Figure 5.

Omniscan-induced cells form mineralized nodules in vitro. PBMCs (150 × 10³) were plated in fibronectin-coated chamber slides or in 6-well culture plates at 5 × 10⁶ cells per well in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B, and treated with a 0.5-mmol/L concentration of Omniscan for 8 days. On day 9 the medium was removed from the wells and replaced with osteogenic differentiation medium with supplements, as mentioned in Materials and Methods. Panels show representative light microscopic images. PBMCs on day zero (A) and after 8 days of Omniscan treatment (B). C–E: Omniscan-treated cells in osteogenic differentiation medium for different days as indicated. Calcium deposition and mineralized nodules as detected by alizarin red (F) and with von Kossa staining (G) after 7, 16, and 21 days, and 5 weeks in osteogenic medium. H: A high-power view showing significantly increased calcium deposition and mineralized nodule formation (alizarin red staining) by Omniscan-induced cells compared with controls. I: A high-power view showing significantly increased calcium deposition and mineralized nodule formation (brownish black by von Kossa staining) by Omniscan-induced cells compared with controls.