Confocal laser scanning microscopic analysis of collagen scaffolding patterns in cranial sutures

Although recent studies indicate that regional dura mater influences the fate of the overlying cranial suture, little is known about the assembly of extracellular matrix (ECM) molecules within the patent and fusing murine cranial suture complexes. Confocal laser scanning microscopy was used to study ECM assembly within patent and fusing cranial suture complexes. Coronal sections (20 microm thick) of patent sagittal (SAG) and fusing posterior frontal (PF) sutures from postnatal 8-, 14-, and 18-day-old Sprague-Dawley rats were scanned in 0.5-microm increments, and images were collected consecutively to create a z-series for three-dimensional reconstruction. Spatial and temporal collagen arrangements were compared between SAG and PF sutures by measuring interfiber distance, fiber thickness, and total collagen surface area at each time point. We demonstrate that on day 8 (before the onset of suture fusion), collagen bundles are randomly arranged in both the SAG and PF sutures. By day 14 (midfusion period), there was a statistically significant reduction in total collagen surface area (80.5% versus 67.4%; P < 0.05) as the collagen bundles were organized into orthogonal lattices along the anterior and endocranial margins of the PF suture. Furthermore, new bone matrix deposition was observed along the edges of these organized collagen bundles. In contrast, collagen within the SAG suture remained randomly arranged and unossified. By day 18 (late fusion period), the PF suture was completely fused except for the posterior-ectocranial portion. This patent section of the PF suture contained a highly organized mineralizing orthogonal collagen lattice. The total collagen surface area in the day-18 PF suture continued to decline compared with the day-8 PF suture (80.5% versus 55.6%; P < 0.05). In the day-18 SAG suture, the collagen bundles remained randomly arranged, and the total surface area did not change. The same analysis was performed in a human pathologic fusing and patent suture. Similar results were observed. The total collagen surface area significantly decreased in the pathologic fusing human suture compared with the patent suture (92.8% versus 60.6%; P < 0.05). Moreover, the pathologically fusing suture contained a highly organized mineralizing orthogonal collagen lattice. This is the first analysis of collagen patterns in patent and fusing cranial sutures.