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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5552_Библиотеки_им_академика_М_И_Перельмана
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Mesenchymal Cell Activation by Biomechanical Stimulation and its Clinical Prospects, 2015, 13-22 13
Nahum Rosenberg (Ed)
All rights reserved-© 2015 Bentham Science Publishers
CHAPTER 2
Experimental Methods for Mechanical Stimulation
of Cells of Mesenchymal Origin
Nahum Rosenberg
*
and Reuben Haber
Department of Orthopaedic Surgery, Rambam Health Care Campus, POB 9602, Haifa 31096,
Israel
Abstract: The metabolic response of mesenchymal cells in mechanically active
environments depends on the interactions between fluid flow, intra and/or extracellular,
with the cytoskeletal components, especially microfilaments. Therefore, in vitro research of
cellular mechanotransduction requires experimental methods that should include two major
components: cultured cells adherent to firm surface that are exposed to controlled
mechanical stimulation. The most commonly used mechanical models for the in vitro study
of cellular responses to mechanical forces at low frequencies (below 5 Hz) utilize
controlled cyclic stretching, when attached to elastic membranes, and exposure of cell
cultures to a controlled fluid flow. For higher frequencies of mechanical stimulation (in the
infrasonic range) application of external vibration is a preferred method. Methods of
mechanical stimulation of mesenchymal cells in vitro are based on their ability to adhere to
plastic surfaces. Therefore the anchorage of these cells to a surface provides the possibility
to transfer mechanical force from the solid out-surface into the cells. Most of the
mesenchymal cell types are metabolically sensitive to the externally applied mechanical
force. Osteoblasts are the best example for cellular sensitivity to mechanical stimulation
therefore we describe the methods for mechanical stimulation of these cells, but the same
methods are also applicable to other mesenchymal cells, such as chondroblasts, fibroblasts,
mesenchymal stem cells, etc.
Keywords: Fluid shear, stretching membrane, vibration.
Bone tissue formation occurs along the vectors of force generated by
muscles and gravity [1]. Accordingly, bone matrix is elaborated by
osteoblasts following exposure to a mechanical force. The process of
mechanotransduction in osteoblasts, i.e., translation of the external
mechanical force into cellular biochemical signals, governs bone
shape maintenance, remodelling and repair. Therefore, understanding
of the mechanotransduction in osteoblast might help in the
development of treatment methods for pathological conditions in
bone, such as osteoporosis and fractures.
*Corresponding author Rosenberg Nahum: Department of Orthopaedic Surgery, Rambam Health Care
Campus, POB 9602, Haifa 31096, Israel; E-mail: nahumrosenberg@hotmail.com
14 Biomechanical Stimulation and its Clinical Prospects Rosenberg and Haber
The metabolic response of osteoblasts in mechanically active
environments depends on the interactions between fluid flow, either
intra and/or extracellular, with the cytoskeletal components, especially
microfilaments [2, 3]. Therefore, in vitro research of mechanotrans-
duction in osteoblasts requires experimental methods that should include
two major components: culture of mature osteoblasts and their exposure
to controlled mechanical stimulation. For this purpose a monolayer
culture of osteoblasts provides a reproducible model of cells exposed to
mechanical stimulation (Fig. 1).
Figure 1: Low power micrograph of human osteoblast-like cells in culture. Multipolar cells are
adherent to a plastic surface (calibration bar - 20μ).
Osteoblasts in two dimensional cultures tend to adhere to plastic
surface of the culture container (flask, well plate, petri dish, etc.) by
protein extensions of the cytoskeleton, which are reflected through the
cell membrane. These proteins include cadhedrins, integrins and
selectins [4]. The adhesion energy of the cell to the surface is at a
magnitude of 10
-16
joules. This energy magnitude is significantly
higher than the total cellular membrane bending energy (10
-18
joules)
[4]. Thus some deformation of the cell is possible without disrupting
Experimental Methods for Mechanical Stimulation Biomechanical Stimulation and its Clinical Prospects 15
the cellular membrane and without interfering with the adhesion of the
cell to the surface. On a theoretical basis, this energetic difference
allows application of up to 4-5% strain to a surface of a simple vesicle
bound by a lipid bilayer membrane and adherent to a plastic surface,
without the membrane distribution [4]. The osteoblast, as a whole cell,
is reinforced by a cytoskeleton and, thus, its mechanical deformation,
without damage to the cellular membrane, might be even higher.
According to these considerations, several methods for the application
of deforming forces on osteoblasts have been developed. In all these
methods, a basic concept of interrelated movements of the surface, the
adherent cells and intra-/extra-cellular fluid was utilized. Therefore,
the logical way of applying a mechanical force to the adherent to the
surface cell would be:
To induce a flow of the fluid surrounding the cells (culture media)
To change the dimensions of the surface holding the adherent cells
To generate a controlled movement of the surface with the adherent
cells
All these methods will cause deformation of the cellular membrane on
its free, not adherent to the supporting surface, interface with the
surrounding fluid (Fig. 2).
