hFOB 1.19

Characterization of the Mineralization Process in Human Fetal Osteoblastic Cell Line (hFOB 1.19 Cells) and Osteosarcoma Cell Line (Saos-2 Cells)

Osteoblast and chondrocyte-mediated mineralization employs matrix vesicles (MVs) rich in TNAP and annexins, essential for apatite formation. Bozycki et al. compared mineralization capabilities of hFOB 1.19 osteoblastic cells and Saos-2 osteosarcoma cells.

Human fetal osteoblastic hFOB 1.19 and osteosarcoma Saos-2 cells were cultured for seven days in media without (Resting, R) or with 50 µg/mL ascorbic acid (AA) and 7.5 mM β-glycerophosphate (β-G) (Stimulated, S). Resting cells were elongated (Fig. 1A and B, arrows). AA and β-GP treatment caused hFOB 1.19 cells to halve their longest axis (Fig. 1C and 1E), while Saos-2 cells' length remained unchanged (Fig. 1D and E). Alizarin red-S/cetyl pyridinium chloride (AR-S/CPC) analysis showed stimulated cells formed more calcium nodules (hFOB 1.19—twice, Saos-2—nearly thrice) versus resting cells (Fig. 2A and B). Levamisole (Lev), a TNAP inhibitor, largely blocked nodule formation (Fig. 2A and B). K201 increased amorphous calcium complexes in resting cells (Fig. 2A and B). TNAP activity was lower in hFOB 1.19 than Saos-2 cells (Fig. 2C and D), increasing in stimulated conditions (Fig. 2C and D). Lev reduced TNAP activity to basal levels, while K201 had minimal impact (Fig. 2C and D).

Fig. 1. Morphology of hFOB 1.19 (A,C) and Saos-2 (B,D) cells in resting (A,B) and stimulated (C,D) conditions (Bozycki L, Mroczek J, et al., 2021).

Fig. 2. Mineralization level of hFOB 1.19 (A,C) and Saos-2 (B,D) cells in resting conditions (white) or after seven-day stimulation with AA and β-GP (red) (Bozycki L, Mroczek J, et al., 2021).

Determination of the Effects of SeMet on Osteogenic Differentiation Using Alizarin Red Dye and Image Analysis

Osteoporosis stands as a widespread condition that causes fractures and bone metabolism factors affect its development. Multiple scientific studies demonstrate that selenium serves as an essential trace element with beneficial effects on bone health. Sahin et al. investigated if selenomethionine (SeMet), a selenium variant, accelerates the differentiation process of osteoblastic hFOB cells.

Two experimental groups were created using SeMet concentrations of 5, 10, 15, and 25 µM with one group including osteogenic differentiation cocktail (OD +) and the other excluding it (OD −). At the end of the 28th day, both models were stained with alizarin red, and calcium deposits were examined. It was observed that calcium deposits increased in a dose-dependent manner in both the OD( +) and OD( −) groups in which SeMet was applied (Fig. 3).When alizarin red stainings were analyzed with the ImageJ program, it was determined that SeMet increased the amount of calcium deposits in a concentration-dependent manner in both experimental setups (SeMet concentrations + OD( +) or OD( −)), and in 5–10-15 and 25 µM SeMet + OD( +) administrations, each concentration was significant compared to the control (p < 0.05). In 5–10-15 and 25 µM SeMet + OD( −) administrations, the last two SeMet concentrations (15–25 µM) were determined to be significant compared to the control (p < 0.05) (Fig. 3).

Fig. 3. Calcium deposit results (alizarin red staining) (Sahin E, Arafat M, et al., 2024).