The emergence of ECM mechanics and cytoskeletal tension
as important regulators of cell function
Shelly R. Peyton Æ Æ Cyrus M. Ghajar Æ Æ
Chirag B. Khatiwala Æ Æ Andrew J. Putnam
Received: 10 July 2006/Accepted: 26 October 2006/Published online: 12 May 2007
? Humana Press Inc. 2007
cells from mammalian tissues represented a critical
advance in biomedical research, enabling individual
cells to be cultured and studied in molecular detail.
However, in these traditional cultures, cells are grown
on rigid glass or polystyrene substrates, the mechanical
properties of which often do not match those of the
in vivo tissue from which the cells were originally
derived. This mechanical mismatch likely contributes
to abrupt changes in cellular phenotype. In fact, it has
been proposed that mechanical changes in the cellular
microenvironment may alone be responsible for driv-
ing specific cellular behaviors. Recent multidisciplinary
efforts from basic scientists and engineers have begun
to address this hypothesis more explicitly by probing
the effects of ECM mechanics on cell and tissue
function. Understanding the consequences of such
mechanical changes is physiologically relevant in the
context of a number of tissues in which altered
mechanics may either correlate with or play an
important role in the onset of pathology. Examples
include changes in the compliance of blood vessels
associated with atherosclerosis and intimal hyperplasia,
as well as changes in the mechanical properties of
developing tumors. Compelling evidence from 2-D
The ability to harvest and maintain viable
in vitro model systems has shown that substrate
mechanical properties induce changes in cell shape,
migration, proliferation, and differentiation, but it
remains to be seen whether or not these same effects
translate to 3-D systems or in vivo. Furthermore, the
which cells respond to changes in ECM mechanics
remain unclear. Here, we provide some historical
context for this emerging area of research, and discuss
recent evidence that regulation of cytoskeletal tension
by changes in ECM mechanics (either directly or
indirectly) may provide a critical switch that controls
Mechanotransduction ? Cytoskeleton ? RhoA
Extracellular matrix ?
In the late 19th century, Julius Wolff first proposed the
idea that bone is deposited and resorbed in response to
mechanical stress . This fundamental premise
motivated numerous mathematical incarnations of
Wolff’s Law, and has had a long history of importance
in orthopedics. Unknowingly to him, Wolff would also
have a huge impact on the field of tissue engineering
into the 21st century, as witnessed by extensive
research focused on the development of bioreactors
capable of applying mechanical forces to engineered
tissues, particularly those designed for musculoskeletal
emerging experimental evidence also suggests that the
intrinsic mechanical properties of the cellular micro-
environment can influence cell function both in vitro
S. R. Peyton ? C. B. Khatiwala ? A. J. Putnam
Department of Chemical Engineering and Materials
Science, The Henry Samueli School of Engineering,
University of California, Irvine, CA 92697-2715, USA
C. M. Ghajar ? A. J. Putnam (&)
Department of Biomedical Engineering, The Henry
Samueli School of Engineering, University of California,
3107 Natural Sciences II, Irvine, CA 92697-2715, USA
Cell Biochem Biophys (2007) 47:300–320
and perhaps in vivo, suggesting that Wolff’s Law may
be extended more generally to the development of
tissues throughout the body.
A better understanding of the role of mechanics,
and the molecular mechanisms by which cells sense
mechanical cues, is critical to translating tissue engi-
neering from an empirical science to a rigorous engi-
neering discipline, potentially enabling the elucidation
of predictive design criteria for synthetic biomaterials
designed to mimic the extracellular matrix (ECM).
Clinically, tissue engineering is motivated by the high
cost of treating patients with organ or tissue failure,
which accounts for approximately 50% of the total
annual health care costs in the US . Surgical
therapies for the treatment of these problems include
organ/tissue transplantation or the use of completely
synthetic devices (heart valves, ex vivo dialysis, etc.),
but the limited availability of transplantable tissues and
the inability of completely synthetic prostheses to
adapt, remodel, and fully restore function limit these
approaches. The field of tissue engineering holds
enormous potential to revolutionize the field of medi-
cine in the long run by providing replacement tissues
for the human body, but may have an even greater
impact in the short-term by providing relevant tissue-
specific model systems in which to study cell physiology
and/or pathology. The interest in tissue engineering of
late is staggering, with global publications rising from
about 200 in 1999 to more than a 1,000 in 2003
(European Molecular Biology Organization Reports,
2004). Despite significant research progress in the
realm of tissue engineering over the past two decades,
functional engineered tissues of any substantial size
have not been realized.
Translating advances in tissue engineering from
bench to bedside requires overcoming many hurdles
(For review, see ). One hurdle relates to controlling
cell, and thus tissue, behavior to create functional
engineered tissues. It is increasingly clear that a variety
of inputs must be provided with precise spatial and
temporal control in order to control cellular pheno-
type. These inputs can be in the form of soluble
chemokines (e.g., growth factors), signals from neigh-
boring cells, cues from insoluble proteins that mediate
cellular adhesion to the ECM, and physical or struc-
tural properties inherent to the ECM. With respect to
these latter ECM-derived cues, others and we have
hypothesized that the bi-directional, reciprocal inter-
actions between cells and the ECM in which they
reside constitute a critical control element. This
hypothesis is supported by extensive recent evidence
suggesting an instructional role for ECM mechanics in
driving cell behavior and tissue development.
This review will focus on this hypothesis in greater
detail, discussing some of the recent evidence high-
lighting the ECM’s mechanical role in regulating cell
adhesion, spreading, and migration. We also discuss
the relevance of ECM mechanics to tissue function
and both normal and pathological development. The
mechanisms by which intrinsic ECM mechanics influ-
ence cell and tissue behavior remain unclear, but are
likely similar to mechanotransduction mechanisms by
which cells respond to applied mechanical forces. Here
we focus on the idea that changing matrix mechanics
influences myosin-driven, actin-mediated contractility,
through either a biophysical disruption of a presump-
tive force balance between the cell and ECM or via
biochemical changes in cell signaling. Finally, given the
apparent importance of ECM mechanics on cell fate,
we summarize the natural protein and synthetic poly-
mer-based systems that are currently in use to study
ECM mechanical influences on cell behavior, and dis-
cuss the particular systems that can be translated to
address this question in 3-D.
