Pt 5025

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.

Human dental pulp stem cells (hDPSCs, Lonza, PT-5025, Basel, swiss) were cultured in M-DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 μg/mL streptomycin at 37 °C in a 5% CO₂ incubator. For the experiments, hDPSCs were cultured in standard odontogenic induction medium, namely, M-DMEM supplemented with 10% FBS, antibiotics, 10 nM dexamethasone (Sigma–Aldrich, D4902, St Louis, MO, USA), 50 ng/mL BMP-2 (Sigma–Aldrich, B3555, St Louis, MO, USA), 20 ng/mL TGF-β1 (Bio Legend, 580704, San Diego, CA, USA), and 5 ng/mL FGF-2 (Bio Legend, 571504, San Diego, CA, USA) for 3 or 5 days at 37 °C in a 5% CO₂ incubator. For drug exposure, MS-275 (Apexbio, A8171, Houston, TX, USA) was initially dissolved in dimethyl sulfoxide (DMSO), then the concentration was adjusted with PBS, and it was added to the media. The medium was changed every 2–3 days. The hDPSCs from passages 3 to 5 were used for the experiments. DMSO (Sigma-Aldrich, D5879, St Louis, MO, USA) was added to the control at an additional 1/5000 to offset the effect of DMSO on the experiment.

Proietics™ human Dental Pulp Stem Cells (DPSCs) were harvested from an adult third molar and cryopreserved in the primary passage (PT‐5025; Lonza, Alpharetta). DPSCs were maintained and expanded in Dulbecco Modified Eagle Medium (DMEM) at 37°C and 5% CO₂. Cells were passaged at 80% confluence, with a medium change taking place every 2‐3 days.

HeLa (ATCC), human foreskin fibroblasts (HFFs)^{28 (link)}, DPSCs (PT-5025) and MSCs derived from bone marrow (MSC, PT-2501) cell lines (Lonza) were cultured with the same media composition as DPSCs later passages (10% FBS and 1% Penicillin-Streptomycin in DMEM). Human embryonic stem cells (hESCs) [Elf-1(NIH_hESCs Registry #0156)] were cultured as previously described in hESCs media^{29 (link)}. Briefly, cells were grown in either mTeSR1 media (StemCell Technologies) or 2iL-I-F media (DMEM/F-12 media supplemented with 20% knock-out serum replacer (KSR), 0.1 mM nonessential amino acids (NEAA), 1 mM sodium pyruvate, penicillin/streptomycin (all from Invitrogen, Carlsbad, CA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), 1 µM GSK3 inhibitor (CHIR99021, Selleckchem), 1 µM of MEK inhibitor (PD0325901, Selleckchem), 10 ng/mL human LIF (Chemicon), 5 ng/mL IGF1 (Peprotech) and 10 ng/mL bFGF. In one differentiation experiment, the DPSCs were primed for short term (4 days) with TeSR media supplemented with a metabolite-cocktail (TeSRmeta) containing kynurenine (KY) and methylnicotinamide (MNA), which was previously shown to be beneficial to stem cells^{29 (link)}.

For the cell cytotoxicity test, hDPSCs purchased from Lonza (PT-5025; Basel, Switzerland) at passage 8 were used. The cytotoxicity of the synthesized powder was evaluated using the MTT assay (Ahn et al., 2020 (link)). To determine the proper concentration of powder, a pre-test was conducted with four different amounts of powder (125, 250, 500, and 1,000 μg/well). hDPSCs were seeded at a density of 1 × 10⁴ cells/well in a 24-well culture plate. The cells were cultured in the material extraction full media-containing α-MEM (Gibco), 10% FBS (Merck), 1% penicillin-streptomycin (Gibco), and 5 μg/ml plasmocin (InvivoGen, San Diego, CA) in a 5% CO₂ incubator at 37°C for 24 h. The culture medium in each well was replaced with 100 μL fresh culture medium containing 10 μL MTT solution (5 mg/ml MTT in sterile phosphate-buffered saline; Sigma-Aldrich, St. Louis, MO, United States) and incubated for 4 h at 37°C and 5% CO₂. The medium was replaced with 100 μL of dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, United States) for 5 min at 37°C. The absorbance was subsequently measured at 570 nm using a microplate reader (Dynex, Lincoln, United Kingdom). After determining the proper powder concentration, the MTT assay was conducted using the same process for 1, 2, 3, and 7 days. All results were obtained from three independent experiments and control wells were also prepared with full media.

The hDPSCs were purchased from Lonza (PT-5025, Basel, Switzerland) and it is isolated from adult third molars collected during the extraction of a donor's ‘wisdom’ teeth. The hDPSCs were maintained in Eagle's minimum essential medium (MEM, Corning, Manassas, VA, USA), supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA), 1% penicillin–streptomycin (Gibco BRL), and 5 μg/mL plasmocin (InvivoGen, San Diego, CA, USA) at 37 °C in a humidified incubator with 5% CO₂, with a medium change twice a week. We used commercially available propofol (Fresenius Kabi Austria GmbH, Hafnerstrabe, Austria), which was dissolved into dimethyl sulfoxide (DMSO, Sigma–Aldrich, St Louis, MO, USA). The cultured cells were treated with propofol (5, 20, 50 and 100 μM).