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 osteoblasts 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.