Sugawara Yasuyo, Kamioka Hiroshi, Honjo Tadashi, Tezuka Ken-ichi, Takano-Yamamoto Teruko
Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata, Okayama City, Okayama 700-8525, Japan.
Bone. 2005 May;36(5):877-83. doi: 10.1016/j.bone.2004.10.008. Epub 2005 Apr 7.
Osteocytes are surrounded by hard bone matrix. Therefore, it has not previously been possible to demonstrate the real architecture of the osteocyte network in bone. We previously reported that it is possible to observe osteocytes in bone by labeling the cells with fluorescence and using confocal laser scanning (CLS) microscopy. In this study, we for the first time conducted an extensive analysis of the morphology and morphometry of the three-dimensional (3D) osteocyte structure using three-dimensionally reconstructed fluorescent images. Sixteen-day-old embryonic chick calvariae were stained with fluorescently labeled phalloidin and observed using a confocal laser scanning microscope. Morphometry of osteocytes in the calvaria was analyzed using extensive three-dimensional reconstructing software IMARIS, process length measuring software NEURON TRACER and cell surface area-/cell volume-analyzing software SURPASS. From the IMARIS-derived images, we found that the average of 10 osteocytes is 52.7 +/- 5.7 processes, and the point-to-point distance between centers of the osteocytes was 24.1 +/- 2.8 microm. In addition, we could calculate that each osteocyte spans an average of 4180 +/- 673 microm3 of bone volume. NEURON TRACER showed that the length of osteocyte processes was 0.26 +/- 0.02 microm per 1 microm3 bone compartment. In addition, SURPASS indicated that the surface area of osteocytes was 0.36 +/- 0.03 microm2 per 1 microm3 bone compartment and that the volume ratio of osteocyte cell body to bone compartment was 9.42% +/- 1.18%. Together, the average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070 +/- 145 microm, 1509 +/- 113 microm2, and 394 +/- 49 microm3, respectively. It is possible to reconstruct the real architecture of the osteocyte network and obtain morphometric data from fluorescently labeled osteocytes in chick calvaria.
骨细胞被坚硬的骨基质所包围。因此，以前无法展示骨中骨细胞网络的真实结构。我们之前报道过，通过用荧光标记细胞并使用共聚焦激光扫描（CLS）显微镜，可以观察骨中的骨细胞。在本研究中，我们首次使用三维重建的荧光图像对三维（3D）骨细胞结构的形态和形态测量进行了广泛分析。用荧光标记的鬼笔环肽对16日龄的胚胎鸡颅骨进行染色，并使用共聚焦激光扫描显微镜进行观察。使用广泛的三维重建软件IMARIS、过程长度测量软件NEURON TRACER和细胞表面积/细胞体积分析软件SURPASS对颅骨中的骨细胞进行形态测量分析。从IMARIS生成的图像中，我们发现10个骨细胞的平均突起数为52.7±5.7个，骨细胞中心之间的点对点距离为24. [+或-] 2.8微米。此外，我们可以计算出每个骨细胞平均跨越4180±673立方微米的骨体积。NEURON TRACER显示，骨细胞突起的长度为每1立方微米骨腔0.26±0.02微米。此外，SURPASS表明，骨细胞的表面积为每1立方微米骨腔0.36±0.03平方微米，骨细胞胞体与骨腔的体积比为9.42%±1.18%。总的来说，一个骨细胞的突起平均总长度、平均表面积和平均体积分别为1070±145微米、1509±113平方微米和394±49立方微米。重建鸡颅骨中荧光标记骨细胞的骨细胞网络真实结构并获得形态测量数据是可能的。
