Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: Implications for suture fusion and patency

Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when no rmal craniofacial growth development goes awry, we must first investigate t he mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birt h provide an ideal model to study these mechanisms. In previous studies, ou r group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. T hese studies have led to the hypothesis that calvarial DM is regionally dif ferentiated and that this differentiation guides the development of the ove rlying suture. To test this hypothesis, we evaluated the messenger RNA (mRN A) expression of osteogenic cytokines (transforming growth factor beta1 [TG F-beta1] and TGF-beta3) and bone-associated extracellular matrix (ECM) mole cules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to f use) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochem ical staining and 5-bromo-2'-deoxyuridine (BrdU) incorporation, respectivel y. We showed that the expression of osteogenic cytokines and bone-associate d ECM molecules is potently up-regulated in the DM associated with the PF s uture. In addition, we showed that cellular proliferation in the DM associa ted with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interest ingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional diffe rentiation of cranial suture-associated dural cells, we established dural c ell cultures from fusing and patent rat cranial sutures in N6 rats and eval uated the expression of osteogenic cytokines (TGF-beta1 and fibroblast grow th factor 2 [FGF-2]) and collagen I. In addition, we analyzed cellular prod uction of proliferating cell nuclear antigen (PCNA). These studies confirme d our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF-beta1, FGF -2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture-derived dural cells produced significantly less PCNA than S AG suture-derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significa nt increase in proliferation (*p < 0.001) in FRCs cocultured with SAG sutur e-derived dural cells as compared with FRCs cocultured alone or with PF sut ure-derived dural cells. Taken together, these data strongly support the hy pothesis that the calvarial DM is regionally differentiated resulting in th e up-regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in c ytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increa se our understanding of the events that lead to premature cranial suture fu sion.