3.7.2. hFOB 1.19 Osteoblast Growth and Differentiation

Foam scaffolds were placed into 24-well culture plates (Nunclon Sphera, Nunc, Thermo Fisher Scientific, Waltham, MA, USA) and stabilized with a tissue adhesive (SuperVet Glue, EVi-MED, Liszki, Poland). The biomaterials were incubated at 34 °C and 5% of CO₂ for 24 h with 1 mL of hFOB 1.19 medium. Following the initial soaking, the medium was aspirated, and 20 µLs of fresh cell suspensions (2 × 10⁵ cells) were spotted at the center of each scaffold. Next, the cells were incubated for 2 h in a humidified 5% CO₂ atmosphere at 34 °C, and then the wells were filled with 1 mL of fresh medium. The cells seeded onto scaffolds were incubated at 34 °C to support cell proliferation.

To visualize immature osteoblasts within PGSs and PGS/HAp, the scaffolds were preincubated with cells for 7 days, washed with PBS and fixed with a 3.7% formaldehyde (Sigma-Aldrich, Saint Louis, MO, USA) solution for 10 min. The samples were mounted in a Frozen Section Media 22 (Leica Biosystems Inc., Buffalo Grove, IL, USA) and sectioned on a cryostat (Leica CM1950, Leica Biosystems Inc., Buffalo Grove, IL, USA). The resulting 20 µm-thick individual sections were stained using the May–Grunwald–Giemsa protocol and analyzed under a light microscope (Nikon ECLIPSE 50i, Nikon Inc., Tokyo, Japan) at the Laboratory of Microscopic Imaging and Specialized Biological Techniques at the University of Lodz.

For parallel experiments, the composites with hFOBs 1.19 were cultured at 39 °C in osteogenic medium, additionally supplemented with 50 μg/mL of ascorbate-2-phosphate (Sigma-Aldrich, Saint Louis, MO, USA), 1 μM of dexamethasone (Sigma-Aldrich, Saint Louis, MO, USA) and 10 mM of β-glycerophosphate (Sigma-Aldrich, Saint Louis, MO, USA).

For both culture conditions, the medium was replaced every three days. Cell culture supernatants were collected after 4, 7, 11, 14, 18 and 21 days of incubation and stored at −80 °C until further evaluation of the soluble markers of osteogenic differentiation.