Hdpscs

The hDPSCs (Lonza, Walkersville, MD, USA) were cultured and expanded with Alpha-minimum eagle’s medium (α-MEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and were supplemented with 1% v/v penicillin-streptomycin (P/S) in physiological conditions held constant (37 °C air temperature, 5% CO₂ atmosphere). The mediums were exchanged every 3 days. Subculturing was conducted when samples reached 70%–80% confluency through dissociation induced with TrypLE™ Select (1×) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Adherent cells on flask-bottoms were separated by 1.5 mL trypsin-EDTA (0.05% trypsin/0.02% EDTA, Life Technologies Co., Waltham, MA, USA) for 5 min at 37 °C in an incubator.
hDPSCs were prepared into cell suspensions and we adjusted cell concentrations to 1 × 10⁶ cells /mL. C-TCP, 3D-PLGA/TCP, and 3D-TCP (N = 8) samples were placed in a 24-well plate, and 15 μL of hDPSCs suspension was seeded onto each cubic disk for pre-culturing for 2 h. After cells were dropped to the scaffold, culture mediums were refilled.

In this study, we used human dental pulp stem cell line (HDPSCs; Royan Institute, Tehran, Iran). HDPSCs were cultured and expanded in low-content glucose Dulbecco’s modified Eagle medium (DMEM/LG; Gibco), with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Biosera). The culture plates were maintained at 37ºC in a humidified atmosphere with 5% CO₂. The cells between passages 3 to 6 were subjected to various experiments.

Human dental pulp stem cells (hDPSCs) (Beijing Tason Biotech) were cultured in Dulbecco’s modified Eagle medium (Gibco) with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere. Lipofectamine 3,000 Transfection Reagent (ThermoFisher) was used for transient transfections. Forty-eight hours after transfection, hDPSCs were fixed with 4% paraformaldehyde, permeabilized using 0.25% Triton-X100(Gibco), stained with 4′,6-diamidino-2-phenylindole(Abcam) and mounted. Images were taken using an LSM 510 Meta confocal microscope (Leica Camera AG, Wetzlar, Germany) with a ×20/2.00 objective lens.