Craniosynostosis-associated Fgfr2(C342Y) mutant bone marrow stromal cells exhibit cell autonomous abnormalities in osteoblast differentiation and bone formation

Abstract

We recently reported that cranial bones of Fgfr2(C342Y/+) craniosynostotic mice are diminished in density when compared to those of wild type mice, and that cranial bone cells isolated from the mutant mice exhibit inhibited late stage osteoblast differentiation. To provide further support for the idea that craniosynostosis-associated Fgfr mutations lead to cell autonomous defects in osteoblast differentiation and mineralized tissue formation, here we tested bone marrow stromal cells isolated from Fgfr2(C342Y/+) mice for their ability to differentiate into osteoblasts. Additionally, to determine if the low bone mass phenotype of Crouzon syndrome includes the appendicular skeleton, long bones were assessed by micro CT. Fgfr2(C342Y/+) cells showed increased osteoblastic gene expression during early osteoblastic differentiation but decreased expression of alkaline phosphatase mRNA and enzyme activity, and decreased mineralization during later stages of differentiation, when cultured under 2D in vitro conditions. Cells isolated from Fgfr2(C342Y/+) mice also formed less bone when allowed to differentiate in a 3D matrix in vivo. Cortical bone parameters were diminished in long bones of Fgfr2(C342Y/+) mice. These results demonstrate that marrow stromal cells of Fgfr2(C342Y/+) mice have an autonomous defect in osteoblast differentiation and bone mineralization, and that the Fgfr2(C342Y) mutation influences both the axial and appendicular skeletons.

Figure 1

Subcutaneous implant placement. This schematic shows the position of six implants that were placed subcutaneously, on the dorsal surface of immunodeficient mice.

Figure 2

The Fgfr2^C342Y mutant bone marrow stromal cells exhibit abnormal osteoblastic gene expression and diminished mineralization in vitro. Bone marrow stromal cells were isolated from Crouzon Fgfr2^C342Y/+ (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 β-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. *P < .05 between genotypes.

Figure 3

Histologic staining of implants. Ossicles formed eight weeks after subcutaneous implantation of cells mixed with a collagenous matrix were stained by trichrome (a and b) or by hematoxylin and eosin (c and d). Note the greater amount of deep blue (a) and light pink (c) staining in ossicles formed by wild type cells, indicative of greater bone formation by these cells. In comparison, note the greater amount of light blue (b) and greyish-pink (d) staining in ossicles formed by Crouzon cells, indicative of greater amounts of original implanted collagen. Also note that ossicles formed by either wild type or mutant cells contain bone marrow.

Figure 4

The Fgfr2^C342Y mutation inhibits mineralized tissue formation in vivo. (a) Radiographic image of representative implants shows increased radiodensity of ossicles formed by wild type (WT), as compared to those formed by Crouzon (Cz) cells. (b) Quantification of radiodense tissue by densitometry confirms that implanted wild type cells formed significantly greater amounts of mineralized tissue compared to mutant cells (n = 12 implants per genotype). *P < .05 versus WT. (c) Homogenized implants formed by wild type cells also have significantly greater levels of alkaline phosphatase enzyme expression than homogenized implants formed by Crouzon cells (n = 3 implants per genotype). *P < .05 versus WT.

Figure 5

Diminished cortical bone volume and density in the long bones of Fgfr2^C342Y/+ mice. Micro-CT analyses demonstrate significantly diminished cortical bone volume/total volume, bone mineral density, and tissue mineral density in tibias of Fgfr2^C342Y/+ (Cz) mice as compared to wild type (WT) mice. *P < .05 between genotypes.

Figure 6

Similar trabecular bone volume and density in the long bones of Fgfr2^C342Y/+ mice. Micro-CT analyses demonstrate no significant differences in bone volume, bone volume/total volume, bone mineral density, and tissue mineral density in tibias of Fgfr2^C342Y/+ (Cz) mice as compared to wild type (WT) mice.