Mechanical regulation of cell adhesion, spreading,
Among the earliest evidence indicating the importance
of mechanical interactions between cells and the
matrix came in the form of pioneering work from
Harris and colleagues, who showed that cell-generated
tractional forces induce the formation of wrinkles in
thin silastic substrates . Although provocative,
these did not make a significant impact in the literature
until 1997 when Wang and colleagues showed that the
migration of NIH 3T3 fibroblasts depends on ECM
stiffness . Using substrates with tunable mechan-
ical properties covalently functionalized with a mono-
layer of matrix protein, they demonstrated that
fibroblasts preferentially migrate from a soft surface to
a stiff surface. They created two polyacrylamide hy-
drogels of different rigidities side-by-side, creating a
sharp interface between soft and stiff. Cells that sensed
this boundary preferentially migrated onto the stiffer
The widespread appeal of Wang’s studies to the field
of mechanobiology was his use of ECM-functionalized
polyacrylamide gels, familiar to arguably every lab
conducting cell and molecular biology research for use
in protein separations, as the underlying cell adhesion
substrate. Intuitive to all of us who have performed
relative amounts of acrylamide and bisacrylamide
Cell Biochem Biophys (2007) 47:300–320 301
straightforward manner. Wang’s studies with these
substrates led to the identification of a new form of
directed cell migration, called durotaxis or mechano-
taxis, in which compliance gradients (analogous to
chemotactic or haptotactic gradients) dictate the
direction of cell movement . Subsequent studies
by other investigators using micropatterned polyacryl-
amide substrates confirmed this form of directional
migration, showing that NIH 3T3s and endothelial cells
preferentially accumulate and persist on stiff regions
after several days of culture . Further, elegant
studies using photopolymerized polyacrylamide gels to
create radial gradients in compliance have shown that
smooth muscle cells (SMCs) sense smooth, subtle
gradients in mechanical compliance . Exploiting
these findings has already led to the development of
two bioengineered devices designed to harness this
new form of directional migration [25, 202], and may
yield additional applications in wound healing and
Extensive studies of cell spreading and morphology
using polyacrylamide substrates have revealed that the
control of cell spreading and morphology by ECM
mechanics depends heavily on cell type. Endothelial
cells  and chondrocytes  have been docu-
mented to abruptly change their spread area with
increasing stiffness, whereas neutrophils do not .
On the other hand, the extent of SMC spreading
depends on both ECM mechanics and ligand density in
a biphasic fashion . This biphasic dependence was
also observed in cell migration studies (Fig. 1). Spe-
MC3T3-E1 cells achieved maximum migration speeds
in vitro on ‘‘optimally stiff’’ substrates, although the
exact values were cell-type dependent [100, 148]. This
observed biphasic dependence on ECM rigidity bears
striking resemblance to the regulation of migration
speed by cell-ECM adhesivity predicted by modeling
and verified experimentally a number of years ago
[47, 141]. Not surprisingly then, the value of the opti-
mal stiffness at which cell speed was maximized could
be shifted by varying the concentration of ECM pro-
tein covalently attached to the substrate [100, 148],
suggesting a strong interplay between ECM chemistry
and mechanics in the regulation of cell migration.
These data imply a tradeoff between substratum ad-
hesivity (the availability of cell–matrix adhesions) and
stiffness (the elasticity of the substrate) in regulating
cell speed. On substrates with high ligand availability,
cell speed reaches a maximum on softer substrates,
while on substrates with lower ligand density a maxi-
mum cell speed requires a stiffer substrate. Computa-
tional modeling of 3-D cell migration by Zaman et al.
predicted a similar orthogonality between ECM
chemistry and mechanics . In support of this
Fig. 1 Cell motility depends on ECM rigidity in a biphasic
manner. (A) In 2-D culture, SMCs reach a maximum migration
speed on substrates of intermediate stiffness. The value of this
intermediate stiffness shifts to softer substrates as ligand density
is increased. The solid curve shows migration speed on a
polyacrylamide gel with 0.8 lg/cm2fibronectin, and the dashed
curve represents a concentration of 8.0 lg/cm2. Asterisks denote
statistical significance from the maximum migration speed on
respective fibronectin concentration (Figure adapted with per-
mission from ). (B) In 3-D cultures, computational models
predict cell motility similarly depends on the balance of cell–
ECM attachment (ligand density, given as an order of magnitude
estimate in [M]) and ECM stiffness. Cells on soft substrates
require more attachment points to generate sufficient traction to
pull the cell body forward and migrate. In contrast, cells on
stiffer substrates and/or substrates that are highly adhesive
generate a large amount of internal traction, making it difficult
for cells to retract their rear edge, therefore reducing their
migration speed. (Figure adapted with permission from )
302 Cell Biochem Biophys (2007) 47:300–320
supporting a role for ECM mechanics in 3-D tumor cell
migration was also recently published by these same
investigators . Specifically, 3-D cell migration as-
says in which Matrigel density, fibronectin concentra-
tion, and integrin adhesiveness were systematically
varied revealed that the speed with which DU-145
carcinoma cells migrate depends on a delicate balanc-
ing act between cell-based tractional and adhesive
forces. In agreement with our earlier 2-D studies using
polyacrylamide substrates, these studies reported that
3-D cell migration speeds depend on Matrigel con-
centration (and in turn, ECM mechanical properties)
in a biphasic fashion. However, when cell adhesiveness
was reduced using an integrin-blocking antibody, the
maximum migration speed shifted to softer substrates,
apparently contradicting the finding on 2-D substrates
that maximal migration speeds can be achieved despite
reduced adhesiveness so long as substrate stiffness is
increased to compensate . However, as discussed
later in this review, manipulating Matrigel density
simultaneously influences adhesive ligand density,
proteolytic sensitivity, and porosity, which in turn may
account for this apparent contradiction. Clearly there
are rich opportunities for future studies to address the
mechanisms underlying these distinct differences be-
tween 2-D and 3-D migration and the correlation with
Nevertheless, despite this and other compelling
evidence from polyacrylamide substrates indicating
that ECM mechanics influence cell spreading and
motility, it has been argued that changing the cross-
linking density of polyacrylamide gels alters the surface
properties of the gels. This may influence the relative
surface hydrophilicity of gels possessing different
moduli values, which may, in turn, affect the covalent
coupling or physical presentation of full-length pro-
teins on the acrylamide surfaces. As a result, observed
differences in cells cultured on polyacrylamide gels of
different mechanical properties may be due to the
indirect changes in ligand presentation. Our own
findings suggest that changing cross-linking density
does not significantly alter the covalent coupling of
ECM proteins [100, 148], but we acknowledge that
more extensive surface characterization is needed to
rule out this possibility. However, perhaps even more
convincing is the ever-increasing number of studies
with a wide variety of cell types (from neurons to
osteoblasts), some of which exploit mechanically tun-
able systems other than polyacrylamide, all which
converge to the same idea: virtually all mammalian
cells are mechanosensitive. We refer the reader to
some excellent recent reviews for additional insights
regarding this universal conclusion [48, 68, 91].
The impact of ECM mechanics on cell phenotype
and tissue development
While there is now extensive evidence that the 2-D
spreading and motility of many different cell types are
influenced by ECM mechanics in vitro, the relevance
of these observations for cell function and tissue
development in vivo is just beginning to be elucidated.
Morphogenesis requires the coordination of commu-
nities of cells and proper cell differentiation, and the
chemical role of the ECM in regulating these is well-
documented . Predicted by the premise of Wolff’s
Law, the importance of mechanical forces on tissue
development has increasingly become recognized over
the past two decades, and it is now widely believed that
the application of external forces to tissues influences
both morphogenesis and homeostasis. This is intuitive
for cardiovascular and musculoskeletal tissues, which
experience dynamic mechanical loading in vivo. As a
result, the development of bioreactor systems capable
of applying physiologic loads has become a major focus
of tissue engineering research. Engineers have devel-
oped in vitro systems to apply mechanical strain to
engineered bone , cartilage , and skeletal
muscle , and have contributed to our under-
standing of the responses of endothelial cells to phys-
iologic shear stresses . A great deal of work has
also focused on the response of vascular SMCs and
tissues to static  and cyclic mechanical strain 
in an effort to mimic the forces experienced by these
cells that result from the pulsatile nature of blood flow.
