Osteogenic differentiation medium

Human periodontal ligament fibroblasts (HPLFs) (ScienCell, Cat. No. 2630) were cultured in fibroblast medium (ScienCell, Cat. No. 2301), which consisted of 100 mL of basal medium, 10 mL of fetal bovine serum (FBS, Cat. No. 0010), 5 mL of fibroblast growth supplement (FGS, Cat. No. 2352), and 5 mL of antibiotic solution (P/S, Cat. No. 0503). After embedding the cells within the collagen scaffold, we cultured them further in osteogenic differentiation medium (ScienCell, Cat. No. 7531) [30 (link)].

BMSCs were cultured in modified MEM Alpha medium for 3 days and then replaced with osteogenic differentiation medium (ScienCell Research Laboratories, United States) for 7 days for alkaline phosphatase (ALP) staining and 14 days for alizarin red (AR) staining. The cells were washed with PBS and fixed with 4% paraformaldehyde at 37°C for 15 min. The cells were stained with an alkaline phosphatase staining kit (Beyotime, China) and stained with 1% AR at room temperature for 15 min. The stained specimens were then observed with an inverted microscope (Olympus Corp, United States) and a digital camera.

Characterization of DPSCs (3rd–5th passage) was examined by multipotential differentiation. Angiogenic differentiation was evaluated using an endothelial tube formation assay (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. In brief, 1.5 × 10⁴ cells cultured in angiogenic induction medium for 10 days were seeded in a 16-well chamber slide (Thermo Fisher Scientific) coated with 50 µL extracellular matrix (ECM) gel. After 4 h, the cells were incubated with 1× Staining solution and imaged by an Olympus FV1000 confocal microscope (Olympus, Center Valley, PA, USA). For odontogenesis, 1.5 × 10⁴ cells were cultured with osteogenic differentiation medium (ScienCell, Carlsbad, CA, USA) in a 6-well plate, and Alizarin Red S staining (Sigma-Aldrich) was used to evaluate the cellular calcium deposit at 10 and 20 days. The cells fixed in 4% formaldehyde were stained with 40 mM Alizarin Red S for 30 min (min) and imaged by an Olympus BX60 microscope (Olympus).