25 cm2 polystyrene culture flask

Isolation and characterization of DPSCs were carried out using the explant culture method [6 (link)]. Pulp tissue was minced into tiny fragments. The pieces were placed in 35 mm polystyrene plastic culture dishes. A sufficient amount of fetal Bovine Serum (FBS) (Gibco, Rockville, MD, USA) was added to the tissues to cover them completely. The tissues were incubated for 24 h at 37 °C and 5% CO₂. The DPSCs culture system was maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 20% FBS and antibiotic-antimycotic solution at the same temperature and CO₂ conditions. The culture medium was replenished twice weekly, and the cell growth, health, and morphology were evaluated regularly with an inverted phase-contrast microscope. At 70–80% confluence, the cells were detached using 0.25% Trypsin-EDTA solution (Invitrogen, Carlsbad, CA, USA) and transferred to a 25-cm² polystyrene culture flask (Nunc, Rochester, NY, USA). Confluent DPSCs were detached using 0.25% Trypsin-EDTA solution, and then continuously passaged in for expansion and further experiments. Cells from passages 2 to 4 were used in the experimentation.

Scientific research (IRB)-College of Dentistry, Jazan University approved the present study (Ref. no. CODJU-19714). After obtaining the informed consent, DPSCs were isolated from a healthy permanent tooth (age: 14–25 years) extracted for orthodontic purposes with appropriate oral hygiene (n = 3) by using the explant culture method described previously [42 (link)]. Pulp tissue was macerated into tiny pieces and were transferred to 35 mm polystyrene plastic culture Petri dishes. An adequate amount of fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) was added to the tissues to cover them completely. A 24 h incubation protocol was performed at 37 °C and 5% CO₂ for the explants immersed in FBS; the DPSCs were further expanded in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 20% FBS and antibiotic-antimycotic at the ambient temperature and CO₂ condition. The culture medium was replenished once in two weeks, and the proliferation and morphological characteristics were ascertained regularly with an inverted phase-contrast microscope. After 70–80% confluence was attained, the cells were detached using 0.25% Trypsin-EDTA solution (Invitrogen, Carlsbad, CA, USA) and transferred to a bigger 25-cm² polystyrene culture flask (Nunc, Rochester, NY, USA). Cells from passage 2 to 4 were used for all the experiments.

Isolation and characterization of DPSCs from the extracted teeth was carried out using the explant culture method [16 (link)]. The sampled pulp tissue was minced into tiny fragments. The pieces were placed in 35 mm polystyrene plastic culture dishes. A sufficient amount of fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) was added to the tissues to cover them completely. The tissues were incubated for 24 h at 37 °C and 5% CO₂. The whole DPSCs culture system was further maintained in DMEM (Invitrogen, Carlsbad, CA, USA). It was supplemented with 20% FBS and antibiotic-antimycotic solution at the same temperature and CO₂ conditions. The culture medium was replenished twice weekly. The cell growth, health and morphology were monitored regularly with an inverted phase-contrast microscope. After 70–80% confluence was attained, the cells were treated with 0.25% Trypsin-EDTA solution (Invitrogen, Carlsbad, CA, USA) for detachment and transferred to a bigger 25-cm² polystyrene culture flask (Nunc, Rochester, NY, USA). Confluent DPSCs were detached using 0.25% Trypsin-EDTA solution and continuously passaged for expansion. Cells from passages 2 and 6 were used for all experimental assays. DPSCs from patients with periodontitis were denoted as pDPSCs, and cells from periodontally healthy subjects were denoted as hDPSCs.