Differentiated SMCs in normal arteries are con-
tractile, and do not respond to growth signals under
normal in vivo conditions. When removed from the
body and placed in 2-D cell culture, these cells revert
from their contractile (differentiated) phenotype to a
synthetic (proliferative) phenotype, typically seen
during vasculogenesis. This transition is influenced in
culture by ECM composition, soluble factors, and
mechanical stress [179, 180]. Using two-dimensional
cell culture systems capable of applying uniaxial or
equibiaxial strain, it has been shown that the transition
of SMCs from the synthetic to the contractile pheno-
type can be transiently induced by removal of serum
components or via mechanical strain [160, 195]. Similar
results have also been reported with 3-D engineered
smooth muscle tissues [94, 101]. This phenotypic con-
version depends on the chemical identity of the ECM,
as does the phenotypic response of engineered smooth
muscle tissues subjected in vitro to physiologic levels of
strain [101, 102, 134, 178]. In general terms, these
findings imply that mechanical forces play a homeo-
static role in maintaining the differentiated SMC
Cell Biochem Biophys (2007) 47:300–320 303
phenotype. This argument is further supported by re-
cent evidence obtained using engineered smooth
muscle tissue models, which indicate that SMCs take
on an osteoblast-like phenotype in the absence of
cyclic mechanical strain by increasing expression of
several bone-associated genes in a manner that mimics
ectopic calcification .
Thus, while it is clear that SMCs and numerous
other cell types respond to applied load, the in vitro
studies using polyacrylamide described in the previous
section also suggest that nearly all cells are responsive
to the intrinsic mechanical properties of their micro-
environment. In vivo, the static mechanical properties
of tissues vary greatly from one tissue to another. For
example, soft neurological tissues (e.g., brain) possess
an equilibrium modulus on the order of 1 kPa, whereas
hard connective tissue (e.g., bone) can have moduli
values in excess of 1 MPa . Do these different
mechanical properties actively instruct cells? The first
insights into this question came in the late 1980s, when
the differentiated phenotype of epithelial cells in vitro
was obtained in a soft, deformable 3-D matrix, but not
on a rigid glass substratum with comparable ligand
More recently, it has been suggested that cells will
adopt tissue-specific fates when cultured on or within
surfaces possessing mechanical properties that mimic
their native in vivo environment, a hypothesis that
makes intuitive teleological sense. Perhaps the best
evidence supporting this hypothesis comes from
Discher and colleagues, who first showed that multi-
nucleated skeletal muscle myotubes form striated actin
and myosin filaments indicative of their optimal dif-
ferentiation on compliant surfaces whose mechanical
properties approximate the equilibrium modulus of
native skeletal muscle (~12 kPa) . Recently, more
compelling data from this same group have shown that
matrix mechanical properties can direct the fate of
bone marrow-derived mesenchymal stem cells, with
soft matrices that mimic brain supporting neurogene-
sis, stiffer matrices that mimic muscle supporting
myogenesis, and rigid matrices that approach the
stiffness of a collagenous osteoid microenvironment
conducting osteogenesis . Importantly, this latter
study also showed that reprogramming of these lin-
eages via the addition of soluble factors is possible if
the culture duration is short (~1 week). However, after
several weeks in culture, the cells commit to the line-
age specified by the mechanical properties of the sub-
Extending this theme, we recently reported that
SMCs regulate the expression and organization of
calponin and caldesmon, two contractile markers
indicative of SMC differentiation, as a function of
substrate mechanics , and that osteoblastic pro-
genitor cells deposit mineralized matrix to an increas-
ing degree as ECM rigidity is increased (Fig. 2) .
In general, tissue morphogenesis in vitro appears to be
regulated by ECM compliance in the same fashion,
with evidence that fibroblasts, kidney cells, and heart
explants all form tissue-like structures on soft sub-
strates but tend to migrate away from each other and
spread out into non-tissue like structures on stiffer
surfaces, just as they do on polystyrene tissue culture
The ECM’s influence on capillary morphoegenesis
is another area in which we and other investigators are
interested . Whether forming through the differ-
entiation of precursorcells
sprouting from an existing vascular network (angio-
genesis), the processes by which endothelial cells (ECs)
assemble into functional tubes within a 3-D environ-
ment have implications in developmental processes
and numerous pathologies . While great progress
has been made in identifying the key chemical medi-
ators governing this phenomenon , Ingber and
Folkman were the first to propose a critical role for
ECM mechanics on EC differentiation into tubular
Fig. 2 Mineral deposition is regulated by polyacrylamide stiff-
ness in pre-osteoblastic cells. These data show that MC3T3-E1
cells, a mouse pre-osteoblastic cell line, deposit increasing
amounts of hydroxyapatite-based mineral as substrate stiffness
is increased (left-to-right). Mineral deposits were detected using
Von Kossa staining and appear as focal black spots. (Scale bar,
100 lm.) (Figure adapted with permission from )
304 Cell Biochem Biophys (2007) 47:300–320
networks [88, 92]. This work built on earlier 2-D
studies demonstrating that while rigid substrates
resisted cell-generated forces and encouraged cell
spreading, more malleable substrates facilitated cell-
rounding . Ingber and Folkman made the correla-
tion to EC differentiation based on matrix density; by
culturing capillary ECs on a rigid substrate, they were
able to induce rapid tube formation by coating the cell
culture plates with intermediate concentrations of ei-
ther fibronectin or collagen type IV. High protein
density led to cell spreading while low ECM density
resulted in less attachment (and hence apoptosis) and
decreased cell–cell contact, which is necessary for tube
formation to occur . The role of substrate
mechanics has also been more explicitly investigated
by culturing human umbilical vein endothelial cells
(HUVEC) on fibrin hydrogels of varying physico-
chemical properties. These studies showed that HU-
VECs only form tube-like structures on fibrin gels of
very low density (0.5 mg/ml), while bovine retinal
endothelial cells required a far more dense substrate to
form capillary-like structures (8.0 mg/ml) . These
results suggest that the native microenvironment of a
particular EC is a determinant in its behavior. Similar
studies using ECM-modified polyacrylamide gels,
which allow the separation of ECM ligand density and
mechanics, demonstrated that keeping the adhesive
ligand density constant while increasing the stiffness of
the substrate was sufficient to drive HUVECs away
from a tube-like phenotype and toward a proliferative
Studies that more appropriately mimic the 3-D
microenvironment experienced by cells in vivo have
just begun to explore the effect of matrix mechanical
properties on capillary morphogenesis. Nehls and
Herrmann created a system in which microcarrier
beads coated with ECs are embedded within fibrin gels.
By varying the physicochemical properties of the
matrix via pH changes, they demonstrated that rela-
tively rigid gels supported uncoordinated EC migration
while the malleable gels, polymerized at higher pH,
supported formation of microvessels . Subsequent
studies have supported the notion that increasing fibrin
gel stiffness, either by increasing cross-linking density
via Factor XIII  or by increasing fibrinogen con-
centration  results in reduced capillary network
formation, respectively (Fig. 3). Others have used
collagen to determine whether floating gels are better
able to support capillary morphogenesis than con-
strained gels. While mechanically constrained collagen
gels led to a slightly lower length of the capillary-like
structures that formed, the constrained gels demon-
strated significantly higher average lumen area, which
was attributed to the inability of ECs to remodel and
contract these matrices vs. their floating counterparts
. Collagen hydrogels have also been used to
demonstrate that neighboring colonies of ECs produce
tractional stresses that remodel the fibrillar network
and cause directional sprouting of neighboring colonies
toward each other . Similar studies using floating
vs. attached 3-D gels have shown that fibroblast
proliferation depends on gel contraction in part due to
changes in the activity of the PDGF receptor, sug-
gesting cross-talk between substrate mechanics and
growth factor signaling .
