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Mesenchymal Cell Activation by Biomechanical Stimulation and its Clinical Prospects, 2016, 23-34 23
Nahum Rosenberg (Ed)
All rights reserved-© 2016 Bentham Science Publishers
CHAPTER 3
Mechanical Stimulation of Mesenchymal Stem Cells
David Nikomarov and Nahum Rosenberg
*
Department of Orthopaedic Surgery, Rambam Health Care Campus, POB 9602, Haifa 31096,
Israel
Abstract: Stem cells’ research reached an advanced stage with clear understanding of
the ability of multipotent mesenchymal stem cells to maturate to different metabolically
mature cells of the connective tissue. In combination with advancement in genetic
research it is now possible to start decoding basic cellular mechanotransduction
pathways in stem cells. This novel research field has the potential to lead to the
discovery of new and important niche in the biomedicine with a prospective of reaching
important clinical advancements. Although important research success has been reached
recently the exact mechanisms of the MSC’s determination of the fate are still elusive
and require further extensive research efforts. In order to reveal the basic mechanisms
of stem cell differentiation two distinct areas of investigation are mostly considered –
the biochemical perspective and the physical or biomechanical perspective. In dealing
with the biomechanical prospective, an attempt to mimic the mechanically active milieu
for the MSCs was introduced using different research methods (see Chapter 2). Because
mesenchymal derived cells differentiate into tissues that usually have mechanically
supportive function, it is only logical to hypothesize that mechanical stimuli might
cause changes on the cellular level. In this chapter we review the effects of mechanical
stimulation of mesenchymal stem cells (MSCs) according to different features and
parameters of the stimulus: shear stress applied to MSCs by fluid, micro-environmental
scale cellular mechanical stimuli, substrate stiffness, cell shape and surface topography
effects, the genomic level control of external mechanical force, micro-environmental
nano-fibres’ stretch and reorientation with the directional effect of the mechanical
stimulation. Clearly the understanding of the numerous mechanical effects with
different mechanical parameters of the stimuli that are applied to MSCs has been
advanced recently but still requires further extensive and thorough research in order to
manipulate these cells biomechanically for a clinical use.
Keywords: Cell differentiation, ECM, mechanical stimuli.
INTRODUCTION
Stem cells’ exciting characteristics hold a great potential for biological research
and high hopes for future medical therapies. The incredible possibility to replace
*Corresponding author Rosenberg Nahum: Department of Orthopaedic Surgery, Rambam Health Care
Campus, POB 9602, Haifa 31096, Israel; E-mail: nahumrosenberg@hotmail.com
24 Biomechanical Stimulation and its Clinical Prospects Nikomarov and Rosenberg
diseased cells and to use stem cells as "repair kit" in pathological situations seems
to light the imagination. Stem cells research is still in its initial phase, and one of
the main fields that requires a special attention is their biochemical response to
different stimuli, humoral and biophysical. Despite the recent rapid advancements
in the field of stem cells research, the deep and exact understanding of the cellular
mechanisms, which are responsible for maturation of stem cells into specific cell
types of connective tissue, are not clear.
A lot of research was directed to discover the humoral factors and their pattern or
regulatory activities involved in maturation of MSC’s, but surpassingly these
activities were revealed more clearly following biophysical stimulation of these
cells. These studies have been conducted in order to explore the effect of
biomechanical stimulation on multipotent cells. Some of the initial examples are
the early studies by Hamilton et al. [1] and by Park et al. [2], who demonstrated
that cyclic unidimensional stretch is a driving factor in the differentiation of bone
marrow-derived mesenchymal stem cells (MSCs) toward smooth muscle cells
(SMCs). Later studies demonstrated some degree of vascular cells (SMC and/or
endothelial cell) differentiation from stem cells as a result of tension and cyclic
stretch [3, 4], shear stress and pressure [5], or combined shear, stretch, and
pressure [6]. Other studies demonstrated that osteogenic differentiation can result
from mechanical stimuli [7-9]. Different studies showed the importance of
different types of mechanical stimulations, e.g. fluid shear stress, matrix stiffness,
surface topography, cell shape, combinations of shear and dynamic compression,
cyclic tensile strain, mechanical forces, etc on the metabolism of stem cells.
