Download Mechanobiology of bone cells

Bone mechanobiology
© Schattauer 2010
Mechanobiology of bone cells
J. Rychly
Arbeitsbereich Zellbiologie, Medizinische Fakultät, Universität Rostock
Keywords
Schlüsselwörter
Bone cells, mechanics, mesenchymal stem
cells, differentiation
Knochenzellen, Mechanik, mesenchymale
Stammzellen, Differenzierung
Summary
Zusammenfassung
Bone mass, morphology and properties of the
bone material are regulated by the functions
of osteoblasts, osteocytes, and osteoclasts.
These cells respond directly or indirectly to
mechanical forces from the environment with
the expression of differentiation markers, proliferation or release of bioactive factors. Osteocytes appear to be an important regulator
for the adaptation of bone to changes in the
mechanical environment. Mesenchymal stem
cells which are located in bone marrow can be
mechanically stimulated to differentiate into
osteoblasts and chondrocytes but not to adipocytes. Integrin receptors are the principal
mediators of mechanical forces and induce a
signal transduction. The conversion of mechanical signals into biochemical signals is facilitated by unfolding of proteins to expose
binding sites. Implant materials offer the opportunity to control the mechanical stimulation of cells by modifying the rigidity,
geometry of adhesion sites, and the 3D-environment.
Knochenmasse, Morphologie und Eigenschaften des Knochens werden durch Osteoblasten, Osteozyten und Osteoklasten reguliert.
Diese Zellen reagieren direkt oder indirekt auf
mechanische Stimuli aus der Umgebung mit
der Expression von Differenzierungsmarkern,
mit Proliferation oder Freisetzung von bioaktiven Faktoren. Osteozyten sind wichtige Regulatoren für die Adaptation des Knochens an
Veränderungen der mechanischen Umgebung. Mesenchymale Stammzellen, die im
Knochenmark lokalisiert sind, können durch
mechanische Stimulation in Osteoblasten und
Chondrozyten, aber nicht in Adipozyten differenzieren. Integrinrezeptoren sind die wesentlichen Mediatoren der mechanischen
Kräfte und induzieren eine Signaltransduktion. Die Umwandlung von mechanischen Signalen in biochemische Signale wird durch das
Entfalten von Proteinen zur Präsentation von
Bindungsstellen ermöglicht. Implantatmaterialien bieten die Möglichkeit, durch Modifikation der Steifigkeit, der geometrischen Anordnung von Adhäsionsmotiven und der Gestaltung der 3D-Umgebung die mechanische
Stimulation der Zellen zu steuern.
Correspondence to
Prof. Dr. Joachim Rychly
Universität Rostock, Medizinische Fakultät,
Arbeitsbereich Zellbiologie
Schillingallee 69, 18057 Rostock
Tel.: 03 81/494 57 30, Fax: 03 81/494 57 39
E-Mail: [email protected]
Mechanobiologie von Knochenzellen
Osteologie 2010; 19: 245–249
received: August 20, 2010
accepted: August 24, 2010
Introduction
Mechanical signals are critical to control the
physiological processes in human. In particular mechanical stimuli are potent regulators of bone mass, morphology and properties of the bone material (22). Clinical evi-
dence has proved the anabolic effect of
mechanical load in bone (32). Experimental
studies have examined strain magnitude
and frequency that determine the osteogenic response (16). During functional
loading normal strains causes volumetric
changes, while shear strains cause angular
deformation. But the functional responses
are ultimately mediated by the cells, which
are able to sense mechanical signals. Bone
remodelling is facilitated by osteoclasts
wich resorb bone and osteoblasts which
produce bone. Osteocytes which develop
from osteoblasts are regarded as the principal sensor cells for mechanical signals in
bone (20). These cells are located in the vicinity of interstitial fluid-filled lacunae and
it has been postulated that when bone tissue
is deformed by mechanical load a fluid
pressure gradient is formed which move
across osteocytes to induce shear stress on
the cells. In addition mesenchymal progenitor cells are becoming of great interest in the
context with mechanical regulation of bone
tissue because these cells are a driving force
in functional engineering of bone tissue. In
this review the biological responses of bone
cells to physical forces, the underlying
mechanisms of sensing and mechanotransduction as well as the mechanical manipulation by substrate materials will be discussed.