Alteration of ECM mechanics in pathophysiology
In addition to the evidence that ECM mechanics
influence cell function and tissue development in vitro,
new correlations between mechanics and pathophysi-
ology are being identified. This is perhaps most clear in
the context of cardiovascular disease, but new insights
regarding the role of ECM mechanics in tumorigenesis
and metastasis are also emerging.
Fig. 3 Fibrin gel density regulates endothelial sprouting in 3-D.
In an in vitro model of capillary morphogenesis, endothelial cells
(ECs) seeded onto dextran microcarrier beads are cultured
within 3-D fibrin gels of increasing density (2.5, 5, and 10 mg/ml
from left-to-right). After 14 days of culture, these data show that
the extent of EC differentiation into capillary-like tubes
decreases with increasing fibrin concentration. However, because
increasing fibrin concentration simultaneously stiffens the
matrix, changes its porosity (and hence proteolytic sensitivity),
and alters the adhesive ligand density, the isolated effects of
these parameters are not easily studied in this system. (Scale bar
500 lm). (Data similar to that in .)
Cell Biochem Biophys (2007) 47:300–320305
Cardiovascular disease accounts for an estimated
$330 billion in health care costs each year, afflicts
61.8 million Americans, and will account for nearly
1.5 million deaths in the US this year alone (Source:
AHA Annual Report). In a variety of cardiovascular
pathologies, such as hypertrophy, hypertension, and
atherosclerosis, the mechanical properties of blood
vessels and small arteries may be dramatically altered.
However, it remains to be seen whether or not these
mechanical alterations are solely a downstream con-
sequence of earlier causal events, or whether or not
they can actively contribute to the onset of pathology.
Some evidence in support of this latter possibility
comes from the observation that hypertensive condi-
tions, which effectively increase the ‘‘zero stress state’’
(the residual intrinsic stress in the absence of blood
pressure), induce the phenotypic transition in SMCs,
characterized by a decrease in the expression of
smooth muscle myosin heavy chain isoforms, a-actin,
h-caldesmon, and calponin [2, 4, 116]. Hypertensive
tissues respond, in part, by remodeling their ECM in
response to the altered mechanical load, as evidenced
by changes in elastin deposition .
In this context, we hypothesize that normal ECM
compliance is critical in the maintenance of healthy
vasculature, and that small changes in the local
mechanical environment of an otherwise healthy artery
provide a set of signals instructing SMCs to switch from
their contractile phenotype to a synthetic phenotype.
These SMCs then act more like fibroblasts, adopting a
proliferativephenotypethatis also capableof
migrating from the media to the intima, and which
correlates with upregulation of fibronectin and Type I
collagen expression while downregulating the produc-
tion of elastin (Fig. 4). All of these events (prolifera-
tion, migration, and misregulated ECM remodeling)
may then contribute to intimal hyperplasia, perhaps
triggered by earlier molecular events.
Tumorigenesis and metastasis
It has been proposed that cancer cells undergo a series
of six physiological changes as they progress toward an
through autocrine signaling, insensitivity to growth
inhibitory signals, disregard of apoptosis cues, unin-
hibited replication, sustained angiogenesis, and, lastly,
enhanced invasiveness and ability to metastasize .
Accompanying these changes are alterations to the
ECM that essentially ‘‘dedifferentiate’’ the surround-
ing tissue. While the great majority of cancer research
has focused on the aforementioned hallmarks and the
differences in signaling and gene expression within
cancer cells that cause these changes, the insoluble
cues conferred by the altered ECM to the tumor and
other cells within the tumor stroma are also critical.
The fact that tumor microenvironments possess an
often radically altered ECM is an underappreciated
concept from the perspective of basic scientists. How-
ever, this concept is likely much more intuitive to cli-
nicians who routinely detect tumors through palpation.
identify tumors are often based on structural alterations
Fig. 4 Changes in vessel wall mechanics may influence the onset
or severity of cardiovascular pathologies. In an atherosclerotic
vessel, the intrinsic mechanical properties of the vessel wall
(indicative of the zero stress state) are often dramatically altered
due to changes in cholesterol deposition, shear stress, and/or
changes in blood pressure. These mechanical cues may alter
SMC phenotype in diseased vessels in one or more of the
following three ways (from left-to-right), (1) by stimulating the
migration of SMCs from the medial to the intimal layer; (2) by
inducing the hyperproliferation (or hypertrophy) of SMCs within
the intimal layer; (3) by triggering alterations in ECM synthesis
and remodeling by SMCs in the intimal layer. These pathological
responses may exacerbate the formation of atherosclerotic
plaques or contribute to arterial restenosis following angioplasty.
(Figure adapted with permission from .)
306 Cell Biochem Biophys (2007) 47:300–320
in the tumor microenvironment. The concentration of
matrix components is drastically increased in the tumor
stroma , and the ratio of components are also af-
fected by tumor-induced vascular permeability causing
increased deposition of fibrin throughout the extra-
cellular space . The immediate implications are
multi-fold. First, the elastic modulus of tumor tissue is
greatly increased while matrix porosity is decreased,
reducing the interstitial mobility of higher molecular
weight molecules . Second, shrinking of the
interstitial space could create a slower out-flux of tu-
mor- and stroma-generated ligands from the microen-
vironment, resulting in increased local concentrations
. Third, localized fibrosis could trigger a response
among stromal fibroblasts to further enhance angio-
genesis and perpetuate this cycle . Still, much of the
current research has treated the ECM as a passive
bystander, focusing on how proteinase (especially
members of the matrix metalloproteinase, MMP,
family) secretion by tumor cells functions to remodel
the matrix. However, given the lack of clinical success
experienced by MMP-targeting drugs in halting late-
stage cancer invasion and metastasis , it is clear that
other avenues must be explored.
Bissell and colleagues were among the first to rec-
ognize the instructive importance of 3-D ECM as a
regulator of tumor formation and growth. They dem-
onstrated that culturing breast epithelial cells in Ma-
trigel resulted in growth arrest and differentiation into
spherical colonies, with protein expression patterns
indicative of polarized structures. In contrast, culturing
carcinomas in these gels resulted in reverse-polarized
or unpolarized structures that would not assume a
quiescient state . This system was later exploited
expression in the normal and malignant cell types, a
difference that led to the significant finding that b1/b4
surface expression ratio essentially modulated pheno-
type; inhibiting intergrin b1in malignant cultures re-
verted these cells to a nearly normal phenotype, while
inhibiting b4in normal cells disrupted their morpho-
genesis . Integrin blocking in malignant cells was
also found to reduce overexpression of EGF receptor
levels, indicating a coupling of these signaling path-
ways that was not previously observed in 2-D .
While these studies explored how cell–matrix
interactions affected the differentiation of normal and
malignant breast epithelial cells, the explicit role
played by matrix mechanics in these phenomena has
only recently been addressed by two seminal papers.