Fluid Shear Stress
Fluid shear stress can be broadly defined as the deforming factor generated by the
movement of fluid on a surface [10]. In particular, fluid shear stress is defined as
the stress (i.e., force per unit of area) that moving fluid applies tangentially to the
solid boundary of its container [11]. The role of fluid shear stress in biology has
been investigated extensively in the context of vascular biology, where the
continuous flow of blood constantly generates shear stress on the endothelial cells
which are lining the inner surface of blood vessels. Early studies showed that the
endothelium is responsive to shear stress [12-15]. This finding initiated a new area
of investigation leading to the current concept that fluid mechanical forces are
essential regulators of vascular function [16, 17]. Following these findings in the
vascular research the role of fluid shear stress, as a regulator of cell function, has
been recognized in other biological systems, where fluid flow is physiologically
present, i.e. from the kidney tubules [18] to the canaliculi in the bone tissue [19].
Mechanical Stimulation of Mesenchymal Stem Cells Biomechanical Stimulation and its Clinical Prospects 25
Adamo et al. extensively reviewed the published data on the fluid shear stress's
ability to direct the differentiation and modulate the function of embryonic and
somatic stem cells [10]. They concluded that the stem cells are not an exception to
the principle that many different cell types are susceptible to the influence of fluid
shear stress. This review demonstrated that fluid shear stress is part of important
biological activities in MSCs. Additionally it was found that a mechanical force
induces cellular differentiation by activating specific genetic and signaling
pathways with promotion of the cellular maturation via gene expression that is
responsible for various cellular activities. The reviewed studies initiated the
understanding of the prospective of using fluid mechanical model as an additional
‘‘differentiation tool’’ for cultured MSCs’ stimulation, in addition to humoral and
extracellular matrix factors.
In the research of the stem cells differentiation following exposure to fluid shear
stresses, several crucial points are still unclear [20, 21], i.e. the exact cellular
effects of shear stress on stem cells are still unclear due to the limited range of
experiments that can reveal the exact biological significance of shear-stress-
mediated differentiation of these cells. Additionally because these studies utilize
different research protocols for expansion and inducing maturation of stem cells,
with the use of different mechanical parameters of applied shear stress (different
parameters of frequency, lengths of the shearing wave, acceleration and duration
of cellular exposure to mechanical stress) there is difficulty for didactic
comparison of results from different studies. Therefore an effort to reach a
consensus on the uniform methods and similar protocols of the stem cells
mechanical manipulation, along with exact determination of the
mechanotransduction pathways, is crucial in order to understand the underling
ability of these cells to alter their metabolic activity in the response mechanical
stimulation.
Mechanical Stimuli in the Cellular Microenvironment
Extensive data are available on stem cell mechanics, especially on the effect of
matrix stiffness, surface topography, cell shape and mechanical forces on the
phenotypical determinations of MSCs and embryonic stem cells [22]. Recent
studies showed that the mechanical properties of the extra cellular milieu and
external application of mechanical forces initiate various cellular pathways that
determine cellular structure and function [23-31]. In addition to the in vitro
studies, there is evidence that mechanical forces affect and regulate tissue three
dimensional structure in vivo [32-36]. While long standing and extensive efforts
have been devoted for the understanding of the pathways of humoral cellular
26 Biomechanical Stimulation and its Clinical Prospects Nikomarov and Rosenberg
function, i.e. how growth factors and cytokines trigger and transduce cellular
signals, new aspects of mechanical factors that determine MSC’s metabolism
have been revealed recently. The overall conclusion from the recent research is
that MSC’s differentiation into specific mature cell type is, at least partially,
influenced by the mechanical microenvironment [22]. Biochemical interactions of
soluble factors and the extracellular matrix (ECM) in the neighbouring cells can
determine the biochemical and mechanical signals and regulate and maintain the
cell cycle.
Surface adhesion receptors, such as integrins and cadherins, mediate cell adhesion
to the ECM scaffold and to the neighbouring cells. These proteins are part of the
cellular mechanism to sense forces, transmit them to the interior of the cell or to
other cells, and transduce them into biochemical signals that are essential for a
spectrum of cellular responses, including motility of cells, differentiation and
regulation of cell proliferation [25, 26, 30, 31, 37, 38].
In the cellular microenvironment, the ECM characteristics, i.e. stiffness,
topography and composition, are involved in the regulation of cells’ behaviour. It
is important to recognize that cells are sensitive to the microenvironment surface
that acts as signal generator. The cells respond to the microenvironment signaling
by their shape adaptation. This process of adaptation generates traction stress and
produce mechanical forces that can be transmitted to neighbouring cells. Exposure
of the cells to a mechanical stimulation, such as fluid shear stress (discussed
above) activates mechano-sensitive ion channels, heterotrimeric G proteins,
protein kinases and other membrane associated signal-transduction molecules.
