSCs, and PDLSCs were used.
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