Calcium responses in flow-treated primary chick bone cells

Objectives: In this study, we examined flow-induced calcium signaling in chick bone cells isolated from fetal chick calvaria. To identify osteocytes and osteoblasts in the same field where the calcium imaging has been performed, we used OB7.3, a chicken osteocyte-specific monoclonal antibody, as well as assessed alkaline phosphate activity. By the combination of calcium imaging and identification of the bone cells, we investigated whether osteocytes are chief cells that respond to fluid shear stress. Methods: Bone cells were isolated from 16-dayÐold embryonic chicken calvaria by the serial treatment with EDTA and collagenase. Cells were subjected to fluid flow (12 dynes/cm2) in flow chamber connected to flow loop apparatus for 5 minutes. Measurement of intracellular calcium (CAi) concentration was performed using calcium indicator dye fluo-3. Relative increase in CAi was assessed using confocal imaging system (Flowview FV300). Results: 1. Bone cells showed different CAi increment during the fluid-flow period to during no-flow period. Peak values were reached within 20 sec after onset of fluid flow. 2. It was possible to decide phenotype of the cells in the field where the calcium imaging has been performed. 3. Flow-induced CAi increment was seen in osteoblasts (32.4%) and lining cells (45.6%), which were negative to both OB7.3 and ALPase activity. However, the percentage of CAi increment seen in osteocytes was 5.5%. 4. Magnitude of CAi increment over baseline did not show any significant differences in osteocytes, osteoblasts, and lining cells. Conclusion: The increment of intracellular calcium was seen flow-treated osteoblasts and lining cells. However, osteocytes were less sensitive to fluid shear stress.