Biological responses of
bone cells to mechanical
forces
Tissue may experience different types of
mechanical forces, like pressure, tensile
forces or shear forces, applied by fluids. In
the bone tensional forces predominate in
response to movement of articulated tissues, when the flexion of tendons and contraction of muscles pulling on bone. For in
vitro experiments bone cells or progenitors
have been mechanically exposed to hydraulic pressure, compression of a carrier,
substrate strain or shear stress. Although
these approaches induce differential mechanical effects to the cell, they all provoke a
deformation of the cell body or changes in
the cell shape. In addition, experiments
were done in which defined adhesion receptors, which are regarded as mechOsteologie 3/2010
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J. Rychly et al.: Mechanobiology of bone cells
anotransducers, have been mechanically
stressed (6, 15, 34). In this case, the shape of
the cell remains nearly unaltered.
Numerous studies analysed the effect of
mechanical stimuli in respect to the anabolic functions of osteoblasts. Applying
different modes of mechanical strain the
expression and secretion of collagen I as the
major component of the bone matrix increased. Similarly, ALP, osteocalcin, osteopontin and matrix mineralization were
stimulated due to cyclic strains (42, 45).
Like cyclic strain, flow stimuli have been reported to induce osteogenic functions,
such as increase in ALP activity (8). This
may be important for designing bioreactors to stimulate osteogenesis. However, the
data are controversial and demonstrate
that interactions of different environmental factors as well as the mode of mechanical
stimuli are important. For example, in
other experiments flow stress can promote
the expression of some osteogenic markers,
but had no effect on the expression of ALP
and osteocalcin, which was dependent on
the adhesion substrate (38). Mechanical
stimulation also stimulates the secretion of
bioactive factors which modulate the biological responses in an autocrine or paracrine manner. The release of prostaglandin
E2 (PGE2) and NO were stimulated in primary osteoblasts due to fluid flow (26, 31).
PEG2 can stimulate IGF-I expression in osteoblasts and NO can induce anabolic
functions in osteoblasts throught the MAPkinase pathway. The direct or indirect
stimulation of growth factors in osteoblasts
due to mechanical loading have been demonstrated (40). Secretion of TGFβ, bFGF,
VEGF and different interleukins were increased in osteoblasts after application of
different modes of strain, which indicates
that mechanical forces also regulate the
physiology of bone by the stimulation of
the cytokine secretion.
Several experiments investigated the
role of mechanical forces for cell proliferation, which is a key factor in bone
formation (40). Most of the studies have
shown that relatively short times of fluid
flow or cyclic strains induced an increased
proliferation of osteoblasts. A constant
fluid flow of 30 minutes was sufficient to
stimulate the proliferation of primary osteoblasts.
Osteocytes are terminally differentiated
osteoblasts and form a syncytial network
throughout bone that is ideally structured
to perceive and respond to site-specific
changes in mechanical loading of the skeleton. They are surrounded by interstitial
fluid-filled lacunae and canaliculi that
when mechanical forces deform bone the
resulting fluid-flow perturbs osteocytes
and their dendrites in the canaliculi. Osteocytes have been implicated as the mechanosensors in bone and appear to be an important regulator for the adaptation of
bone to changes in the mechanical environment (17). According to the concept of
Frost’s mechanostat bone regulates forming and resorbing of bone in response to
the magnitude of the external mechanical
load. In mice, weightlessness induced
apoptosis of osteocytes followed by increased bone resorption 14 days later. Together with supporting studies it was concluded that apoptosis of osteocytes is the
consequence of disuse and is required for
stimulation of osteoclastogenesis and bone
resorption (1). While a lack of mechanical
loading leads to apoptosis of osteocytes,
animal studies as well as in vitro experiments demonstrated that mechanical load
prevent apoptosis of osteocytes (17). A
mechanism for this effect might be an increased expression of Bcl-2, which has antiapoptotic activity. Thus, if bone receives
sufficient mechanical stimuli osteocytes remain viable and bone mass is maintained.
