Wang Liyun, Wang Yilin, Han Yuefeng, Henderson Scott C, Majeska Robert J, Weinbaum Sheldon, Schaffler Mitchell B
Leni and Peter W. May Department of Orthopaedics, Mount Sinai School of Medicine, New York, NY 10029, USA.
Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11911-6. doi: 10.1073/pnas.0505193102. Epub 2005 Aug 8.
Solute transport through the bone lacunar-canalicular system is believed to be essential for osteocyte survival and function but has proved difficult to measure. We report an approach that permits direct measurement of real-time solute movement in intact bones. By using fluorescence recovery after photobleaching, the movement of a vitally injected fluorescent dye (sodium fluorescein) among individual osteocytic lacunae was visualized in situ beneath the periosteal surface of mouse cortical bone at depths up to 50 microm with laser scanning confocal microscopy. Transport was analyzed by using a two-compartment mathematical model of solute diffusion that accounted for the characteristic anatomical features of the lacunar-canalicular system. The diffusion coefficient of fluorescein (376 Da) was determined to be 3.3 +/- 0.6 x 10(-6) cm2/sec, which is 62% of its diffusion coefficient in water and is similar to diffusion coefficients measured for comparably sized molecules in cartilage. The diffusion of fluorescein in bone is also consistent with the presence of an osteocyte pericellular matrix whose structure resembles that proposed for the endothelial glycocalyx [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239-255]. To our knowledge, this is the first instance where the dynamics of molecular movement has been measured directly in the bone lacunar-canalicular system. This in situ imaging approach should also facilitate the analysis of convection-based transport mechanisms in bones of living animals.
溶质通过骨陷窝 - 小管系统的运输被认为对骨细胞的存活和功能至关重要，但事实证明难以测量。我们报告了一种能够直接测量完整骨骼中溶质实时运动的方法。通过光漂白后荧光恢复技术，利用激光扫描共聚焦显微镜，在小鼠皮质骨骨膜表面下方深度达50微米处，原位观察到一种经活体注射的荧光染料（荧光素钠）在各个骨细胞陷窝之间的运动。使用溶质扩散的双室数学模型对运输进行分析，该模型考虑了陷窝 - 小管系统的特征性解剖结构。确定荧光素（376道尔顿）的扩散系数为3.3±0.6×10⁻⁶平方厘米/秒，这是其在水中扩散系数的62%，并且与在软骨中测量的大小相当的分子的扩散系数相似。荧光素在骨中的扩散也与骨细胞周细胞基质的存在相一致，其结构类似于所提出的内皮糖萼结构[Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239 - 255]。据我们所知，这是首次直接测量骨陷窝 - 小管系统中分子运动的动力学。这种原位成像方法也应有助于分析活体动物骨骼中基于对流的运输机制。