First, Wozniak et al. examined how breast epithelial
cell differentiation is mediated by the physical prop-
erties of 3-D collagen gels . Specifically, while
breast epithelial cells would differentiate into polarized
duct-like structures in floating collagen gels, they
would not form these structures in constrained gels,
instead assuming a proliferative phenotype. Increasing
collagen concentration over a narrow range in the
floating gels also inhibited tubulogenesis. Second,
Paszek et al. systematically measured the stiffness of
malignant breast tissue in transgenic mice and com-
pared it to normal tissue. They found that normal
breast tissue was extremely soft, whereas the micro-
environment surrounding tumor and the tumor itself
was significantly stiffer . Whether matrix stiffening
actively causes cancer is uncertain, as is the precise
mechanism by which cancer cells sense stiffening
within the tumor stroma. However, as the circum-
stantial evidence continues to mount, the notion of the
ECM as a passive bystander in tumor pathogenesis is
fading. The result may possibly be a new class of cancer
drugs focused on disrupting the mechanosensing
portion of cell circuitry.
Mechanisms underlying cell responses
to mechanical stress
mechanics can control cell fate, the underlying mech-
anisms by which this occurs remain under debate. As a
result, the study of mechanotransduction, the process
by which cells and tissues sense and respond to
mechanical changes in their environment, has attracted
a great deal of attention in the past decade. Ultimately,
mechanotransduction involves a cascade of events that
eventually converts a mechanical signal into an inte-
grated cellular response [30, 88, 168]. It is generally
well-accepted that a preferred pathway for mechano-
transduction involves integrins, a class of heterodi-
meric cell surface receptors responsible for the
adhesive interactions between cells and the ECM.
Integrins form a direct physical linkage between the
ECM and the cytoskeleton, and also activate bio-
chemical signaling networks by nucleating signaling
proteins on the cytoplasmic side of the plasma mem-
brane [70, 86, 165]. Given this integrated biochemical
and biomechanical role for integrins, two dominant
paradigms have emerged to explain the responses of
cells to changes in the mechanical microenvironment.
The first paradigm is based on the fact that integrins
mediate a bi-directional, reciprocal, and dynamic force
balance between cells and the ECM. The second
paradigm suggests that changes in the mechanics of the
ECM trigger a cascade of soluble signaling events
initiated from the adhesion sites.
Cell Biochem Biophys (2007) 47:300–320307
The force balance paradigm: outside-in
On one hand, the force balance paradigm implies that
externally applied mechanical forces can be directly
transduced from the ECM to the underlying cytoskel-
eton, imparting changes in cytoskeletal assembly and
organization . This idea, pioneered by Ingber and
colleagues, is based on models of tensegrity architec-
ture first put forth by Fuller [63, 87, 186]. Applied to
cells, the concept of tensegrity suggests the cytoskele-
ton exists in a state of pre-stress that provides a
homeostatic balance of forces between the cell and its
surroundings [87, 89, 90]. (The implications of pre-
stress are discussed in more depth later in this section.)
Externally applied mechanical forces directly trans-
duced to the underlying cytoskeleton via integrins
would be super-imposed on this pre-existing homeo-
static balance, and thereby change the assembly and
organization of cytoskeletal elements to compensate
. These changes in filament assembly may then
directly or indirectly alter cellular gene expression by
influencing a variety of signaling pathways [30, 85].
Consistent with this paradigm, changes in microtubule
assembly occur in SMCs subjected to step-changes in
applied strain on the time-scale of minutes [153, 155].
In response to the much faster dynamic forces gener-
ated by actin–myosin contraction in beating cardio-
myocytes, microtubules undergo periodic buckling and
unbuckling consistent with the idea that they are under
compression . There is also evidence that applied
strain changes the size of focal adhesions  as well
their dynamic growth [11, 133], which may ultimately
control cell function . Results from these and other
studies have led to the development of predictive
models regarding adhesion formation  and cell
strengthening and organization  in response to
Often over-looked in critiques of the tensegrity
concept is the active role of the ECM in the pre-
existing force balance. This issue was recently ad-
dressed in an elegant study that measured the elastic
recoil of actin stress fibers upon laser incision .
Inhibiting myosin-generated tension in the cytoskele-
ton slowed the recoil of the fibers. Furthermore, on
stiff substrates, disruption of multiple stress fibers did
not result in appreciable changes in cell shape. How-
ever, on soft substrates, disruption of single fibers in-
duced significant changes in cell shape and produced
ECM retraction many microns away form the original
laser incision. These data show that a rigid ECM is stiff
enough to bear the increased load that results from
severing the stress fibers, whereas a compliant ECM is
not. Importantly, these studies strengthen the idea that
the cytoskeleton–integrin–ECM linkage provides a
physical continuum for the reciprocal transmission of
How might the force balance paradigm explain the
data described earlier showing that cell motility is
regulated by ECM mechanics in a biphasic fashion?
Force balance arguments imply that cells tune the
magnitude of tractional stresses generated by myosin
motors moving on actin filaments based, in part, on the
rigidity of the ECM. Cells adhering to soft substrates
would be unable to generate sufficient tractional forces
to propel the cell forward. On the other hand, cells
adhering to rigid substrates would generate so much
tractional stress that they would be incapable of
movement, locked in a state of cellular ‘‘rigor mortis’’
characterized by a high degree of actin filaments and
large, robust focal adhesions, both of which would need
to be remodeled to yield locomotion. On substrates of
intermediate stiffness, the state of actin-mediated
traction is optimized in such a way that motility itself is
optimized. In support of this model, it has been shown
that agents that disrupt actin contractility retard cell
motility on substrates of intermediate stiffness .
Several prior studies regarding substrate compliance
in 2-D have noted the large, well-defined focal adhe-
sion structures in many different cell types anchored to
stiff substrates, in contrast to the small, ill-defined
adhesions in cells on softer substrates [53, 100, 144, 145,
148] (Fig. 5). The amount of force generated by these
adhesive contacts has been directly measured using
traction force microscopy, a powerful technique in
which the displacement of fluorescent microbeads
embedded within polyacyrlamide substrates is used in
conjunction with a computational algorithm to derive
the maximum likelihood estimate of traction stresses
. Therefore, this technique allows determination
of traction stress maps in cells cultured on compliant
substrates. However, measurements of the amount of
force generated by different types of adhesion struc-
tures have been contradictory. There is some evidence
that tractional forces generated by small, nascent cell
adhesions in lamellipodia are in fact greater than those
generated by larger, more mature adhesions [14, 129].
However, other studies have shown that smaller focal
complexes formed at the leading edge of a motile cell
exert smaller forces than focal adhesions .
Nevertheless, it is clear that the force generating
capacity of the cell is highly dependent on the
mechanical properties of the ECM, and that tension in
the actin cytoskeleton provides an important feedback
signal governing cell motility. Within this theoretical
framework, studies measuring cell motility as a
308 Cell Biochem Biophys (2007) 47:300–320
function of substrate adhesiveness or ECM mechanics
can now be unified under the umbrella of actin con-
tractility, a parameter that can be tuned in one of three
ways to optimize motility: (1) by manipulating the
strength of each integrin–ECM interaction; (2) by
changing the total number of interactions; and (3) by
selecting a substrate with an optimal stiffness capable
of maximally supporting migration speed.
