Strain magnitude dependent intracellular calcium signaling response to uniaxial stretch in osteoblastic cells

Abstract

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