Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures

Lamellar bone is a major component of most mammalian skeletons. A prominent component of individual lamellae are parallel arrays of mineralized type I collagen fibrils, organized in a plywood like motif. Here we use a dual beam microscope and the serial surface view (SSV) method to investigate the three dimensional collagen organization of circumferential lamellar bone from rat tibiae after demineralization and osmium staining. Fast Fourier transform analysis is used to quantitatively identify the mean collagen array orientations and local collagen fibril dispersion. Based on collagen fibril array orientations and variations in fibril dispersion, we identify 3 distinct sub-lamellar structural motifs: a plywood-like fanning sub-lamella, a unidirectional sub-lamella and a disordered sub-lamella. We also show that the disordered sub-lamella is less mineralized than the other sub-lamellae. The hubs and junctions of the canalicular network, which connect radially oriented canaliculi, are intimately associated with the disordered sub-lamella. We also note considerable variations in the proportions of these 3 sub-lamellar structural elements among different lamellae. This new application of Serial Surface View opens the way to quantitatively compare lamellar bone from different sources, and to clarify the 3-dimensional structures of other bone types, as well as other biological structural materials.

► Lamellae of rat circumferential lamellar bone comprise fanning, unidirectional and disordered structural motifs.
► Disordered sub-lamellae are relatively thin (0.1-0.5 µm), poorly mineralized and open structures that may well have important mechanical functions.
► Part of the canalicular network is in the proximity to this disordered structure.
► SSV method using FIB-SEM can be used to compare collagen networks in 3D in different collagen-containing tissues.