Review

Effects of extracellular matrix viscoelasticity

on cellular behaviour

https://doi.org/10.1038/s41586-020-2612-2 Ovijit Chaudhuri1 ✉, Justin Cooper-White2 ✉, Paul A. Janmey3,4 ✉, David J. Mooney5 ✉

& Vivek B. Shenoy6 ✉
Received: 19 December 2019

Accepted: 17 June 2020

Substantial research over the past two decades has established that extracellular
Published online: 26 August 2020
matrix (ECM) elasticity, or stiffness, affects fundamental cellular processes,
Check for updates
including spreading, growth, proliferation, migration, differentiation and organoid
formation. Linearly elastic polyacrylamide hydrogels and polydimethylsiloxane
(PDMS) elastomers coated with ECM proteins are widely used to assess the role of
stiffness, and results from such experiments are often assumed to reproduce the
effect of the mechanical environment experienced by cells in vivo. However, tissues
and ECMs are not linearly elastic materials—they exhibit far more complex
mechanical behaviours, including viscoelasticity (a time-dependent response to
loading or deformation), as well as mechanical plasticity and nonlinear elasticity.
Here we review the complex mechanical behaviours of tissues and ECMs, discuss the
effect of ECM viscoelasticity on cells, and describe the potential use of viscoelastic
biomaterials in regenerative medicine. Recent work has revealed that matrix
viscoelasticity regulates these same fundamental cell processes, and can promote
behaviours that are not observed with elastic hydrogels in both two- and
three-dimensional culture microenvironments. These findings have provided
insights into cell–matrix interactions and how these interactions differentially
modulate mechano-sensitive molecular pathways in cells. Moreover, these results
suggest design guidelines for the next generation of biomaterials, with the goal of
matching tissue and ECM mechanics for in vitro tissue models and applications in
regenerative medicine.

Although indications of the effects of the mechanical properties of associated signalling, conformational changes in mechanosensitive
culture substrates on cell behaviours have long been observed, it is proteins such as talin, vinculin or lamin, activation of mechanosen-
only recently that this concept has become widely accepted by the sitive ion channels (such as piezo1), and downstream activation of
scientific community. Earlier studies demonstrating the effect of sub- transcription factor activity7–10. Although changes in ECM mechanics
strate mechanics on cell structure and proliferation were overshad- are sensed by cells over short timescales, these can affect long-term
owed by an emphasis on genetics and biochemistry in cell biology1,2. cellular processes such as differentiation, fibrosis and malignancy
Then, in the late 1990s, using polyacrylamide hydrogels of varying through continued sensing, mechanical memory and changes in the
elastic moduli coated with ECM proteins as the cell culture substrates, epigenome11–13. Reported tissue elastic moduli vary from hundreds of
Pelham and Wang showed that substrate stiffness affected cell–ECM pascals in brain and fat tissue all the way up to tens of gigapascals in
adhesion, cell spreading and migration3. Since this study, numerous bone14,15. Furthermore, alterations in tissue mechanics are observed
groups have used polyacrylamide gels, and a variety of other material in development and in various diseases and have been linked to cell
systems with tunable elastic moduli, to show that substrate stiffness phenotype in these contexts16,17. Thus, the current consensus is that
affects various other processes, including proliferation and apopto- ECM stiffness plays a key part in regulating development, homeostasis,
sis, stem cell differentiation, breast cancer progression and response regenerative processes and disease progression.
to drugs4–6. Mechanistically, the current view is that cells exert trac- Living tissues and organisms appear to be macroscopically solid
tion forces using actomyosin-based contractility when coupled to objects, but they behave very differently to what one would expect
substrates through integrin-based adhesions, or other cell–surface of a perfectly elastic, or Hookean, solid when put under pressure or
links, and they sense variations in substrate stiffness through differ- stretched. For example, although our skin and fat tissues do recover their
ing magnitudes or extents of integrin and syndecan clustering and shape after they are pinched or compressed, or after a wearable device is
1
Department of Mechanical Engineering, Stanford University, Stanford, CA, USA. 2School of Chemical Engineering and Australian Institute of Bioengineering and Nanotechnology, The
University of Queensland, Brisbane, Queensland, Australia. 3Institute for Medicine and Engineering, Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, USA.
4
Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA. 5School of Engineering and Applied Sciences and Wyss Institute, Harvard University, Cambridge, MA, USA.
6
Center for Engineering Mechanobiology and Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA, USA. ✉e-mail: [email protected];
[email protected]; [email protected]; [email protected]; [email protected]

