Dynamic studies on living cells with an atomic force fluorescence microscope

Analysis of the reaction of bone structure to mechanical stimulation is a key issue in understanding the origins of osteoporosis and mechanical adaptation of living bone to external forces. This is thought to be regulated on a cellular level. We have investigated quantitative mechanical stimulation of single bone cells and their immediate intracellular calcium responses using a combination of an atomic force microscope (AFM) and a fluorescence microscope, developed in our laboratory. The force stimulation system can apply quantified forces in the pico- and nano-newton regime on exactly defined positions of a cell. We present here the first measurements using this system on the mechanically induced free calcium response of primary osteoblasts. The threshold forces for stimulation and the temporal behavior of the free calcium response are analyzed and the signal transmission to adjacent cells is investigated. An immediate increase of the intracellular calcium concentration is observed after dynamic stimulation with forces between 110nN and 550nN. Forces exceeding 700nN destroy the cell and the free calcium concentration of the neighboring cells react as a consequence.

[1]  T D Brown,et al.  Techniques for mechanical stimulation of cells in vitro: a review. , 2000, Journal of biomechanics.

[2]  M. Berridge.  Inositol trisphosphate and calcium signalling , 1993, Nature.

[3]  Peter Lipp,et al.  Calcium - a life and death signal , 1998, Nature.

[4]  R Y Tsien,et al.  Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. , 1985, Cell calcium.

[5]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[6]  P. Hansma,et al.  A nondestructive method for determining the spring constant of cantilevers for scanning force microscopy , 1993 .

[7]  J. Ando,et al.  Intracellular calcium response to directly applied mechanical shearing force in cultured vascular endothelial cells. , 1994, Biorheology.

[8]  C. Hung,et al.  Intracellular Ca2+ stores and extracellular Ca2+ are required in the real-time Ca2+ response of bone cells experiencing fluid flow. , 1996, Journal of biomechanics.

[9]  Sverre Myhra,et al.  Determination of the spring constants of probes for force microscopy/spectroscopy , 1996 .

[10]  B. Nebe,et al.  Stimulation of integrin receptors using a magnetic drag force device induces an intracellular free calcium response. , 1996, European journal of cell biology.

[11]  M. Grattarola,et al.  Preliminary results on the electrostatic double-layer force between two surfaces with high surface potentials , 1998 .

[12]  S. Grinstein,et al.  Bromo-A23187: a nonfluorescent calcium ionophore for use with fluorescent probes. , 1985, Analytical biochemistry.

[13]  H. Butt,et al.  Direct Measurement of Particle−Bubble Interactions in Aqueous Electrolyte: Dependence on Surfactant , 1998 .

[14]  R. Duncan,et al.  Calcium signals and calcium channels in osteoblastic cells. , 1998, Seminars in nephrology.

[15]  S. Okuma,et al.  A method for determining the spring constant of cantilevers for atomic force microscopy , 1996 .

[16]  L E Lanyon,et al.  Static vs dynamic loads as an influence on bone remodelling. , 1984, Journal of biomechanics.

[17]  H J Donahue,et al.  Differential effect of steady versus oscillating flow on bone cells. , 1998, Journal of biomechanics.