Hmscs

The hMSCs derived from bone marrow were purchased from ATCC (Manassas, VA, USA), Human dental pulp stem cells (namely: “old donor stem cells” from 15 to 30 years old) were purchased from Lonza, for the young donor group, stem cells derived from human exfoliated deciduous teeth stem cells (Creative bioarray) were utilized. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza S.r.l., Milano, Italy) supplemented with 10% fetal bovine serum (FBS) (Bidachem S.p.A., Milano, Italy) and 1% penicillin/streptomycin (P/S) (EuroClone, Milan, Italy) to form complete DMEM (cDMEM) in T25 flasks.

hMSCs (PromoCell) were expanded in basic cell culture media (αMEM supplemented with 0.2 mM ascorbic acid, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% FBS) and the cultures were kept at 5% CO₂ in a humidified atmosphere at 37 °C. hMSCs at passage 4 were seeded in 12-well cell culture plates at 5000 cells per cm². For experiments on nanoparticle films, hMSCs were seeded at the same seeding density in a non-treated 6-well culture plate. hMSCs were allowed to adhere for 24 h.
Human osteoblast cells (FOB) were purchased from ATCC (hFOB 1.19). Frozen cells were thawed and expanded in specific media consisting of a 1:1 mixture of Ham’s F12 Medium Dulbecco’s Modified Eagle’s Medium and 2.5 mM L-glutamine along with 10% of G418 fetal bovine serum (ATCC), and the cultures were kept at 5% CO₂ in a humidified atmosphere at 37 °C. Human umbilical vein endothelial cells (HUVEC) purchased from Bio-connect were expanded in endothelial cell growth medium 211–500 (Sigma-Aldrich, St. Louis, MO, USA), and the cultures were kept at 5% CO₂ in a humidified atmosphere at 37 °C. FOB at passage 4 and HUVEC at passage 6 was seeded in a 96-well plate at 5000 cells per cm². Both FOB and HUVEC were allowed to adhere for 24 h before cytotoxicity experiments.