Fig 3.

Effect of menaquinone‐4 (MK‐4) and 25‐hydroxyvitamin D₃ [25(OH)D₃], alone and combined, on periostin and collagen type I (COL‐1) expression in 3D and 2D cultures of primary human osteoblasts (hOBs): Relative mRNA expression levels for POSTN and COL1A1 in 3D osteospheres (A) and 2D cultures of primary hOBs (B) cultured with 10μM MK‐4 (M) or 0.01μM 25(OH)D₃ (D), alone and in combination (MD) at different days after vitamin addition. Relative mRNA expression levels were normalized to reference gene ACTB (2D cultures) and ACTB and B2M (3D cultures) and presented as fold‐change relative to unexposed controls. Values represent the mean ± SD. (C) Immunofluorescence characterization of cell nuclei (blue), periostin (green), and COL‐1 (red) in selected areas of frozen sections of 21‐day‐old mineralized osteospheres without treatment and treated with 10 μM MK‐4, 10μM MK‐4 and 0.01 μM 25(OH)D₃, and 0.01μM 25(OH)D₃ (scale bar = 50 μm). Mean intensities were quantified in five random regions of interest in each whole section. Significant differences were analyzed with SigmaPlot t test. Significant different from control at p < 0.05, p < 0.01, and p < 0.001.