Systematic study on the osteocyte network formation and their mechanical responsibility

目的：通过对成骨细胞向骨细胞转化过程中力学性能的检测，分析成骨细胞向骨细胞转化的过程。此外，在鸡颅骨骨细胞之间的缝隙连接通讯进行了检查，使用FRAP分析。材料与方法：采用荧光标记鬼笔环肽和鸡骨细胞特异性单克隆抗体OB7.3对鸡胚颅骨和分离培养的骨细胞进行鉴定。用原子力显微镜分析活细胞的弹性模量。采用光漂白后荧光替代法（FRAP）分析了pH、[Ca^<2+>]_e和PTH对鸡颅骨骨细胞GJIC的影响。结果：3个细胞群外周区的弹性模量均显著高于核区，细胞核区的弹性模量显著高于核区。口周区的弹性模量 关于我们 成骨细胞为12053±934 Pa，类骨细胞为7971±422 Pa，成熟骨细胞为4471±198 Pa。在大多数骨细胞突起中检测到Cx43免疫反应。FRAP分析显示骨细胞间存在染料偶联。在未处理的骨细胞中，荧光强度在光漂白后5 min内恢复43.7±2.2%。用18 α-GA预处理骨细胞，骨细胞增殖明显降低。当pH_0降低时，荧光恢复率显著降低。相反，当pH_0增加时，荧光恢复率显著增加。当[Ca^<2+>]_e从1增加到25 mM时，荧光恢复率显著降低。在骨碎片暴露于1.0至10 nM rPTH 3小时，荧光的替代显着增加。螯合胞内钙离子影响[Ca^<2+>]_e和PTH对GJIC的调节。结论：骨细胞的弹性模量力学特性和局部粘附存在动态变化。此外，我们的研究首次表明，骨细胞之间的GJIC是由细胞外环境和激素刺激在骨重建过程中调节。这种方法可能比目前的方法更具有生物学相关性。成骨细胞过程可能具有与骨细胞树突网络不同的功能作用。少

Aim: We examined mechanical property to analyze the change from osteoblasts to osteocytes. Furthermore, gap junctional intercellular communication among osteocytes in chick calvaria was examined using FRAP analysis. We established 3D cultures of osteoblast and osteocytes.Materials & Methods: Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody. The elastic modulus of living cells was analyzed with Atomic Force Microscopy. Using Fluorescence Replacement After Photobleaching (FRAP) analysis we examined the effect of changes in pH_0, [Ca^<2+>]_e, and addition of PTH on GJIC in osteocytes in chick calvaria. Anti-Connexin43 (Cx43) immunolabelling was used to localize gap junctions.Results: The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of os … More teoblasts was 12053±934 Pa, that of osteoid osteocytes was 7971±422 Pa and that of mature osteocytes was 4471±198 Pa. Cx43 immunoreactivity was detected in most of the osteocyte processes. FRAP analysis showed dye-coupling among osteocytes. In untreated osteocytes, fluorescence intensity recovered 43.7±2.2% within 5 min after photobleaching. Pretreatment of osteocytes with 18 α-GA, significantly decreased. When pH_0 was decreased, fluorescence recovery significantly decreased. Conversely, when pH_0 was increased, fluorescence recovery was significantly increased. When [Ca^<2+>]_e was increased from 1 to 25 mM, fluorescence recovery was significantly decreased. In bone fragments exposed to 1.0 to 10 nM rPTH for 3 h, replacement of fluorescence was significantly increased. Chelating intracellular calcium ions affected GJIC regulation by [Ca^<2+>]_e and PTH. There were significant differences between actin and microtubule cytoskeletons in the process of MC3T3-E1 cells and primary osteocytes.Conclusions: There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells. Furthermore, our study showed for the first time that GJIC among osteocytes is regulated by the extracellular environment and by hormonal stimulation during bone remodeling. This method may be more biologically relevant to living bone than current methods. Osteoblasts processes may have a different functional role than the osteocyte dendritic network. Less