The force balance paradigm: inside-out
On the other hand, the reciprocal dynamic nature of
the proposed force balance also implies that cell-gen-
erated forces can be transmitted across integrins to the
supporting ECM. A striking example comes from the
aforementioned work of Harris and colleagues who
showed that cells cultured on thin silicon rubber sub-
strates visibly wrinkle the substrate as a result of cell-
based tractional forces . The physical cytoskeleton–
integrin–ECM continuum, and the ECM’s ability to
resist cell-based tractional forces in particular, also
appear to be critical factors in controlling cell shape
which, for a number of cell types, is essential to regu-
late the balance between cell growth, differentiation,
and death [29, 45, 59, 92, 127]. Cell-based forces are
also involved in ECM remodeling, which can further
modify the mechanical context in which cells reside
[30, 31, 33, 65, 79]. In smooth muscle, bone, and other
connective tissues, cells remodel their ECM in re-
sponse to altered mechanical loading [31–33, 98].
Furthermore, the extent to which fibronectin can be
bundled into fibrils is actin-mediated and depends on
ECM stiffness . Pharmacologic agents that disrupt
actin-based contractility disrupt the proper assembly of
fibronectin , lending further support to the idea
that alterations in the dynamic balance of forces
between the cytoskeleton and the ECM are important
during normal and pathologic tissue development
Extending the force balance paradigm to 3-D
matrices in order to predictably control cell fate in
environments that more closely mimic those found
in vivo remains a sizable challenge. Actin polymeri-
zation and focal adhesion formation are entirely dif-
ferent in 3-D compared to 2-D , and assessing the
degree of actin contractility is not as straightforward.
There is clearly a need for the 3-D equivalent of
traction force microscopy. Macroscopic methods to
measure cell-based tractional forces in 3-D collagen
gels have shown the deformation of the surrounding
matrix depends on the initial concentration of the
collagen . In agreement with the force balance
predictions, this suggests that the amount of tractional
stress generated by the cells depend on the contractile
resistance of the ECM. However, more direct mea-
surements of contractile force per fibroblast showed no
dependence on the stiffness of collagen-GAG con-
structs in which they were cultured, even though the
contractile displacement of the matrices still increased
with matrix compliance . In our opinion, these
findings are not contradictory to simple force-balance
models, but instead reflect the cell’s attempt to achieve
‘‘tensional homeostasis’’ by maintaining a constant
Fig. 5 Changes in ECM mechanics influence actin cytoskeletal
assembly. The ability of cells to form actin stress fibers and focal
adhesions, indicative of an overall increase in contractility,
increases as ECM rigidity is increased from left-to-right. In these
data, MC3T3-E1 pre-osteoblastic cells cultured on stiffer
substrates show an increase in F-actin (top row) and focal
adhesions (denoted by vinculin staining, bottom row). Images
are 100· with 10 lg/ml collagen functionalized to surface of
polyacrylamide gels. (Data similar to that in .)
Cell Biochem Biophys (2007) 47:300–320309
level of force consistent with the state of cellular pre-
stress. To maintain tensional homeostasis in a compli-
ant matrix, more of the resistance from the actin-
mediated contractility must be provided by the
microtubules or other compression-resistant materials.
Maintaining a constant force in a stiffer 3-D matrix
could be achieved with reduced load on the microtu-
The force balance paradigm: cytoskeletal pre-stress
The evidence summarized thus far depicts a strong link
between substrate stiffness and cell-generated con-
tractility. As briefly mentioned earlier, the idea that a
cell exists in a pre-existing force balance between
the cytoskeleton and the ECM is consistent with the
tensegrity-based hypothesis. This implies that the
cytoskeleton maintains a certain degree of prestress,
which acts as a determinant of its initial stiffness and
assures that the cell will respond immediately when
mechanically perturbed (For review, see ). The
magnitude of this cytoskeletal pre-stress governs the
amount of traction a cell can apply to the ECM ,
as well as the relative stiffness of the cell itself  as
revealed by elegant experimental measurements using
magnetic twisting cytometry . Altering this pre-
stress by modulating actomyosin contractility pharma-
cologically has also been shown to alter both cell
mechanical properties  and cell traction ,
reinforcing the idea that a discrete structural connec-
tion between the cytoskeleton and ECM provides a
preferred path for the transmission of mechanical
information (both outside-in and inside-out). More-
over, both the microtubule and intermediate filaments
seem to have a role in determining cell pre-stress, as
disruption of the microtubule network increases cell
traction significantly , and intermediate filaments
regulate the cellular response to twisting and shear (for
review, see ). Collectively, all of these data are
predicted by mathematical models of tensegrity [174,
175]. These models also predict that mechanical forces
can be propagated over long distances via cytoskeletal
pre-stress , even to the nucleus, a concept for
which there is now convincing experimental evidence
[83, 121]. Furthermore, the distance at which these
cytoskeletal forces can be propagated within a cyto-
plasm is tightly regulated by cell pre-stress, shown via
the application of contractile and relaxing agents .
Other experimental evidence has shown that twist-
ing integrin receptors using RGD-coated magnetic
beads induces endothelin-1 gene expression in endo-
thelial cells, and that this response can be blocked by
disrupting tension in the actin network . For a more
thorough treatment of the proposed mechanisms by
which changes in cytoskeletal pre-stress may dictate
cell function, either directly or indirectly, we refer the
reader to an excellent set of companion articles pub-
lished in 2003 [89, 90].
The signaling paradigm
Besides the direct physical connection, there is also
compelling evidence that applied mechanical forces
trigger changes in cellular biochemistry likely derived
from the cluster of signaling molecules at focal adhe-
sion sites [28, 168] or from ion fluxes via mechano-
sensitive ion channels [12, 49, 181]. In the case of focal
adhesion signaling, focal adhesion kinase (FAK) has
been shown to be mechanosensitive in numerous
studies [100, 114, 200]. Its activation is believed to
initiate by changes in integrin clustering and the sub-
sequent recruitment of structural proteins (such as
tensin, a-actinin, talin, and vinculin), which together
form an immature focal complex. The maturation of
these complexes to more well-defined and fully devel-
oped focal adhesion structures is dependent on the
cell’s ability to apply force to the nascent adhesions
[11, 66]. This is turn coincides with a cascade of
intracellular signals from FAK to numerous targets,
including integrin-linked kinase (ILK), Src, PI3-K,
PKC, PTP-PEST, p130Cas, and paxillin, among others
(For review, see ). These FAK-initiated signals
trigger a diverse array of cellular responses (such as
membrane protrusion, adhesion, migration, cell cycle
progression, and even differentiation) by linking to
other downstream effectors, including the Rho-family
GTPases and the Ras-MEK-ERK pathway (For
reviews, see [194, 200]).
