Fig. 4.

The formation and structure of the cancellous bone (layer 4) that forms the outer layer of the vertebral body. (A) Micrograph of a transverse section of a vertebra embedded in methacrylate and stained with toluidine blue, smolt stage. Layer 4 (L4) consists of cancellous bone with cavities, and is characterized by a woven collagen matrix and a high osteocyte content. This layer is formed outside layers 2 (L2) and 3 (L3). Layer 2, which is the thinnest layer of the amphicoel, has a lamellar structure and is acellular. Layer 3, which makes up the bulk of the compact lamellar bone of the amphicoel, has very low osteocyte content. (B) SEM micrograph of the cancellous bone in layer 4. The matrix structure is porous, non-laminar and woven. (C,D) Images of holospondylous vertebrae from the caudal region of the trunk. (C) SEM micrograph of a vertebra from a specimen at 1000 d°. The first structures to form within layer 4 are longitudinal plate-like trabeculae on the lateral sides of the vertebra. (D) Digital 3D reconstruction of micro-CT data of a vertebra at the smolt stage. At this stage layer 4 has a cancellous structure with both longitudinal and transverse trabeculae that occupies the concavity between the biconoid surfaces of the compact bone of the amphicoel. Layer 4 grows through branching and extension of the trabecular plates. The cranial and caudal rims of the vertebra are made up of the compact bone of layers 2 and 3, and a demarcation zone between layer 3 and 4 runs parallel to the rims. The cancellous bone of layer 4 is continuous with the bone of the neural and haemal arches. (E) Transverse methacrylate section of the midregion of a vertebra, stained with toluidine blue. Stage: 2200 d°. Within layer 4 the trabeculae grow through deposition of osteoid from dense aggregations of osteoblasts on the apical surfaces (arrow). As the trabeculae extend they also grow in thickness, which results in a club-shaped cross-sectional profile. The lateral surfaces of the trabeculae are covered with osteoblasts with a squamous morphology (arrowhead). The space between the trabeculae is filled with adipose tissue. The notochord (n) is indicated. (F) Cross-section of a vertebra at the smolt stage reconstructed from the same X-ray micro-CT data-set as in D. Note the open structure of the vertebra, and at this stage there are six main trabeculae on each side in addition to the neural and haemal arches. The notochord (n) is indicated. Scale bars (A) 100 µm; (B) 50 µm; (C) 100 µm; (D) 1 mm; (E) 100 µm; (F) 1 mm.