Tissue interactions between craniosynostotic dura mater and bone

Abstract

Background: Cells within the dura mater have been implicated in the determination of suture patency and fusion. Craniosynostosis (CS), the premature fusion of 1 or more of the cranial sutures, could result from abnormal control over the differentiation of osteoprogenitor cells from the dura mater. This study tested whether dura mater cells derived from rabbits with congenital CS were different from cells derived from normal rabbits and investigated the effects that CS dura mater had on osteogenic differentiation in vitro and in vivo.

Methods: Cells were derived from the dura mater from wild-type rabbits (WT; n = 23) or CS rabbits (n = 16). Cells were stimulated with bone morphogenetic protein 4, and alkaline phosphatase (ALP) expression and cell proliferation were assessed. Dura mater-derived cells were also cocultured with primary rabbit bone-derived cells, and ALP was assessed. Finally, interactions between the dura mater and overlying tissues were manipulated in vivo.

Results: Craniosynostotic dura mater-derived cells proliferated faster than did WT cells but were not more ALP positive. Coculture experiments showed that CS dura mater cells induced increased ALP activity in CS bone-derived cells, but not in WT bone-derived cells. In vivo experiments showed that a physical barrier successfully inhibited dura mater-derived osteogenesis.

Conclusions: Coculture of CS bone- and CS dura mater-derived cells evoked an abnormal phenotype in vitro. Covering the CS dura mater led to decreased bone formation in vivo. Further investigations will focus on the signaling molecules involved in the communication between these 2 CS tissue types in vitro and in vivo.

Figure 1

Schematic diagram of the surgical procedure used. Note that the suturectomy site on the synostosed side was treated with: 1) nothing (suturectomy control), 2) patent suture from the opposite side of the same animal (suture only), 3) PTFE membrane (barrier only), or with 4) a patent suture and a PTFE membrane (suture and membrane).

Figure 2

Graph showing the mean absorbance (+/− SEM) of WT or CS dura mater-derived cells cultured in one of three different media. PM = proliferation medium (control); BMP = proliferation medium + BMP4; OM = osteogenic medium. Note that CS dura mater-derived cells had more cells in each of the media compared to WT.

Figure 3

Graph (above) and histological images (below; ALP stain) showing ALP activity of WT and CS dura mater cells derived from suture-associated (Suture) and non-suture-associated (Non-suture) regions. Notice that, overall, dura mater-derived cells from regions under bone (WT and CS non-suture, CS suture [fused]) were different from dura mater-derived cells from regions under a patent suture (WT suture).

Figure 4

Histological images (left) and graph (right) of ALP activity assessed in bone cells cultured alone (control) or co-cultured with WT dura mater- or CS dura mater-derived cells. Note that co-culture with CS dura mater-derived cells increased the ALP activity of CS bone-derived cells only. All other co-culture conditions showed lower ALP activity compared to controls.

Figure 5

Histological analysis of calvarial bone suturectomy site healing. Note that transplantation of a patent suture leads to fusion of the transplanted suture (Suture Only), implantation of a PTFE membrane inhibited bone growth across the suturectomy site (Barrier Only), and implantation of the PTFE membrane underlying the transplanted patent suture was capable of maintaining suture patency (Suture and Barrier).