On compliant polyacrylamide substrates, the me-
chanotactic migration of fibroblasts depends on FAK
activation, as FAK-null cells showed a reduction in cell
speed and directional persistence . In pre-osteo-
blastic MC3T3-E1 cells, FAK phosphorylation also
increases as ECM rigidity increases, coinciding with an
increase in mineral deposition . Due to FAK’s
association with integrins, it may be especially critical
in transducing mechanical cues to other downstream
signaling molecules. One such candidate molecule is
Src, a known target of FAK. A seminal study using
optical tweezers showed Src’s involvement in regulat-
ing adhesion strength in response to applied forces [35,
58]. A more recent study using optical tweezers and a
FRET-based reporter showed Src is activated locally in
response to an applied force, and that waves of Src
activity can propagate over long distances inside the
cell . Other studies have shown that applying
310 Cell Biochem Biophys (2007) 47:300–320
mechanical loads to smooth muscle tissue increases
ERK 1/2 and FAK phosphorylation alongside Src
activation . Stretching of smooth muscle also in-
creases influx of calcium and sodium, which contributes
to these signaling responses [116, 137]. Phosphoryla-
tion of PDGF  and ERK  have also been
shown to be required for fibroblast-mediated contrac-
tion of 3-D collagen gels. The addition of TGF-b to
these matrices increased contraction, suggesting that
soluble biochemical signals can synergize with bio-
physical changes in ECM to yield a coordinated cel-
lular response [74, 184].
Despite evidence supporting both the purely bio-
chemical signaling and force-balance models, it is more
likely that these paradigms are not mutually exclusive
and that there is some form of integration or cross-talk.
How then might force-induced changes in cellular
biochemistry be linked with changes in the biophysical
properties of the cytoskeleton?
RhoA as a mechano-sensitive switch linking
cytoskeletal tension and cell function
One likely candidate that repeatedly emerges as an
integrator of cellular biochemistry and biophysics is
RhoA, a small GTPase whose activation enhances ac-
tin contractility by stimulating the formation of stress
fibers and focal adhesions . Part of the larger Ras
superfamily, the Rho-family GTPases (which include
Rac1, Cdc42, RhoA, B, C, and other isoforms) have
been widely implicated in integrin-mediated signaling
[37, 159, 166] and in the control of cell migration [57,
78, 196]. RhoA in particular plays a critical role in the
assembly of actin stress fibers in response to various
soluble stimuli, including serum, growth factors, lyso-
phosphatidic acid (LPA) [34, 36, 135], and to insoluble
adhesion ligands such as fibronectin [13, 159].
A definitive connection between cell contractility
and Rho-family GTPases has been elucidated over the
past decade. Increased contractility following depoly-
merization of microtubules using various pharmaco-
logical agents (e.g., nocodazole and colchicine) was
first shown to occur in 1989 . This increased con-
tractility was later shown to be the result of greater
myosin light chain phosphorylation  mediated via
activation of RhoA [56, 119]. RhoA’s influence on
actin contractility is mediated in part by one of its
downstream effectors (Rho-associated protein kinase,
or ROCK). ROCK in turn regulates the phosphoryla-
tion of myosin light chain, and therefore the interaction
between actin and myosin II that contributes to actin-
mediated contractility in numerous cell types .
Other studies have demonstrated that RhoA is
required for the selective stabilization of microtubules
 via a mechanism that involves another down-
stream effector, the Diaphanous-related formin, mDia
. The activity of RhoA has also been shown to be
downregulated by Src’s kinase activity, which stimu-
lates the activity of p190RhoGAP, a GTPase-activat-
ing protein that inactivates RhoA [9, 10]. Thus, RhoA
potentially provides an essential mechano-sensitive
link integrating the force-induced changes in bio-
chemical signaling with biophysical changes in the
Countless recent studies have implicated the Rho
GTPases in the cellular response to mechanical stress
and the maintenance of tensional homeostasis [3, 8, 97,
125, 136, 154] (For review, see ). There is also con-
vincing data from two studies supporting a role for
RhoA in cell fate, one of which shows that RhoA
regulates the switch between adipogenesis and myo-
genesis  and one which shows that osteogenic
commitment also relies on RhoA . The latter of
these two studies used a micropatterning approach to
confine cell shape, and found that mesenchymal stem
cells constrained to small adhesive islands adopted an
adipogenic fate, while those permitted to spread over
much larger islands were pushed toward an osteogenic
fate . The osteogenic fate could be rescued in
unspread cells by expressing constitutively active
RhoA; likewise, the osteogenic fate could be subverted
in spread cells by expressing dominant-negative RhoA.
Agents that disrupted actin-mediated contractility also
inhibited osteogenic differentiation in spread cells,
suggesting that RhoA’s effects appeared not to be
mediated by its activity per se, but rather its effects on
cytoskeletal tension. Given that manipulating ECM
mechanics similarly regulates cytoskeletal tension,
others and we are working to address the hypothesis
that tuning substrate compliance can control stem cell
fates. This concept is shown schematically in Fig. 6.
Further evidence supporting a critical role for
RhoA-mediated tension in the control of cell fate
comes from the aforementioned studies of Wozniak
et al.  and Paszek et al.  regarding tumori-
genesis. In both studies, misregulation of contractility
mediated by RhoA-ROCK signaling correlated with
changes in cell proliferation and morphology induced
by altering ECM mechanics. This is particularly inter-
esting given that some Rho GTPases are overexpres-
sed in human tumors . Another recent study
showed that the regulation of histone acetylation by
cell adhesion to the ECM is mediated by intracellular
contractility . In particular, suspended gastric
carcinoma cells showed increased levels of acetylation
Cell Biochem Biophys (2007) 47:300–320311
when held in suspension relative to the levels in cells
attached to fibronectin. Treatment of cells with tri-
chostatin A significantly increased histone acetylation
in suspended, but not fibronectin-adherent, cells.
Inhibition of either myosin light chain kinase or ROCK
decreased both the basal and trichostin A-stimulated
levels of acetylation. Excitingly, this suggests a possible
link between cytoskeletal tension and gene expression.
Finally, evidence that RhoA is required for proper
fibronectin assembly in fibroblasts , and that
pharmacologic agents that disrupt actin-based con-
tractility disrupt the proper assembly of fibronectin
, suggest that RhoA and its effects on cytoskeletal
tension provide critical closure to the feedback loop
between ECM mechanics and both normal and path-
ologic tissue development.
Addressing the role of ECM mechanics
Recent efforts to decipher the role of the ECM in
determining cell and tissue function have exploited
several natural and synthetic biomaterials to create
ECM analogs. Using such systems requires increased
multidisciplinary collaborations between engineers or
physical scientists, who often focus on synthesis and
characterization of these materials, and the biological
scientists who use them as in vitro cellular environ-
ments. In the context of tissue engineering, a healthy
debate continues on whether to use naturally derived
polymers (those directly taken from animal tissue) or
completely synthetic systems (engineered from the
ground-up) as ECM analogs . In the case of nat-
ural polymers, nearly all cell types can be immediately
and directly used on or within these materials, since
they contain native functionalities to bind integrins.
Synthetic ECM analogs, on the other hand, either
support cell adhesion indirectly (through the passive
adsorption of adhesive proteins) or must be chemically
modified to contain specific adhesive peptides or pro-
teins. Both naturally derived and synthetic materials
have been used to address the impact of ECM
mechanics on cell function.
