Lateral bone ridge expansion and internal tissue replacement for vertebral body growth in Pacific bluefin tuna Thunnus orientalis.

Vertebral growth is an essential developmental process to support the expansion of the vertebrate body. In teleosts, the lateral side of the vertebral bodies develops to form different structures among species in the late stages of vertebral growth, although lateral structures are not apparent in the early stages. Lateral structures are one of the structural features that determine the diversity of teleost vertebrae. However, explanations for the formation of lateral structures are conflicting because few reports have investigated the growth of teleost vertebral bodies. To clarify the growth process, we analyzed the morphological changes in the vertebral body of Pacific bluefin tuna Thunnus orientalis at different developmental stages using micro-computed tomography (CT) scans. The micro-CT scans showed that the vertebral centrum formed a plate-like ridge on the lateral side along the cranial-caudal direction and extended laterally with increasing thickness. Simultaneously, the proximal region of the lateral ridges became porous as the vertebrae grew to form bone marrow cavities. Furthermore, we used histological observations to describe the relationship between these morphological changes and osteoblast and osteoclast activities. Osteoblasts accumulated on the distal edges of the lateral ridges, whereas osteoclasts were distributed in the bone marrow cavities. These observations suggest that bone resorption occurs proximally to form bone marrow cavities in addition to bone synthesis at the edges of the lateral ridges. The bone marrow cavities were occupied by blood vessels, extracellular matrix, and adipocytes, and the internal tissue composition changed to increase the area of adipose tissue. Because the ratio of bone volume decreases in large vertebrae, bone formation and resorption are regulated to separate the external cortical and internal trabecular bones to support the vertebrae. This study is the first to report the formation of lateral structures and can be applied to similar lateral structures in the vertebrae of other teleost species.

Three-dimensional topology optimization model to simulate the external shapes of bone.

Elucidation of the mechanism by which the shape of bones is formed is essential for understanding vertebrate development. Bones support the body of vertebrates by withstanding external loads, such as those imposed by gravity and muscle tension. Many studies have reported that bone formation varies in response to external loads. An increased external load induces bone synthesis, whereas a decreased external load induces bone resorption. This relationship led to the hypothesis that bone shape adapts to external load. In fact, by simulating this relationship through topology optimization, the internal trabecular structure of bones can be successfully reproduced, thereby facilitating the study of bone diseases. In contrast, there have been few attempts to simulate the external structure of bones, which determines vertebrate morphology. However, the external shape of bones may be reproduced through topology optimization because cells of the same type form both the internal and external structures of bones. Here, we constructed a three-dimensional topology optimization model to attempt the reproduction of the external shape of teleost vertebrae. In teleosts, the internal structure of the vertebral bodies is invariable, exhibiting an hourglass shape, whereas the lateral structure supporting the internal structure differs among species. Based on the anatomical observations, we applied different external loads to the hourglass-shaped part. The simulations produced a variety of three-dimensional structures, some of which exhibited several structural features similar to those of actual teleost vertebrae. In addition, by adjusting the geometric parameters, such as the width of the hourglass shape, we reproduced the variation in the teleost vertebrae shapes. These results suggest that a simulation using topology optimization can successfully reproduce the external shapes of teleost vertebrae. By applying our topology optimization model to various bones of vertebrates, we can understand how the external shape of bones adapts to external loads.

Comparative morphological examination of vertebral bodies of teleost fish using high-resolution micro-CT scans.

Vertebral bodies of teleost fish are formed by the sclerotomal bone covering the chordacentrum. The internal part of the sclerotomal bone is composed of an amphicoelous hourglass shaped autocentrum, which is common in most fish species. In contrast, the external shape of the sclerotomal bone varies extensively among species. There are multiple hypotheses regarding the composition and formation of the external structure. However, as they are based on studies of few extant or extinct species, their applicability to other species remains to be clarified. To understand the morphology, formation, and composition of vertebral bodies in teleosts, we performed a comparative analysis using micro-CT scans of 32 species from 10 orders of Teleostei and investigated the detailed morphology of the sclerotomal bone, especially its plate-like ridge and trabeculae. We discovered two structural characteristics that are shared among most of the examined species. One was the sheet-like trabeculae that extend radially from the center of the vertebral body with a constant thickness. The other was the presence of hollow spaces on the internal parts of the lateral ridge and trabeculae. The combination of different arrangements of sheet-like trabeculae and internal hollow spaces formed different shapes of the lateral structure of the vertebral body. The properties of these two characteristics suggest that the external part of the sclerotomal bone grows outward by deposition at the bone tip, and that, concurrently, bone absorption occurs in the internal part of the sclerotomal bone. The vertebral arches were also formed by the sheet-like trabeculae, indicating that both, the vertebral body and the arches, are formed by the same component. The micro-CT scanning data were uploaded to a public database so they can be used for future studies on fish vertebrae.