Pt 3005

Human dental pulp stem cells (hDPSCs) were purchased from Lonza (PT-5025, Lonza, Basel, Switzerland) in a commercially-available human dental pulp stem cell bullet kit (PT-3005, Lonza, Basel, Switzerland). New culture medium was replaced twice a day in a 37 °C humidified atmosphere with 5% CO₂. The cells started the experiment when the culture reached passage 3–6; the 3D scaffolds were pretreated with 75% ethanol for 1h, then subjected to ultraviolet light exposure for 30 min before the cell study. After the various duration of cultures, the PrestoBlue assay (Invitrogen, Carlsbad, CA, USA) was utilized, following the manufacturer’s manual instructions to experiment. Internal mitochondrial activity was investigated according to the color intensity of the reagent. The viability value was measured by a multi-well spectrophotometer (Infinite Pro M200, Tecan, Männedorf, Switzerland) at 570 nm with a reference wavelength of 600 nm. The only hDPSCs seeding was the control group.

In this study, we used human dental pulp stem cells (hDPSCs, PT-5025; Lonza, passages 3–8, Basel, Switzerland) to evaluate the biological properties of all 3D-printed scaffolds, and the cells were further cultured in a commercially available human dental pulp stem cell bullet kit (PT-3005; Lonza, Basel, Switzerland) under 5% CO₂ at 37 °C. The hDPSCs grew on the tissue flask, in which the medium was changed every 2 days. In the osteogenic and angiogenic assay, the hDPSCs were measured using osteogenesis/angiogenesis assay kits (StemPro™ osteogenesis differentiation kit/angiogenesis starter kit; Invitrogen) for estimation of cell differentiation.

Commercial human DPSCs (PT-5025, Lonza, Basel, Switzerland) were purchased. These cells were characterized and certified by the manufacturer.¹⁷ DPSCs were cultured in a growth medium (DPSC Bullet Kit PT-3005, Lonza, Basel, Switzerland), according to the manufacturer’s instructions.¹⁷ Cryopreserved cells were thawed, transferred to culture vessels (STARLAB, Hamburg, Germany), and incubated at 37°C with 5% CO₂. The medium was changed every two days. Subculturing was performed when the cells reached about 80% confluency. Cells at passage 3 were used for all assays. $\text{Cells (}\left.3\times {10}^4/\text{well}\right)$ were cultured in 24-well plates in the growth medium for 24 hours for each assay. Untreated DPSCs cultured in the growth medium served as a control for the experiments. All experiments were conducted in triplicate.