These activate the downstream signalling cascades and lead to force-dependent
changes in gene expression [39], which are mediated by the distortion of the
specific adhesion receptors that link to the cytoskeleton. This process is
accompanied by deformation of the lipid bilayer that, in lesser extent but also
contributes to the mechanical signaling pathways [30].
Substrate Stiffness
The stiffness of the microenvironment determines the MSC’s fate characteristics
[22]. The importance of sensing the elastic properties of the ECM had been shown
in studies with different cell types [29, 40, 41]. Engler et al. made the initial
attempt to evaluate the role of matrix stiffness in determining the human MSCs
maturation [23]. He used polyacrylamide gels coated with collagen as an artificial
matrix for cell attachment in vitro [41]. The elastic properties of the matrix ranged
from soft to a relatively rigid, depending on the extent of chemical crosslinking.
Mechanical Stimulation of Mesenchymal Stem Cells Biomechanical Stimulation and its Clinical Prospects 27
The effects of various degrees of matrix elasticity on the hMSCs were tested. The
results of this study suggested that cell differentiation was determined by the
matrix material properties, i.e. degree of stiffness. When the cells were cultured
on a relatively soft surface, similar to the elasticity properties of brain tissue (0.1–
1 kPa), they acquired a neuronal phenotype. When external surface had
intermediate stiffness properties, similar to muscle (8–17 kPa), myogenic
phenotype predominated, and on the rigid matrices, similar to cartilage and bone
(25–40 kPa), osteoblastic differentiation has been revealed. Molecular analysis
(microarray) showed that the expression of the neuron, muscle or bone
characteristic markers was increased four to six-folds when exposed to a specific
enhancing surface substrate. Immunofluorescence studies showed that the lineage-
specific markers of neural tissue, muscle, bone and cartilage reached 50% level in
comparison to corresponding differentiated cultured cells. This implies that the
supporting matrix material properties are one but not the only factor for
determination of the final differentiation of cells. This mechanical factor is highly
efficient in the partial induction of MSCs’ differentiation into a specific
mesenchymal lineage.
It was also found that the inhibition of non-muscle myosin II (NMMII) blocked
all the elasticity-directed lineage determinations, without significant effect on
many other aspects of cell function, indicating on the existence of distinct cellular
pathways that are involved in cell-supporting substrate interactions that direct
cellular differentiation [23]. This observation was supported by others showing
that regulation by matrix stiffness was the essential and important part of the
regulatory cellular pathways in addition to the growth factors that are responsible
the MSCs’ differentiation induction. Zemel et al. demonstrated that the alignment
of NMMII based stress fibres in the MSCs is depended on the matrix martial
properties. These authors found that maximal effect was reached at similar
rigidity properties of both, the cell and the matrix. From this observation it is
logical to suggest that mechanical interactions between the supporting matrix and
cytoskeleton organization are part of the cellular maturation process [42].
The exact signalling pathways by which micro-environment determines the cell
maturation fate are still unclear and should be further determined. Huebsch et al.
evaluated the impact of the supporting matrix mechanical properties on the
mesenchymal progenitor cells’ maturation in the three dimensional cultures,
which more resemble the in vivo conditions [43]. They used a model of
encapsulated in 3D hydrogel synthetic ECM murine MSCs. In this experimental
model the matrix level of elasticity had a significant effect on MSCs maturation
28 Biomechanical Stimulation and its Clinical Prospects Nikomarov and Rosenberg
determination, i.e. osteogenesis was observed at the intermediate elasticity levels
of 11–30 kPa, while the adipogenesis occurred in a more elastic supporting matrix
of 2.5–5.0 kPa. Therefore the observations of the effect of matrix material
properties on the MSCs’ maturation are actually similar in two and three
dimensional cultures [23]. Interestingly, the level of the cell differentiation in the
three dimensional environment was not dependent on cellular morphology. The
latter is not in an agreement with a previous report on a monolayer cultures [27].
