Craniosynostotic rabbit dura mater inhibits osteogenic differentiation of bone-derived cells

Objectives: Molecular interactions between cranial sutures and the underlying dura mater have been implicated in the pathogenesis of the premature fusion of one or more of the cranial sutures, termed craniosynostosis. Here, we utilized a rabbit model of nonsyndromic craniosynostosis to derive cells from the region of the fused coronal suture. Bone-derived cells were treated with osteogenic stimuli either in the presence or absence of dura mater-derived cells to test the hypothesis that factors expressed by the dura mater underlying fused coronal sutures would increase the osteogenic response of bone-derived cells.

Methods: Calvarial bones (from 69 day-old) and dura mater (from 10 day-old) were harvested from New Zealand White rabbits with prenatal coronal suture synostosis. Bone-derived cells were stimulated either through the addition of BMP4 to the culture medium (10, 50, or 200ng/ml) or by culturing them in osteogenic medium. Along with osteogenic stimulation, the effect of dura mater-derived cells on osteogenic differentiation was tested using alkaline phosphatase (ALP) staining and quantitative ALP assays (Sigma). Matrix mineralization was assessed using alizarin red staining. Mean differences in ALP expression were analyzed using a 2x3 (culture group by BMP dose) 2-way ANOVA.

Results: BMP4 induced the osteogenic differentiation of bone-derived cells in a dose-dependent manner and culture in osteogenic medium led to the deposition of a mineralized matrix. Co-culture with dura mater cells inhibited ALP activity below baseline levels and inhibited the deposition of a mineralized matrix.

Conclusions: These data do not support the hypothesis that the dura mater from neonatal craniosynostotic rabbits will enhance or induce the osteogenic differentiation of bone-derived cells. Surprisingly, we found that co-culture with dura mater cells actually inhibited osteogenic differentiation through unknown mechanisms. Because these results differ from other published results, we believe this model warrants further investigation.