Figure 2: The mutant bone marrow stromal cells exhibit abnormal osteoblastic gene expression and diminished mineralization in vitro. Bone marrow stromal cells were isolated from Crouzon (Cz) and wild type (WT) littermates and then cultured with ascorbate for the indicated number of days to induce osteoblast differentiation. Runx2, bone sialoprotein (BSP), osteocalcin (OCN), and tissue non-specific alkaline phosphatase (TNAP) and collagen type 1 alpha1 (col1a1) mRNA levels were measured by real-time PCR. Black lines represent wild type; grey lines represent Crouzon (a, b, c, d, and e). Results are presented as normalized to Hprt1. Cells were cultured with ascorbate and -glycerophosphate to induce mineralization for 18 days (f). Mineralized nodules were stained by Von Kossa and quantified by densitometry. Cells were cultured with ascorbate for 18 days, and alkaline phosphatase (Tnap/Alpl/Akp2) enzyme activity was quantified by incubation of cells with a colorimetric substrate. Enzyme activity was quantified by densitometry (g). Results shown are means ± standard deviations from triplicate experiments for all data shown. between genotypes.