Systematic study on the osteocyte network formation and their mechanical responsibility

目的：通过力学性能的检测，分析成骨细胞向成骨细胞的转变。此外，FRAP分析还检测了鸡颅骨中骨细胞间的缝隙连接通讯。材料与方法：用荧光标记的鬼臼蛋白和鸡成骨细胞特异性单抗OB7.3鉴定鸡胚颅盖骨和分离培养的骨细胞。用原子力显微镜分析活细胞的弹性模数。采用荧光置换后光漂白(FRAP)分析方法，研究了pH_0、[Ca~(2+)]_e变化和PTH的加入对鸡颅骨细胞GJIC的影响。结果：3个群体细胞周边区的弹性模量值均显著高于核区。Os…周边区的弹性模数成骨细胞数为12053±934pA，类骨样骨细胞数为7971±422pA，成熟骨细胞数为4471±198pA。Cx43免疫反应阳性表达于大多数骨细胞突起。FRAP分析显示骨细胞间存在染料偶联。在未经处理的骨细胞中，光漂白后5min内荧光强度恢复到43.7±2.2%。经18α-GA处理后，骨细胞数量明显减少。当pH_0降低时，荧光回收率显著降低。相反，随着pH_0的升高，荧光回收率显著增加。当[Ca^&lt；2+&gt；]_e从1 mM增加到25 mM时，荧光回收率显著降低。在1.0~10 nM rPTH作用3h的骨碎片中，荧光替换显著增加。细胞内钙离子的螯合作用影响[Ca~(2+)]_e和甲状旁腺素对GJIC的调节。肌动蛋白和微管细胞骨架在MC3T3-E1细胞和原代成骨细胞的培养过程中有显著差异。结论：骨细胞的弹性模量力学性质和局部黏附存在动态变化。此外，我们的研究首次表明，在骨重建过程中，骨细胞间的GJIC受到细胞外环境和激素刺激的调节。这种方法可能比目前的方法更符合活骨的生物学特性。成骨细胞过程可能具有与骨细胞树突状网络不同的功能。较少

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