Figure 3.

Effects of ascorbic acid supplementation on osteogenic markers expression in human cells cultured in pro-osteogenic cell culture media. Human AoSMC (A) and human dermal fibroblasts (B) were cultured in 5% FBS/DMEM supplemented with 5 mM beta-Glycerophosphate and 25 mcM Forskolin for four weeks. Osteogenic markers expression was measured by immunoassay as described in Materials and Methods. AoSMC (A) and human dermal fibroblasts (B) were seeded in separate 96 well plastic plates and grown to confluence. Cells were cultured in 2% FBS/DMEM supplemented or not with 200 mcM ascorbic acid for four weeks. Cell layers were washed with PBS and fixed with 3% formaldehyde. Immunoasssay for osteogenic markers on fixed cells was done by 2 hours incubation with primary monoclonal antibodies (R&D Systems) followed by 1 hour incubation with secondary goat anti-mouse IgG antibodies labeled with horse radish peroxidase (HRP). Retained peroxidase activity was measured as optical density at 450 nm and expressed as percentage of control cell samples incubated in unsupplemented 2% FBS/DMEM. For more details refer to Materials and Methods section. *, indicate significant differences from ascorbate-free samples (P<0.05) in two-tailed t-test.