In the three dimensional culture conditions the integrin binding, as well as the
reorganization of the adhesion ligands on the nanoscale, is determining the matrix
elasticity. These molecular activities were found to be dependent on mechanical
stimulation by traction and correlated with the MSCs’ osteogenic commitment
[23]. It was noticed that matrix degree of elasticity, additionally to its ability to
determine the cellular maturation, also efficiently regulates the self-renewal of
mature MSCs [44], i.e. it was found that in muscle progenitors, which were
cultured on rigid plastic surface,~106 kPa, lost their regeneration ability and
subsequentially decrease in regenerative potential, while in muscle stem cells,
which were cultured on softer surfaces that are similar to the elasticity of muscle
(12 kPa), a cellular and muscle tissue regeneration was observed in a murine
model with successful in vivo incorporation. These findings further demonstrated
that the degree of matrix stiffness/elasticity contributes to the determination of the
MSCs’ differentiation fate.
Cell Shape
Cell shape also can affect the of MSCs’ fate determination [22], i.e. there is
extensive research evidence that cell shape is a part of several mechanical factors
that are involved in the cellular pathways, such as cellular proliferation, viability
and maturation [25, 45, 46]. In particular, studies showed that variability in cell
shape affects the fate determination of hMSCs [27]. These studies utilized a
micro-patterning technique in order to change the cell shape and to determine the
degree of cell spreading in cultures. It was shown that by controlling these
parameters in the adherent to the surface hMSCs, after being flatten and spread, it
was possible to induce osteogenic characteristics in these cells, while when the
cells were kept round they differentiated into adipocytes.
On the subcellular level there is experimental evidence that the cell shape controls
the activity of Rho family of GTPases that regulate intracellular actin dynamics
[47]. From this evidence the question whether actin cytoskeleton regulators, such
as a RhoA, are involved in lineage specification can be arisen. And indeed it was
shown that the cellular shape controls the fate determination of hMSCs via RhoA
Mechanical Stimulation of Mesenchymal Stem Cells Biomechanical Stimulation and its Clinical Prospects 29
activity, e.g. cell cultures with predominantly negative RhoA caused
adipogenesis, while the cells with active RhoA were directed into oteoblastic
lineage. The RhoA-controlled commitment cellular pathway requires the actin–
myosin generated structural tension. This pathway bypasses the other cellular
signals that involve humoral growth factors.
Therefore cytoskeletal mechanics, cell shape and RhoA signalling are crucial and
independent parts of the determination process of MSCs’ maturation commitment.
Surface Topography
As was mentioned above the superficial topographic characteristics of ECM also
affect the MSCs’ maturation fate [22]. The ECM surface has a complex mixture
of pores, ridges and fibres in the nano-metric scale. These surface properties of
the supporting matrix was found to determine cells’ behaviour [48, 49]. By using
the method of electron beam lithography (EBL) for the precise construction of
nano topographies the abilities to investigate the interactions between cells and
supporting surface was significantly facilitated. And indeed it was shown that the
cultured MSCs respond particularly to the nanoscale topographic characteristics
[50]. Naturally, the in vivo supporting surfaces have highly random surface
characteristics in the contrast to the high order of topographic organization and
symmetry or “ordered disorganization” following EBL on artificial surfaces that
are investigated in vitro. Therefore the constructed by the EBL surfaces are
suitable for designing the research of cell-surface interactions with reduced
number of variables. It was shown that on the surfaces which were imprinted with
120nm in diameter 100nm deep pits, in different patterns ( all with either absolute
or average center–center spacing of 300 nm in square array (SQ), disordered
square array with dots displaced randomly by up to 50 nm on both axes from their
position in a true square (DSQ50), disordered square array with dots displaced
randomly by up to 20 nm on both axes from their position in a true square
(DSQ20) and pits that are placed randomly over a 150µm by 150 µm field
repeated to fill a 1 cm
2
area (RAND)) and seeded with MSCs, district areas of
intense cell aggregation and secretion of bone-specific ECM proteins (osteopontin
and osteocalcin) with calcific nodule formation was observed only on the DSQ50
patterned surface. The use of this specific nanoscale pattern caused a similar rate
of MSCs’ osteogenic maturation as in MSCs that were cultured with osteogenic
media. This cellular phenomenon has a distinct differentiation profile implying on
existence of different maturation mechanisms or cellular pathways for the
induction of this type of cellular differentiation.
