Hfobs

The influence of the scaffolds on cell viability was evaluated via the reduction reaction of the tetrazolium salt MTT (3(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), purchased from Sigma Aldrich, Germany. Scaffolds were exposed to the UV light for 30 min for sterilization. The samples were soaked into 3 mL of Advanced DMEM supplemented with 5% FBS and incubated for 24 h at 37°C in an atmosphere containing 5% CO₂. The human bone tissue derived osteoblast cells (hFOBs, ATCC, UK) (10,000 cells/well) were seeded into a 96-well microtitre plate with a final volume of 100 μL of Advanced DMEM with 5% FBS. The material samples (supernatants of the starting samples) were added to the cells after 24 h of incubation at 37°C in 5 wt.% CO₂ in four parallels and in several dilutions (undiluted, 1:2, 1:4, 1:8 and 1:16). As control, Advanced ADMEM and 5% FBS was added to the cells. After 24 h of treatment, cell viability was determined using the MTT test (Maver et al., 2018 (link); Stergar et al., 2017 (link)). For this purpose, 10 wt.% reagent was added to the medium and discarded after 4 h. Then, 100 μL of DMSO was added and after 5 min, the absorbance was measured spectrophotometrically at 570 nm using the Varioskan multiplate reader (ThermoFisher, Germany).

A human fetal osteoblast cell line (hFOBs; American Type Culture Collection—ATCC^®, Manassas, VA, USA) was cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12), without phenol red (PAN-Biotech GmbH, Aidenbach, Germany), containing 2.5 mM L-glutamine (PanBiotech^®, Aidenbach, Germany), 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 0.3 mg/mL geneticin (G418; PAN-Biotech GmbH, Aidenbach, Germany). The hFOBs were used between 12 and 14 passages.
Human periodontal ligament fibroblasts (hPLFs) were isolated from healthy human periodontal tissue and purchased from Innoprot^®, Spain. They were cultured in DMEM-F12 culture medium supplemented with stable glutamine and 1.2 g/L NaHCO₃ (PAN-Biotech GmbH, Aidenbach, Germany) with 10% FBS (Sigma-Aldrich) and 1% (v/v) penicillin–streptomycin (PAN-Biotech GmbH, Aidenbach, Germany). The hPLF cells were used at the third passage. Both hFOBs and hPLFs were maintained at 37 °C in a humidified atmosphere of 5% CO₂, and the culture medium was changed twice a week.

The human fetal osteoblast cell line (hFOBs) was purchased from the American Type Culture Collection (CRL-11372, ATCC^®, Manassas, VA, USA). The cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM:F12, 1:1) without phenol red (PAN-Biotech GmbH, Aidenbach, Germany), containing 10% (v/v) fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 2.5 mM L-glutamine (PAN-Biotech^®, Germany) and 0.3 mg/mL antibiotic G418 (PAN-Biotech GmbH, Germany).
The human periodontal ligament fibroblasts (hPLFs) were acquired from Innoprot^® (P10867, Innoprot^®, Derio, Spain). The cells were cultured in culture medium composed of DMEM:F12 Mix (1:1) with stable glutamine and 1.2 g/L NaHCO₃ (PAN-Biotech^®, Germany), supplemented with 1% (v/v) penicillin/streptomycin and 10% (v/v) FBS.
Both the osteoblasts and fibroblasts were maintained in an incubator at 37 °C and 5% (v/v) CO₂, in a humidified atmosphere of 100% relative humidity. The medium of both cell lines was changed twice a week, and both cells were observed every day using an inverted microscope (Kern^®, Lohmar, Germany) at 10× magnification to detect morphologic changes.