Nature | Vol 584 | 27 August 2020 | 535

Review
removed, they take some time to do so. Tendons, when stretched slowly, of the ratio of the loss modulus to the storage modulus in viscoelastic
are able to extend and then recoil back to their original size and shape; materials typically depends on the frequency. Viscoelastic solids are
however, when they are rapidly extended, they can further strain-stiffen differentiated from viscoelastic fluids by maintaining stress or elastic
and eventually rupture18. Tissues are therefore not purely elastic materi- resistance at long times under a constant deformation, or by reaching
als, like a rubber ball or a spring, because they exhibit a time-dependent an equilibrium deformation under loading at long times. Everyday
mechanical response and dissipate a fraction of the energy it took examples of viscoelastic solids include Jello (gelatin), a ‘stress ball’ and
to deform them, a property called viscoelasticity or poroelasticity, bread dough, and Silly Putty is an example of a viscoelastic fluid. One
depending on the molecular mechanism. Macroscopically, loss of the of the softest and most dissipative viscoelastic tissues in mammals is
ability to recover shape after applied mechanical stress or stretch is the brain, which has been extensively studied at timescales and defor-
often a sign of injury, disease or ageing: for example, affected tissues mation magnitudes that span the fast (millisecond) and high-stress
may not recover their shape after a bone break, a skin tear, or the droop- (megapascals) range relevant to blasts and concussions to the slow
ing of facial skin after decades of gravitational stress19. However, even (weeks) and low-stress (tens of pascals) deformation caused by tumour
when tissues globally recover shape, local regions may not do so after growth. Depending on the timescale and deformation, brain tissue
the applied forces are removed, experiencing irreversible or plastic can dissipate at least as much energy as it stores in elastically recover-
deformations. Plastic deformation of the ECM is implicated in contrib- able deformation27, and at very long timescales it appears to flow like
uting to the conversion of an originally isotropic network of collagen a glass or liquid28. Furthermore, dissipation (and viscoelasticity) can
fibres to a more aligned pattern that is often seen around tumours20–22, distinguish not only grey matter from white matter, but also differ-
and irreversible changes at cell–cell boundaries caused by cell-derived ent regions of the brain29. Other soft tissues are also viscoelastic, with
forces at junction sites have recently been shown to be essential rheological analysis showing that soft tissues generally exhibit loss,
features of pattern formation during development in Drosophila23 or viscous, moduli that are usually around 10% to 20% of their storage,
and Caenorhabditis elegans24. Many soft tissues also exhibit nonlin- or elastic, moduli at 1 Hz (Fig. 2a). Stress relaxation tests reveal that
ear elasticity by strain-stiffening, or become increasingly difficult to soft tissues—including liver, breast, muscle, skin and adipose tissues—
extend as they are deformed, which may be advantageous in preventing substantially relax their resistance to a deformation over timescales
large deformations that damage tissue25. For example, in blood vessel from tens to hundreds of seconds30–36 (Fig. 2b). Even stiffer skeletal
walls, distensibility at low strains accommodates pulsatile blood flow tissues—including bone, tendon, ligaments and cartilage—are viscoe-
whereas increased stiffness at high strains provides elastic stability lastic, with loss moduli at about 10% of the storage moduli. Embryos
to prevent vessel rupture26. Biological tissues and ECMs thus exhibit at various stages of development37, and regenerative structures such
complex, time- and rate-dependent mechanical behaviours, including a as fracture haematomas30 or blood clots38 also exhibit viscoelasticity.
combination of viscoelasticity, poroelasticity, plasticity and nonlinear Importantly, changes in viscoelasticity have been associated with
elasticity (Box 1). disease progression. Determination of elastic moduli, the basis of pal-
Given that cells interact with ECMs through dynamic processes that pation to identify stiff tumours, is not efficient for identifying most