When mechanical strains are low, apoptosis
of osteocytes will induce bone loss. Several
soluble factors which have been shown to
be modified due to mechanical loading of
osteocytes may be involved in the regulatory function of these cells. Mechanical
strain regulates the expression of RANKL
which recruit osteoclast precursor cells and
osteoprotegerin which blocks osteoclastogenesis (49). The expression of sclerostin in
osteocytes which is regulated by mechanical strain is required for bone loss. Sclerostin inhibits bone formation by inhibiting Wnt-signaling (27).
Only few data exist, how mechanical forces
affect osteoclasts. Osteoclasts are mainly regulated by mechanically loaded osteoblastic cells
which release RANKL or PGE2. A direct
mechanical load to osteoclasts revealed a decreased resorption activity (23).
In recent years main attention was directed to the mechanical regulation of mesenchymal stem cells for bone formation
(28, 35). These cells reside in stem cell
niches, among other in the bone marrow.
Because of their multipotency, i.e. they are
able to differentiate into various cell lines of
the mesenchym, these cells are attractive
for the regenerative medicine. In the last
years much have been learned, how the
complex nature of the microenvironment
in a stem cell niche controls the biology of
the cells. Beside soluble factors, mechanical
stimulation mediated by the extracellular
matrix or in contact with other cells contributes to the regulation of mesenchymal
stem cells. Numerous in vitro experiments
convincingly demonstrated that mechanical loading of mesenchymal precursor
cells stimulates the differentiation to the
osteogenic pathway (35). Different types of
mechanical loads, including fluid flow,
stretching induced increased expression of
osteogenic markers, like osterix, ALP, collagen I within 24 hours and ostecalcin after
two weeks. Other experiments have shown
that mechanical loading stimulates the expression of chondrogenic markers, like
Sox9, collagen II and aggrecan (35). While
mechanical load to mesenchymal stem cells
stimulated the differentiation both to osteoblasts and chondrocytes, the adipogenic
pathway was inhibited (41). Application of
mechanical strain for six hours decreased
the expression of PPARγ, a marker for adipogenesis. In these experiments mechanical strain stimulated the expression of
β-catenin and its translocation to the nucleus, which caused the inhibition of adipogenic differentiation.
A key question is how the direction of
differentiation can be controlled by mechanical loading. There is evidence that the
modulation of the loading pattern, like amplitude, duration, frequency determines
the effects (35). Chondrocyte differentiation depends on the load magnitude and
duration of mechanical stimulation. Another factor is the integration of signals
from the adhesion substrate and soluble
growth factors with the mechanically induced signals. In our experiments we
stimulated the expression of collagen I in
mesenchymal stem cells by a short-time
mechanical stress on the β1-integrin recep-
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J. Rychly et al.: Mechanobiology of bone cells
tors (25). However, this effect was only inducible when the cells were cultured on fibronectin and RGD peptides and depended on the culture medium. Thus, the
complex interaction between different environmental signals determines the biological outcome. In addition to their ability
to differentiate into multiple directions,
growing evidence exists that mesenchymal
stem cells play an important role in the
regulation of regenerative processes by the
release of a variety of bioactive factors (5).
Studies demonstrated that these functions
are equally regulated by mechanical forces.
In osteogenic precursor cells cyclic mechanical stretching stimulated the expression
of osteoprotegerin, which controls the
function of osteoclasts (48). We demonstrated that mechanical loading of integrins in mesenchymal stem cells increased
the expression and release of VEGF, a factor
which stimulates angiogenesis (25). In
these experiments again the combined effect of the adhesion substrate and mechanical signals was obvious. VEGF was stimulated when the cells were cultured on tissue
culture polystyrene but not on covalently
immobilized fibronectin. To understand
the complex interaction of different factors
we have to explore the intracellular mechanisms which govern the mechanical control of cells.
