Local membrane deformation and micro-injury lead to qualitatively different responses in osteoblasts

Micro-damage of bone tissue is known to regulate bone turnover. However, it is unknown if individual bone cells can differentiate between membrane deformation and micro-injury. We generated osteoblasts from mouse bone marrow or bone morphogenetic protein 2-transfected C2C12 cells. Single cells were mechanically stimulated by indentation with the atomic force microscopy probe with variable force load either resulting in membrane deformation only, or leading to membrane penetration and micro-injury. Changes in the cytosolic free calcium concentration ([Ca (2+)] (i)) in fluo4-AM loaded cells were analyzed. When deformation only was induced, it resulted in an immediate elevation of [Ca (2+)] (i) which was localized to the probe periphery. Multiple consecutive local Ca (2+) responses were induced by sequential application of low level forces, with characteristic recovery time of ~2 s. The duration of [Ca (2+)] (i) elevations was directly proportional to the tip-cell contact time. In contrast, cell micro-injury resulted in transient global elevations of [Ca (2+)] (i), the magnitude of which was independent of the tip-cell contact time. Sequential micro-injury of the same cell did not induce Ca (2+) response within 30 s of the first stimulation. Both local and global Ca (2+)elevations were blocked in Ca (2+)-free media or in the presence of stretch-activated channel blocker Gd (3+). In addition, amount of Ca (2+) released during global responses was significantly reduced in the presence of PLC inhibitor Et-18-OCH (3). Thus, we found qualitative differences in calcium responses to mechanical forces inducing only membrane deformation or deformation leading to micro-injury.