span a range of forces, from piconewtons up to hundreds of nanonew- types of brain tumours but instead changes in their dissipative proper-
tons for individual cells, and span a range of timescales, from millisec- ties, as revealed by magnetic resonance elastography, can identify the
onds to hours, we should expect time-dependent and strain-dependent margins of gliomas and other types of brain tumour in situ39. Further-
mechanical responses in ECMs to affect cell–matrix interactions and more, changes in brain viscoelasticity have been linked to ageing40 and
mechanotransduction (Fig. 1). Indeed, an emerging body of evidence multiple sclerosis41. Similarly, breast cancer progression is associated
has demonstrated that these more complex mechanical characteristics with changes in both stiffness and energy dissipation42. Changes in
of tissues and ECMs affect cells, sometimes in ways not anticipated from viscoelasticity are likely to be associated with other types of cancers or
our previous understanding of mechanotransduction, which was based other diseases, particularly those involving fibrosis or inflammation,
on purely elastic substrates. Here we review the complex mechanical as well as injuries, but data on these are largely missing, representing
behaviours of tissues and ECMs, discuss recent work elucidating the a critical gap in our knowledge.
effect of ECM viscoelasticity on cells, and describe the potential for the Materials that exhibit viscoplasticity represent a subset of
use of viscoelastic biomaterials in regenerative medicine. viscoelastic materials, in that they exhibit permanent deformations
when the applied stress exceeds a material ‘yield stress’ and remain
at least partially deformed when the stress is removed. The response
Tissue and ECM mechanics are complex of these materials is viscoelastic to loads or deformations below their
Viscoelasticity has been found to be a near-universal characteristic yield stress. For instance, moulding clay and toothpaste are both visco-
of living tissues and ECMs. In response to a mechanical perturbation, plastic. Reconstituted ECM materials used for cell culture—including
viscoelastic materials exhibit an instantaneous elastic response, which common formulations of type-1 collagen gels, reconstituted basement
is characteristic of purely elastic solids, followed by a time-dependent membrane matrix and fibrin gels—are typically viscoplastic22,43 unless
mechanical response and energy dissipation or loss, both of which they are sufficiently crosslinked covalently by enzymes such as Factor
characteristic are of viscous liquids. Viscoelastic materials will ‘creep’, XIIIa or lysyloxidase44,45. Tissue viscoplasticity has been characterized
or deform in a time-dependent manner, in response to the application even less than tissue viscoelasticity, representing another critical gap
of an external step stress or load, and undergo ‘stress relaxation’, or in knowledge.
reduce stress levels in a time-dependent manner, in response to a step Numerous mechanisms underlie the dissipative properties of
deformation. Furthermore, under an imposed sinusoidal deformation, tissues and ECMs, with some of these mechanisms also leading to
stress and strain are completely in-phase for a purely elastic mate- viscoplasticity. Tissues consist of cells, ECM and extracellular fluid. The
rial, because all of the input deformation energy can be ‘stored’ and ECM, composed of fibrous protein polymer networks, typically type-1
‘recovered’ during each cycle without any loss, whereas for a purely collagen fibre networks, interspersed with highly hydrated, flexible
viscous fluid they are completely out-of-phase, a result of all of the input polysaccharides and other large molecules, is thought to be a key regu-
deformation energy being dissipated or ‘lost’ by internal friction in the lator of tissue mechanics and viscoelasticity46,47. Dissipation in networks
system as it flows. Viscoelastic materials exhibit a response between of collagen or fibrin fibres depends on the nature of the bonds that link
these two extremes, with the in-phase component of the response one fibre to another43,48. Most network crosslinks are non-covalent and
described as the storage, or elastic, modulus and the out-of-phase arise from numerous weak bonds with dissociation rates fast enough to
response described as the loss, or viscous, modulus. The magnitude allow stresses to relax, or allow material creep, on a relevant timescale.

