Abstract
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.
Highlights
Mechanical stimulation of bone is well-known to regulate bone volume, structure and composition[1,2]
When we compared the percentage of cells exhibiting local [Ca2+]i transients in response to indentation performed at low and high speeds, we found that a higher percentage of C2C12-derived osteoblasts responded to the mechanical stimulation compared to primary osteoblasts (Figure 3B)
We have found that qualitatively similar responses were induced in primary and C2C12-derived osteoblasts (Figure 6A, B), the response rate was significantly lower for the primary osteoblasts (Figure 6C)
Summary
Mechanical stimulation of bone is well-known to regulate bone volume, structure and composition[1,2]. Because of the complexity of the bone environment in vivo, several models have been developed to understand the effects of mechanical stimulation on bone cells in vitro[5] These models include application of hydrostatic pressure, longitudinal substrate stretch and fluid shear. Ca2+ signaling induced by mechanical stimulation in turn influences numerous bone cell functions such as cytoskeletal reorganization[7], gene expression[10], proliferation and differentiation[6]. These studies identified significant complex signaling interactions between multiple cells[6], making it difficult to de-convolute the responses of single cells to mechanical stimulation. These techniques do not allow exact control of forces applied to individual cells, nor do they report single cell micro-injury
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call