Type-I collagen is one of the most popular naturally
derived materials used to study the effects of ECM
mechanics on cell function. Collagen contains multiple
cell-binding domains for cells, including multiple RGD
Fig. 6 Hypothetical model predicting the ability of ECM
chemistry and mechanics to direct cell fate. A cell residing in
an ECM with baseline levels of adhesivity (number and/or
availability of attachment points) and rigidity (stiffness of the
surrounding matrix) is depicted in the center of the schematic. In
response to increased adhesivity or rigidity (top), cell-generated
tractional stress is increased either by actin cables pulling with
the same amount force against a larger number of cell–ECM
adhesions, or by increasing the amount of force generated by the
actin fibers at each adhesion. This is triggered by, or correlates
with, changes in the activity of RhoA. On the other hand, when
adhesivity or rigidity is decreased, RhoA activity decreases as
does cell-generated tractional forces. We propose that these
alterations in RhoA and cytoskeletal tension induced by
chemical and mechanical cues present in the ECM may
ultimately influence cell phenotype
312 Cell Biochem Biophys (2007) 47:300–320
sites  that were originally identified in fibronectin
 and which also exist in many other adhesive
proteins [138, 156, 164]. RGD has been shown to bind
a2b1and avb3integrins in osteoblasts , but it is
unclear if this sequence is available for cell binding
in collagen-I, or instead is cryptic and only exposed
under certain conditions . Furthermore, collagen-I
(like most ECM proteins) contains numerous other
sequences that may synergize with RGD to provide
integrin-binding specificity. These sequences include
DGEA, which can engage a2b1 integrin , and
GFOGER (and similar -GER analogs), which prefer-
entially engages a1b1and a2b1integrins [103, 106, 107,
161]. Nevertheless, collagen-I will readily form gels
when heated to 37?C, making it easy to create 3-D
tissue constructs at physiological conditions. Naturally
derived fibrin gels are also attractive materials for
studies on ECM mechanics, particularly in the context
of angiogenesis . Fibrin, the major structural pro-
tein of the provisional wound-healing matrix, is derived
from the proteolytic cleavage of fibrinogen by throm-
bin. Another alternative widely used to create 3-D
cultures is Matrigel, originally derived from the stro-
mal matrix of Engelbreth-Holm-Swarm (EHS) tumors
. Since its primary constituents include laminin,
collagen IV, hepain sulfate proteoglycancs, and ent-
actin, it is well-suited as a basement membrane analog.
However, Matrigel’s properties are difficult to pre-
dictably control due to the fact that it remains a poorly
The mechanical properties of all of these native
biopolymer gels can be tuned by simply increasing the
overall concentration of the protein; however, this also
increases the ligand density, as the two variables can-
not be decoupled. As alluded to earlier, one way to
manipulate the mechanical environment using these
naturally derived biopolymers is to selectively detach,
or float, the gels and compare the results to gels of the
same composition that remain anchored to the culture
dish. Anchorage to the substrate provides a physical
restraint to cell-based tractional forces at the edges of
the construct. However, it is difficult to determine how
far this mechanical effect propagates through the
construct and whether cells in the center of the gel can
sense these changes in anchorage. In addition, although
the elastic moduli (a material property) of these
floating vs. attached constructs remains constant, their
effective stiffness may differ dramatically due to chan-
ges in anchorage. Thus it may be difficult to isolate the
effects of gel mechanics from those of the surrounding
Given the limitations of native biopolymer gels,
numerous investigators are exploring strategies to
engineer synthetic ECM analogs in which the chemical
and mechanical properties can be independently tuned.
Such systems would enable ECM mechanical proper-
ties to be systematically varied in order to study their
isolated influence on cell behavior. Polyacrylamide
substrates remain the standard for 2-D culture studies
focused on mechanobiology. Their attraction is due to
the ease with which their mechanical properties can be
manipulated, and the relatively simple chemistry used
to functionalize them with cell adhesive ligands .
However, polyacrylamide gels are not conducive to
long-term 3-D studies in vitro, nor in vivo, due to the
fact that they are not biodegradable and may retain
some neurotoxicity associated with unpolymerized
acrylamide. Similar arguments about 3-D transference
and toxicity also limit the use of polydimethyl siloxane
(PDMS), a polymer popular for micropatterning and
microfluidics [20, 29] that has also been used for 2-D
studies on substrate mechanics . Alginate (a linear
polysaccharide derived from seaweed) has also been
used for its mechanically tunable properties and its
ability to be functionalized with bioactive peptide se-
quences . The drawback to this system is that most
methods for gel formation involve the use of calcium
cross-linking. Since calcium is a ubiquitous second
messenger and (among other things) regulates smooth
muscle cell contraction by directly binding to calmod-
ulin, efforts to introduce alternative cross-linking
schemes are needed to use alginate in this fashion
As an alternative to these other systems, hydrogels
based on poly(ethylene glycol) (PEG) have more
recently been exploited for mechanobiology studies
. PEG gels have been widely used and charac-
terized as synthetic ECM analogs for tissue engineer-
ing applications [21, 22, 24, 52, 122, 123]. The
incorporation of acrylate end groups on PEG allows
cross-linking and the formation of hydrogels by UV
photo-polymerization . Numerous studies have
shown that cells can be encapsulated in 3-D within
photopolymerized PEG gels with high viability, indi-
cating that the short exposure (~1 min) to UV light
does not adversely affect the cells . Furthermore,
controlling the amount of acrylated PEG monomers
represents a practical approach to controlling the
mechanical properties of PEG hydrogels. In addition,
the same hydrophilic characteristics of PEG that allow
it to form hydrogels also account for its resistance to
protein adsorption. Therefore, PEG gels provide a
blank slate template upon which key functionalities of
native ECM can be conferred.
The covalent attachment of specific cell adhesion
peptides into PEG hydrogels allows cell–ECM inter-
Cell Biochem Biophys (2007) 47:300–320 313
actions to be controlled , and thereby permits
decoupling adhesion site density from gel mechanical
properties. PEG gels have been used to study the
influence of substrate mechanics in both 2-D and 3-D
for neurons , smooth muscle cells [122, 149], and
chondrocytes . However, because the tightly cross-
linked gels have a very small pore size (on the order of
a few nanonmeters), the inclusion of cross-links sensi-
tive to proteolysis [6, 46, 124, 157] or hydrolysis 
will be necessary to extend studies of ECM mechanics
to the 3-D environment. Inclusion of peptides sensitive
to cell-secreted proteases (MMPs and plasmin) into
these gels has already provided an ideal system in
which to study 3-D cell migration, enabling compari-
sons between proteolytically and non-enzymatically
mediated migratory mechanisms [71, 157].
The influence of ECM mechanics on cell behavior and
tissue development is robust, with evidence that the
adhesion, spreading, and migration of several cell types
are all affected by substrate compliance in 2-D and
3-D. Here we have highlighted evidence suggesting
that changes in ECM mechanics exert their influence
on cells by tuning the degree of tension in the actin
cytoskeleton, either directly by maintaining a balance
of forces or indirectly through intracellular signaling
mechanisms. In turn, this tension switch appears to be
a critical regulatory signal governing cell fate and
thereby influencing both normal (e.g., osteogenesis,
capillary morphogenesis) and pathological (e.g., car-
diovascular disease and cancer) tissue development.
Future efforts to attain a better understanding of the
molecular mechanisms by which cells sense and
respond to mechanical cues through the use of well-
defined synthetic ECM analogs will perhaps one day
enhance efforts in regenerative medicine and in the
treatment of numerous diseases.
cial support from the National Institutes of Health (NIDCR:
DE-016117) and the American Heart Association (Western
States Affiliate: 0465111Y) to A.J.P. and fellowships from the
Achievement Rewards for College Scientists (ARCS) Founda-
tion to S.R.P. and C.M.G.
The authors gratefully acknowledge finan-
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