The dura mater has been causally implicated in the premature fusion of calvarial sutures, termed craniosynostosis (CS). In order to better understand the capacity of the dura to influence osteogenic differentiation, the effects of co-culture were examined with dura mater cells derived from either CS or wild-type (WT) rabbits on BMP4-induced osteogenic differentiation of a murine myoblast cell line (C2C12).
C2C12 cells were stimulated with medium containing BMP4 (0ng/ml, 10ng/ml, or 50ng/ml) in culture alone or in co-culture with dural cells isolated from a unique New Zealand White rabbit model of congenital nonsyndromic CS or from wild-type control rabbits. After 3 days, the C2C12 cells were analyzed for osteogenic induction via alkaline phosphatase (ALP) staining and a quantitative ALP assay. The quantitative ALP assay was performed with nine CS samples and three WT control sample. Effects of co-culture on normalized ALP activity were assessed using independent sample t-tests for each BMP dose.
Co-culture with dural cells derived from rabbits with CS significantly increased the normalized ALP activity of C2C12 cells by roughly two-fold at the 10ng/ml (p<0.05) and three-fold at the 50ng/ml dose (p<0.05) of BMP4 in all CS samples. The effects of co-culture with WT dural cells at any BMP4 dose tested on ALP activity showed no statistical significance.
Results suggest that soluble factors expressed by CS dural-derived cells, but not by WT dural-derived cells, can enhance BMP4’s osteogenic effects on C2C12 cells. These results, obtained in a unique pathological CS model, support the assertion that the increased bone formation in CS may result from signals expressed by the dura mater. Future studies will focus on elucidating the molecular mediators responsible for the observed osteogenic effect of craniosynostotic dura mater.