Murshid Sakhr A, Kamioka Hiroshi, Ishihara Yoshihito, Ando Ryoko, Sugawara Yasuyo, Takano-Yamamoto Teruko
Division of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Sendai 980-8574, Japan.
J Bone Miner Metab. 2007;25(3):151-8. doi: 10.1007/s00774-006-0745-5. Epub 2007 Apr 20.
Cell shape is the most critical determinant of cell function and is potentially influenced by the organization of a cell's cytoskeletal components. It has been reported that three-dimensionally cultured osteoblasts have a morphology that closely resembles that of osteocytes, most notably including formation of processes. We have previously shown the critical differences between cytoskeletal components in osteoblasts and osteocytes in two-dimensional culture. We have now extended that investigation to the cytoskeletal components of 3D-cultured osteoblasts and osteocytes using 3D cultures of the osteoblast cell line, MC3T3-E1, and primary osteocytes grown in collagen gel. Three-dimensional fluorescent image reconstructions for actin, fimbrin, alpha-actinin, myosin, tropomyosin, and microtubules were made using IMARIS software. Actin, fimbrin, alpha-actinin, myosin, and tropomyosin all appeared in the processes of both cell types, but fimbrin and myosin showed differences in their distribution patterns between cell types. Microtubules were limited in distribution to the proximal region of osteocyte processes but extended the entire length of MC3T3-E1 cell processes. Microtubules were essential for the integrity and formation of MC3T3-E1 cell processes, but osteocyte processes were dependent on actin. These results showed that there are significant differences between the actin and microtubule cytoskeletons in the processes of 3D-cultured MC3T3-E1 cells and in the processes of 3D-cultured primary osteocytes. These differences in the cytoskeleton of the processes of 3D-cultured osteoblasts and of osteocyte dendrites suggest that osteoblast processes may have a different functional role than the osteocyte dendritic network.
细胞形状是细胞功能的最关键决定因素，并且可能受到细胞细胞骨架成分组织的影响。据报道，三维培养的成骨细胞具有与骨细胞非常相似的形态，最显著的是包括形成突起。我们之前已经展示了二维培养中成骨细胞和骨细胞细胞骨架成分之间的关键差异。现在，我们使用成骨细胞系MC3T3-E1的三维培养物以及在胶原凝胶中生长的原代骨细胞，将该研究扩展到三维培养的成骨细胞和骨细胞的细胞骨架成分。使用IMARIS软件对肌动蛋白、丝束蛋白、α-辅肌动蛋白、肌球蛋白、原肌球蛋白和微管进行三维荧光图像重建。肌动蛋白、丝束蛋白、α-辅肌动蛋白、肌球蛋白和原肌球蛋白在两种细胞类型的突起中均有出现，但丝束蛋白和肌球蛋白在细胞类型之间的分布模式存在差异。微管的分布局限于骨细胞突起的近端区域，但延伸至MC3T3-E1细胞突起的全长。微管对于MC3T3-E1细胞突起的完整性和形成至关重要，但骨细胞突起依赖于肌动蛋白。这些结果表明，三维培养的MC3T3-E1细胞突起和三维培养的原代骨细胞突起中的肌动蛋白和微管细胞骨架之间存在显著差异。三维培养的成骨细胞突起和骨细胞树突的细胞骨架中的这些差异表明，成骨细胞突起可能具有与骨细胞树突网络不同的功能作用。