Mechanisms of
mechanotransduction
In adherent cells the mechanical interaction with the environment is bidirectional. Cells are able to sense mechanical
stress from outside and generate mechanical forces on the adhesion substrate. The
cytoskeleton which consists of a network of
microfilaments and microtubules stabilizes
the entire cell architecture and generates
forces by contractile myosin. The concept
of tensegrity predicts a hard wired cytoskeleton which generates an internal prestress (19). Thus, external mechanical loads
are imposed on a pres-stressed structure.
Cells are able to sense mechanical forces by
components of the cell membrane. Patchclamp techniques have demonstrated the
presence of at least three classes of mechanosensitive ion channels (10). Cyclic
strain can modulate the activity of certain
channels. Mechanical stimulation of the
stretch activated potassium channel by
antibody coated magnetic particles enhanced the expression of osteogenic
marker proteins in mesenchymal stem cells
(24). Lipid rafts represent an organizational structure that supports the compartimentalization of signals in the membrane. These structures have been shown to
be critical for mechanical signal transduction because their disruption abolished
stretch induced activation of MAP-kinases
(36). Integrin receptors in the cell membrane play a key role in transduction of
mechanical forces (18). These heterodimeric receptors consist of a β subunit
which in combination with one of the different α subunits binds specifically to
extracellular matrix proteins, like collagens
or fibronectin. Integrins physically connect
the internal actin cytoskeleton with the
extracellular matrix outside the cell and can
function as mechanotranducers. Application of forces directly to integrins on the
apical cell surface using antibody coated
magnetic particles induces a signal transduction (33). The forces provoked an intracellular calcium response within minutes.
The calcium signal originated in the vicinity of receptors, which were stressed by
beads. This indicates that the cell is able to
sense a mechanical load locally. The calcium wave which spread over the cell and
entered the nucleus was necessary for
further down stream signalling as for
example activation of MAP-kinases (39).
Experiments with triton-100 extraction
demonstrated that mechanical load on
β1-integrin induced a physical link of signalling proteins, like activated focal adhesion kinase to the actin cytoskeleton
(33). Thus, mechanical stimulation induces a physical association between integrins and the cytoskeleton, as well as a close
association of different signalling protein
to facilitate biochemical reactions. Imaging
of these signalling complexes by fluorescence revealed a strong accumulation of
cytoskeletally associated proteins located
near the mechanically stressed integrins
below the apical cell surface (37). Notably,
these protein accumulations were also visible, although weaker, near the basal site of
the cell, which means at a distance of 4 μm
from the stressed receptors. Thus, transduction of mechanical signals was possible
over distances of several micrometers.
A key question is how mechanical signals can be converted to biochemical signals which in turn give rise to a biological
response of the cell. It was demonstrated
that a number of proteins can be physically
stretched and alter their functional states
(44). As we have found, upon physical stress
to integrins the focal adhesion proteins
talin and vinculin are accumulated (37).
Experiments now demonstrated that application of physicals forces causes stretching
of a single talin molecule that exposed
cryptic binding sites for vinculin (11). Detailed analyses have shown that forces applied by cells not only stretch but also unfold fibrillar fibronectin (4). Fibronectin
has many recognition sites and mechanical
force might switch them off and on which
allow a broad range of forces to be translated into biochemical changes. Mechanical forces can also be regulated by the
mechanical properties of the fibronectin fibrils. For example, old fibres differ in their
mechanical properties from new ones and
become increasingly more unfolded with
age (2).
Beside a switch between mechanical signals to biochemical signals in signalling
complexes near the cell membrane, forces
can also induce a direct mechanical transduction from the extracellular matrix to
the nucleus which is mediate by integrins
and the cytoskeletal elements (46). On the
outer nuclear membrane the proteins nesprin 1 and nesprin 2 can bind to actin filaments and further connect to lamin on the
nuclear scaffold. This connectivity can
transduce mechanical signals to the nucleus within milliseconds and induce gene
expression. In the nucleus chromatin remodelling was shown to play a role in gene
expression induced by applied tensional
forces (21).