Figure 2: Schematic representation of a cell adherent to a solid surface and subjected to
deformation in a sagittal plane. The resting cell is bounded by a solid line and the deformed cell,
following movement in two directions, is represented by an interrupted lines.
movement
16 Biomechanical Stimulation and its Clinical Prospects Rosenberg and Haber
Various research methods have shown that osteoblast metabolic activity and
proliferation may be enhanced by mechanical and biochemical manipulations in
vitro (Table 1). The most commonly used mechanical model for the in vitro study
of osteoblast responses to mechanical forces is by the controlled cyclic stretching,
when attached to elastic membranes [5]. By this method, the cells are seeded on a
deformable surface, such as silicone. This surface is stretched by externally
applied unidirectional or radial forces. The deformation is achieved either by
bending of the supporting surface or by its peripheral stretching with subsequent
change in the surface area (Fig. 3).
Figure 3: A method for mechanical stimulation of cultured osteoblasts adherent to a stretchable
membrane. Osteoblasts mechanically deformed due to a radial extension of the supporting surface.
The arrows show the direction of stretching movement.
This procedure, aside from its need for sophisticated hardware with complex
experimental protocols, is unable to produce uniform stretching forces on all the
osteoblasts in culture [5]. Usually, a maximal strain on cells on bending surfaces
is around the bending axis, and on stretchable membranes at their periphery. The
maximal strain in this method does not exceed 20% [6]. The strains are
diminished with the distance from the maximal deformation area according to the
surface material properties and the bending curvature.
Exposure of osteoblast cultures to a controlled fluid flow is another popular model
for the in vitro research of mechanotransduction (Fig. 4). By this method a cyclic
or continuous cell deformation can be achieved by controlled inflow of media
from an external pump into the culture container with adherent to a surface
monolayer of osteoblasts [7]. The flow can be continuous, pulsatile or oscillatory.
This method usually utilizes a closed circuit of flowing media.
Stretchable membrane Cultured osteoblasts
Experimental Methods for Mechanical Stimulation Biomechanical Stimulation and its Clinical Prospects 17
Figure
4: Schematic representation of an experimental method for induction of strain in cultured
osteoblast adherent to a surface by controlled flow of media. The arrows represent the direction of
the media flow.
Both methods described above, i.e. stretchable surfaces and fluid flow application,
are designed to provide controlled oscillating strain in frequencies up to 5 Hz, in
particular mostly in the range of 0.1–2 Hz [5, 8-10]. The design of these devices
does not allow for raising the frequencies of the mechanical deformation of the
cell toward the higher infrasonic range of 20-60 Hz. The reason for preferring the
low frequency stimulation in experiments on mechanotransduction in cultured
osteoblasts is the assumption that, due to the normal human body locomotion in
the range of frequencies of 05-1.5 Hz, the bone cells, embedded in the canaliculi
in the bone matrix, are exposed to this range of mechanical stimulation.
Therefore, the experimental devices mentioned above are designed to mimic the
normal mechanical conditions in vivo.
Following the in vivo evidence that bone mass can be increased by vibration in the
infrasonic range of frequencies [11] and according to the existence of spontaneous
contractions of resting skeletal muscles in the frequency range of 20-60 Hz [12]
that might be an additional physiological source of mechanical stimulation, a need
for an experimental setup for research of these phenomena on cultured osteoblasts
arose. For this purpose an additional method for mechanical activation of
osteoblasts that utilizes the application of external mechanical vibration directly to
a cell culture plate with the ability to provide controlled and tuneable vibration of
the supporting surface of the cultures was developed [13]. This method is based
on the same assumption that cytoskeletal activation is caused by mechanical
deformation of the cells adherent to the culture plate surface [3]. The deforming
force is presumably transferred uniformly to all cultured cells, because the
vibration movement of the solid supporting surface is equally transferred to all the
adherent to it cells. This method is also based on a hypothesis that intracellular
pump
cell culture
18 Biomechanical Stimulation and its Clinical Prospects Rosenberg and Haber
metabolic pathways are influenced by the excitation of cytoskeletal components
by mechanical cell deformation induced by the external fluid flow [7]. This
method should have overcome the technical difficulties of the non-uniform strains
provided by the use of elastic membranes, and can provide a controlled movement
in one axis, and should be relatively simple in its technical handling. It should
also generate a uniform deformation force to a large number of cultured cells. The
basic design of this method utilized a horizontal surface with mounted and firmly
fixed plastic culture container with adherent osteoblasts. The surface moves under
the control of mechanical actuator (shaker). The generated controlled vibration
movement is tuneable in all ranges of vibration frequencies up to 60 Hz, and the
surface movement is recorded by an accelerometer (piezoelectric) and
displacement transducer (LVDT), which provide a real time recording of
mechanical parameters of the surface movement, e.g., amplitude of displacement,
acceleration, frequency (Fig. 5).