30 Biomechanical Stimulation and its Clinical Prospects Nikomarov and Rosenberg
Genomic Control Induction by Mechanical Forces
External mechanical forces control MSCs’ gene expression [22]. In order to
investigate the effects of mechanical forces or strains on MSCs’ differentiation an
original method a micro-patterned cellular alignment was used. By this method
the MSCs were aligned in the direction of uniform vector of the externally applied
mechanical force that generated a uniformly directed strain in these cells [51]. By
using this experimental setup it was found that expression of calponin 1, a marker
for smooth muscle cells, was increased, while the cartilage matrix markers’
expression was diminished. When the cells were aligned perpendicularly to the
direction of the force vector, the osteoblastic expression was reduced [51]. These
findings imply that not only existence of mechanical strain determine the MSCs’
differentiation but also the direction of such strain is important for determining
these cells’ maturation fate. In an additional study there was evidence that
combination of osteogenic cytokines (TGF- ß1) and of cyclic mechanical external
cellular stimulation have a synergistic up regulation effect of calponin 1 gene
expression [52]. From these findings rises a logical conclusion that, both, the
humoral growth factors and mechanical strain interrelate in gene expression that
governs MSCs’ differentiation.
Micro-Environmental Fibres’ Stretch and Reorientation
There is also experimental evidence that fibre stretch and reorientation modulate
mesenchymal stem cell morphology and fibrous gene expression on specific
nano-fibrous microenvironments [53]. Because differentiation of MSCs is
mediated by the incorporation of soluble signalling factors, physical signals,
substrate morphology and external mechanical stimulation, the recognition of the
structure of the micro-patterned external supporting surface should contribute to
gain control of modulation and cellular maturation, with cellular fate
determination, in order to determine the generation of specific functional type of a
connective tissue.
When the MSCs are aligned on nano-fibrous microstructure, the cellular and
nuclear deformations vary depending to the scaffold orientation. Re-orientation of
the aligned MSCs corresponds to the vectors of applied strains. Therefore cellular
deformations at the nanoscale correspond with scaffold orientation, with changes
in nuclear shape, depend on the direction of surface microstructure and the
direction of applied strain. Additionally these deformations modulate gene
expression that is also dependent on scaffold and cell orientations. These findings
support the theory that surface microstructure cause a two dimension mechano-
Mechanical Stimulation of Mesenchymal Stem Cells Biomechanical Stimulation and its Clinical Prospects 31
sensitivity in the MSCs and therefore enable a possibility of experimental design
control for exploration of the mechanotranduction in these cells.
Directional Effect of Mechanical Force Stimuli
Clearly the crucial subject of interest is the enigma of the MSCs’ ability to
determine the phenotypic direction of maturation by biomechanical stimulation
[54] The common hypothesis is, as it has been already mentioned in this chapter,
that the direction of the force stimulation applied to MSCs might have a central
role in determining these cells’ differentiation fate.
Following several studies on the role of the specific growth factors in
mesenchymal stem cell differentiation, progress toward determination of
dedicated research protocols for cartilage, bone and adipose tissue generation was
achieved. But these research methods tend to ignore the important role of
mechanosensibility of these cells in their final fate determination. Therefore in
several studies mechanical stimulation was indeed applied to constructs with
mesenchymal stem cells showing induction of cellular maturation and
differentiation. The example of such pivotal fate determination in MSCs
following mechanical stimulation is their progression either into cartilage or bone
lineage. Force propagation across joints exhorts compression, tension and
translation of the articular surfaces. Therefore it is logical to suggest that all these
force vectors should be utilized in the research of cartilage stimulation in vitro.
Accordingly the role of shear stresses’ role in determination of the fate of stem
cell differentiation was investigated [54]. Moreover the combined effect of
surface shear and cyclic axial compression in chondrogenic differentiation of
human bone marrow-derived stem cells was investigated as well. By using a
loading compression device combination of both stimuli were applied onto fibrin /
polyurethane composites with embedded human mesenchymal stem cells, while
no exogenous growth-factors were added to the culture medium. Compression or
shear alone were insufficient for the chondrogenic induction of hMSCs, but the
exposure of the cells to a shear stress and dynamic compression caused a
significant increase in a cartilage specific gene expression. These findings were
also supported by histological analysis, which presented the sulphated
glycosaminoglycan and the collagen II staining only following the compression
and shear application on the hMSCs. These findings provide important evidence
that supports the need of specific type of mechanical stimulation for a specific
type of differentiation of MSCs and suggest the need of the specific post-
operative mechanical protocols when cell therapy with MSCs is considered.
.
32 Biomechanical Stimulation and its Clinical Prospects Nikomarov and Rosenberg
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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