Dispase 2

BCTC, AMTB, and fura-2AM were purchased from TOCRIS Bioscience (Bristol, United Kingdom). CPZ and icilin were procured from Cayman Chemical Company (Ann Arbor, Michigan, U.S.A.). Medium and supplements for cell culture were ordered from Life Technologies Invitrogen (Karlsruhe, Germany) or Biochrom AG (Berlin, Germany). Melanocyte Growth Medium M2 was obtained from Promocell (Heidelberg, Germany). Dispase II was ordered from Boehringer (Ingelheim, Germany) and accutase was provided by PAA Laboratories (Pasching, Austria). Unless otherwise stated, all other reagents were procured from Sigma (Deisenhofen, Germany).

Human third molars with immature roots were obtained from healthy donors aged 12 to 15 years in the clinic at the School of Stomatology affiliated with China Medical University. Informed consent was obtained from all patients and their parents. The apical papilla was gently separated and digested with dispase II (Boehringer Ingelheim, Mannheim, Germany) and collagenase type I (Worthington Biochemical Co., Lakewood, CO, USA). Single-cell suspensions were seeded and cultured in alpha-minimum essential medium (α-MEM, HyClone, Logan, UT, USA) supplemented with 15% (v/v) foetal bovine serum (FBS, MRC, Uruguay), 1% (v/v) penicillin-streptomycin solution (HyClone), 2 mM L-glutamine (BioSource/Invitrogen, USA), and 0.1 mM L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 °C with 5% CO₂. The expression of MSC surface markers, including CD31, CD34, CD45, CD73, CD90, and CD105, was detected by flow cytometry. The multipotent differentiation potential of SCAP, including the potential for osteogenesis and adipogenesis, was evaluated using osteogenic and adipogenic differentiation media for 4 weeks. Alizarin red S and oil red O staining were used to detect the formation of mineralized nodules and lipid droplets, respectively.