The identification of principal mechanisms of mechanically induced signal
transduction implies the question, how
these mechanisms can be modified to induce a broad diversity of biological responses. The control of cell shape is one factor to determine the direction of cell differentiation. Using micropatterned islands to
control cell spreading, mesenchymal stem
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247
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J. Rychly et al.: Mechanobiology of bone cells
cells differentiated to adipocytes with a
round shape and flat stem cells differentiated
to osteoblasts (30). The osteo-adipogenic
switch due to changes in spreading required
the generation of tension through RhoA-dependent acto-myosin contractibility. Another factor is the mechanical property of
the cell itself. Local mechanical stress to
mouse stem cells induced spreading but not
in differentiated cells (9). The reason was
that the rigidity of the cells was different and
that a threshold of cell deformation is
required to trigger cell spreading. In human
mesenchymal stem cells the Young’s modulus for the rigidity was 3.2 kPa and became
1.7 in fully differentiated osteoblasts (43).
Similarly, the membrane elasticity changed
during differentiation. These differences in
cell elasticity result primarily from differential actin cytoskeleton organization.
Mechanical control by
material properties
The design of implant materials which are
used in the clinical practice offers a great
opportunity to control the mechanical
stimulation of cells in the tissues. These
materials, which range from polymers to
metals provide an artificial matrix which
regulates the properties of the extracellular
matrix proteins which in consequence determines the cell function. In particular, designing materials to direct the fate of stem
cells is of primary interest in regenerative
medicine and tissue engineering. Materials
can be fabricated into structures that exhibit a wide range of mechanical properties. The mechanical properties of materials dictate the direction of differentiation of
mesenchymal stem cells. Cells cultured on
hydrogels with different mechanical properties revealed that mesenchymal stem cells
on a soft material differentiated to neurons,
on hydrogels with an intermediate elasticity towards muscle cells and on a rigid substrate to osteoblasts (13). Importantly, the
differentiation of stem cells correlated with
the matrix elasticity in vivo, i. e. soft neuronal tissue and rigid bone. The generation
of materials with a mechanical gradient
may be used to stimulate migration of stem
cells to a target tissue for tissue regeneration, a process which is called durotaxis
(12). The stiffness of the matrix can also be
used to control the proliferation of stem
cells. Mesenchymal stem cells were kept
quiescent on a soft material and re-entered
the cell cycle on a stiffer substrate (47).
As mentioned above the fabrication of
adhesive islands on different materials which
control the cell shape can be applied to modify mesenchymal cell differentiation. In a elegant study the rigidity of the substrate was
coupled with the geometry of the substrate
(14). Arrays were fabricated which presented
elastomeric microposts in different geometries. The stiffness of the posts was controlled by different post heights. These arrays
impacted morphology, cytoskeletal contractility and stem cell differentiation. Surface
patterning of bioactive materials at the nanoscale level has proven to be a useful strategy to mimick physical cues for cell adhesion
and fate (7). This strategy makes use of the
observation that cell signalling events initiated through interaction with the extracellular matrix are initiated through clustering of
adhesion receptors. As shown, not the total
number of adhesion sites on the substrate
but rather the spatial confinement determines cell adhesion and spreading, whereas a
minimal distance of 58 nm between adhesion sites is required for cell adhesion (3).
The impact of these nanotechniques to control stem cell fate has to be further elucidated.
To mimick the environment of a stem cell
niche for regulating stem cells, progress is
being made in technologies to study cell
regulation in a 3D-environment (29). Application of hydrogel engineering suggests that
the construction of complex microenvironments in three dimensions will be possible
and will allow the control over the dynamic
processes. Mesenchymal stem cells were
shown to respond to locally induced network
changes in stiffness and cell adhesion properties in a cross-linked gel (29).
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