Osteoblasts change their intracellular calcium ion concentration in response to mechanical stimuli. Although it has been reported that osteoblasts sense and respond to stretching of a substrate on which osteoblastic cells have adhered, the details of the dynamic characteristics of their calcium signaling response remain unclear. Motion artifacts such as loss of focus during stretch application make it difficult to conduct precise time-course observations of calcium signaling responses. Therefore, in this study, we observed intracellular calcium signaling responses to stretch in a single osteoblastic cell by video rate temporal resolution. Our originally developed cell-stretching microdevice enables <i>in situ</i> observation of a stretched cell without excessive motion artifacts such as focus drift. Residual minor effects of motion artifacts were corrected by the fluorescence ratiometric method with fluorescent calcium indicator Fluo 8H and fluorescent cytoplasm dye calcein red-orange. We succeeded to detect the intracellular calcium signaling response to stretch by video rate temporal resolution. The results revealed a time lag from stretch application to initiation of the intracellular calcium signaling response. We compared two time lags measured at two different cell areas: central and peripheral regions of the cell. The time lag in the central region of the cell was shorter than that in the peripheral region. This result suggests that the osteoblastic calcium signaling response to stretching stimuli initiates around the central region of the cell.

Keywords

Cell biomechanics, Mechanotransduction, Osteoblasts, Calcium signaling, Stretch stimuli

Paper information

[Advance Publication] (Proper information for citation will be announced after formal publication)

In osteoblast cells, cells change their intracellular Ca2+ concentration ([Ca2+]i) as the response to mechanical stimuli. Although it has been reported that osteoblast cells responded to many kinds of mechanical stimuli including stretch of substrate, shear stress in fluid flow, direct indentation of glass microneedle and hydrostatic pressure etc., the detail of the characteristics of intracellular calcium signaling response to substrate stretch still remains unclear because motion artifact during stretch application causes out of focus plane and observation area, and complicates the in situ time lapse observation of change in [Ca2+]i in a single cell level. In this study, we combined our originally developed cell stretching MEMS device with the ratiometric microscopy method with two kinds of visible wavelength calcium indicator dyes. The cell stretching micro device and the ratiometric method reduce the influence of motion artifact during stretch application, and enable us to quantitatively evaluate the characteristics of cellular calcium signaling response to stretch. MC3T3-E1 osteoblastic cells were plated onto the cell stretching micro device and fluorescently labeled by Ca2+ indicator Fluo 3 and Fura Red. A uniaxial stretch with three magnitudes of strain 5%, 10% and 15% with constant strain rate were applied to the cells, and in situ time lapse observation of cellular calcium signaling response to stretch was conducted with high temporal and spatial resolution. We succeeded in obtaining time lapse fluorescent image sequences during stretch application without excessive out of focus and blank time. The results revealed that MC3T3-E1 cells change the intensity of calcium signaling response to stretch according to the stretch strain magnitude. As stretch strain magnitude was increased, the amount of change in fluorescent ratio value of Ca2+ indicators in stretched cells also increased. This result suggests the possibility that osteoblastic cells can sense the magnitude of mechanical stimuli at upstream of mechanotransduction pathway such as influx of extracellular Ca2+.

Keywords

Cell biomechanics, Mechanotransduction, Osteoblast, Calcium signaling, Stretch stimuli

Paper information

Katsuya SATO, Yuki OGAWA, Shin-ichi ITO, Shoichiro FUJISAWA, Kazuyuki MINAMI, “Strain magnitude dependent intracellular calcium signaling response to uniaxial stretch in osteoblastic cells”, Journal of Biomechanical Science and Engineering, Vol.10, No.3 (2015), p.15-00242. doi:10.1299/jbse.15-00242. Final Version Released on Nov. 13, 2015, Advance Publication Released on Sep 03, 2015.