In the bone, collagen fibrils form a lamellar structure called the "twisted plywood-like model." Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.
Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded osteocyte network in chick calvariae and observed the flow-induced Ca signaling in osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca ([Ca]) significantly increased in developmentally mature osteocytes in comparison with young osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature osteocytes in detail, we evaluated the expression of osteocyte-related genes using the osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c-Fos, Cx43, Panx3, Col1a1, and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature osteocytes are more responsive to mechanical stress than young osteocytes and have important functions in bone formation and remodeling.
The collagen network acts as a scaffold for calcification and its three-dimensional structure influences bone strength. It is therefore important to observe the collagen network in detail and three-dimensionally. In this study, we observed the collagen network of chick embryonic calvariae in membranous bone three-dimensionally using orthogonally arranged FIB-SEM. A 25 脳 25 渭m area of chick embryonic calvaria was observed at a high resolution (25 nm per pixel). The inside of the bone (i.e. the primary calcified tissue), the bone cells (i.e. the osteoblasts and the osteocytes), the organelles, and the collagen fibrils were observed in detail. These structures were observed three-dimensionally using the Amira software program. In addition, the collagen fibrils of the bone were automatically extracted using the XTracing extension software program, and three-dimensional morphometry was performed. Almost all of the collagen fibrils ran along the longitudinal axis of the trabecular bone. We found that the regularity of the collagen fibril orientation was less remarkable in the osteoblast layer, which contained numerous osteoblasts. The collagen fibril orientation started to show regularity toward the central bone layer, which contained few bone cells.
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