Osteogenic Differentiation of hFOB Cells

The osteogenic differentiation was assessed quantitatively (ELISA tests) and qualitatively (CLSM observations) using human osteoblasts. The hFOB 1.19 cells were seeded directly on the biomaterial surfaces at a concentration of 1 × 10⁵ cells/sample and cultured for 7, 14, and 21 days at 37 °C. The osteogenic medium was changed every third day of the experiment. The ELISA tests (ELISA Kit for Collagen Type I—COL1, ELISA Kit for Bone Alkaline Phosphatase—bALP, and ELISA Kit for Osteocalcin—OC, Cloud-Clone Corp., Wuhan, Hubei) were performed using cell lysates, which were prepared according to the protocol described in detail by Przekora and Ginalska [38 (link)]. Moreover, after 21-day incubation, osteogenic markers were visualized using immunofluorescence staining according to procedure described previously [34 (link)]. In this case, rabbit polyclonal anti-collagen I antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-osteocalcin antibody (Bioss, ThermoFisher Scientific, Waltham, MA, USA), and goat anti-rabbit IgG (H + L) antibody-conjugated with AlexaFluor^® 488 (Abcam, Cambridge, UK) were used. Cell nuclei were dyed with Hoechst 33342 (Sigma-Aldrich, Warsaw, Poland). The cells were observed under CLSM (Olympus Fluoview equipped with FV1000, Shinjuku, Japan).