The important advantage of this method, in addition to the ability to control the
mechanical stimulation of cell in a wide range of frequencies, with subsequential
tuning of cellular deformation by changing the amplitude of the movement, and
also the ability to expose a large number of cells to this stimulation, is recording
of both the excitation parameters that are applied to the actuator and the resultant
mechanical parameters of a movement of the supporting platform of a culture. In
other words, it is possible to record not only the planned parameters but the actual
movement data. It was presented previously that these two types of parameters,
which should be as close as possible in order to obtain reliable data from the
experiments, may be significantly different in the stretching membrane method
and might require sophisticated calibrating [5].
The source of the difference might be the stretching membrane material
characteristics with significant “fatigue” factor of the stretchable surface. In the
vibration model these differences are mainly due to the friction between the moving
parts of the supporting platform. The effort to reduce the friction may reduce the
difference between the excitation and the actual mechanical parameters (Fig. 6).
The differences in the experimental approaches used cause undesirable difficulties
in comparing the results from different experiments. In order to compare data
from different experimental methods, the mechanical stimulation should be
uniformly characterized by the mechanical parameters of the resultant
displacement, frequency and acceleration (or wave shape of the applied force).
Experimental Methods for Mechanical Stimulation Biomechanical Stimulation and its Clinical Prospects 19
A
B
Figure 5: Experimental setup for vibration of cultured osteoblasts. The well plate with cultured
cells is mounted on a horizontally oriented shaker. The vibrating movement of the shaker is
controlled by an amplifier. The acceleration is measured by a piezoelectric accelerometer and the
displacement by a special displacement transducer (LVDT). A- schematic representation, B- the
device used for experiments.
In most of the studies, neither all this information nor even common optimal
stimulation parameters for osteoblast-like cells activation are provided. Future
efforts should be directed to standardizing the protocols of the in vitro mechanical
stimulation of osteoblasts, regardless of the experimental method used. This
standardization should provide all the essential biomechanical information, such
as exact specification of the mechanical force applied and whether it is cyclic or
oscillating, the resultant movement wave specifications, and information on the
cell deformation (strain). If this information is provided, the optimal parameters of
the mechanical stimulation can be determined and the present existing
controversy regarding the accuracy of these parameters can be settled. The
standardization of the report of the resultant mechanical parameters in the
different experimental methods is crucial for designing and executing reliable
experiments on mechanotransduction.
0.5 m/sec
2
25 m
SINE
2 mV
20 Hz
ACCELEROMETER
WELL PLATE
SHAKER
AMPLIFIER / PULSE GENERATOR
DISPLACEMENT
TRANSDUCER
20 Biomechanical Stimulation and its Clinical Prospects Rosenberg and Haber
A:
B:
4
3
2
1
0
-1
-2
-3
-4
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
The Exitation Sine wave
Amplitude
Time.[sec]
004
003
002
001
0
-001
-002
-003
-004
0 0.00 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Accelerometer reading
Amplitude
Time.[sec]
Experimental Methods for Mechanical Stimulation Biomechanical Stimulation and its Clinical Prospects 21
C:
Figure 6: A: Excitation profile applied to the vibration actuator – 20 Hz; B: Resultant mechanical
vibration movement parameters showing basic 20 Hz frequency of vibration and displacement
amplitude of 20-30µ; C: Spectrum of the frequencies of the resultant vibration moments showing
the high proportion of vibration with frequency of 20 Hz.
In this experimental setup, it is apparent that
most of the excitation parameters were expressed in the resultant vibration movement.
Table 1. Parametric data of mechanical stimulation of osteoblast-like cells in vitro in several
studies.
Reference: Frequency
Hz
Displacement
mm
Acceleration
mm/sec
2
Time
hours
Wave
shape
Cells Origin Parameters
investigated
[14] 0.05 0.009
a
0.00089
b
48 square osteoblasts chicks DNA,Cell No.
[15] 1 0.0003
a
0.0112
b
1.2 NI osteoblasts
h
uman Cell No.
[16] 1 0.0003
a
0.0112
b
0.07 NI osteoblasts bullock Cell No.
[17] 0.5 0.003
a
0.03
b
6 square MC3T3E1 rat DNA
[13] 20 0.025 450-550 0.13 sine osteoblasts human DNA
NI - not indicated
a
- average displacement estimated from strains applied to cells, assuming that the diameters of osteoblasts are in the range
of 20–40 m
b
- calculated with approximation to sine shaped vibration
We provided here a general overview of the existing concepts for the use of
experimental methods of mechanical stimulation of cultured osteoblasts. It is
Spectrum of accelerometer reading
20
0
-20
-40
-60
0102030405060708090
-20
-40
0
20
-60
0 50 100 150 200 250
Amplitude
Amplitude
HZ
22 Biomechanical Stimulation and its Clinical Prospects Rosenberg and Haber
obvious that the optimal method for the particular experiment should be designed
according to the research hypothesis and might be “tailored” according to the
principles that we described above.
CONFLICT OF INTEREST
The author confirms that author has no conflict of interest to declare for this
publication.
ACKNOWLEDGEMENTS
Declared None.
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