536 | Nature | Vol 584 | 27 August 2020

Box 1

Linking material structure to functional responses under load

Materials can be categorized by how they deform (or change Constitutive equations describe the relationship between stress and
shape) in response to mechanical loading, typically in a stress– strain for a given material. Biological tissues and ECMs can exhibit
strain test. Mechanical stress is defined as the force per unit area, a combination of nonlinear elasticity, viscoelasticity, poroelasticity
with units of pascals (newtons per square metre) and can be in and plasticity. Materials that are both viscoelastic and plastic are
shear or normal. Strain is a normalized measure of deformation. considered to be viscoplastic.

Property Mechanical tests Molecular origins Examples

a Linear elasticity
Loading

Unloading

=E
Resistance of bonds Steel and Glass Concrete
Strain, in material to stretching other metals

b Nonlinear 1. Re-orientation of load-bearing elements

elasticity
Deformation

2. Entropic elasticity
Deformation

Strain, Loose rope (’soft’) Taut rope (’stiff’)

c Viscoelasticity Loss Storage 1. Breaking of weak crosslinks
= E(t)
Loss modulus
modulus

Loading
Storage

Unloading
2. Entanglement release
Strain, Jello/gelatin (viscoelastic solid)
Frequency,
Stress relaxation test Creep test
Strain Stress
Stress Strain

Fluid 3. Protein unfolding 1h

Solid
Solid
Fluid

Time Time Silly Putty (viscoelastic fluid)

d Poroelasticity Stress relaxation (compression)

Stress

Hydrogel Compression
or matrix Water
pushed out

Time Movement of water into or out of matrix

Sponge

e Plasticity Loading
Yield Initial Loading Loading No load
Initial New
bonds bonds
Unloading
Plastic Damage Matrix Irreversible
deformation (bond breakage) flow deformation
Strain, Moulding clay

Box 1 Figure | Mechanical behaviours relevant to biological relationship during loading and unloading (σ = E(t)ε). The ratio
tissues and extracellular matrices. a, In linear elastic materials, of loss to storage is dependent on time. Viscoelastic materials
stress (σ) is linearly related to strain (ε) for small strains (σ = Eε; exhibit stress relaxation in response to a constant deformation,
where E is the elastic modulus), with no loss of mechanical and increased strain, or creep, in response to a constant stress.
energy and reversible deformations (that is, the loading and d, Poroelastic materials exhibit a time-dependent mechanical
unloading curves follow the same path). b, In nonlinear elastic response due to water flow into or out of a porous network when
materials, stress is nonlinearly related to strain for even small a deformation induces change in volume (σ = E(ΔV, t)ε; where ΔV
strains (σ = E(ε)ε). c, Viscoelastic materials exhibit a combination is the change in volume). e, Mechanical plasticity refers to the
of storage of elastic energy, as a solid, and loss of mechanical irreversible deformation of a material following application of
energy, as a fluid. This is reflected by hysteresis in the stress–strain mechanical loading (ε(t∞) > 0).

Nature | Vol 584 | 27 August 2020 | 537

Review
Lumen Epithelium
Actomyosin
fibre

Microtubules
Adhesion
complex Actin cortex

Basement Integrins Actin

membrane polymerization
Pull

Push
Cell

Stromal extracellular matrix

Fig. 1 | Mechanical interactions between cells and extracellular matrices. adhesions, and by pushing, often through actin polymerization and
Cells interact with ECMs mechanically, including by pulling, often through microtubules. The mechanical properties of ECMs mediate these interactions,
actomyosin-based contractility coupled to the ECM through integrin-based resulting in cell mechanotransduction and affecting cell behaviours.

These weak bonds can also exhibit load-dependent dynamics43, and the over short periods. Both slipping of bonds between collagen fibres
breaking of weak bonds under mechanical deformation or loading dissi- and sliding of collagen fibrils have been observed in vivo for tissues
pates energy. Reformation of weak bonds following matrix deformation under load, for example in skin49 and tendon50 tissues, respectively.
can stabilize the deformed state of the material, leading to plastic defor- Polymer entanglements may function similarly to weak crosslinks, as
mations. Using a theoretical fibre network model of collagen, a phase release of an entanglement dissipates energy and allows the matrix to
diagram has been derived that classified the dominant mechanisms of flow. These weak crosslinks or entanglement interactions coexist with
plasticity on the basis of the rate and magnitude of deformation and the more stable covalent crosslinks, which act to diminish liquid-like flow
mechanical properties of individual fibres21. It has been shown that the and mechanical plasticity of the matrix overall, but do not eliminate
experimentally observed viscoplasticity of collagen networks is caused dissipation by unbinding of the weak bonds or by deformations that
by the formation of new crosslinks if moderate strains are applied at can change sample volume. Elastin fibres also act to promote elastic
low rates or by permanent fibre elongation if large strains are applied recovery at the tissue scale51,52. Protein unfolding is another mechanism

a Fluids Skeletal b
Bone
Strain

Cartilage
Ligaments 0
Tendon
1010 Viscoelastic Meniscus
Soft tissues
Loss modulus at 1 Hz (Pa) or ‘viscosity’

109 1.0
Soft tissues Elastic hydrogel
Stress (normalized) or resistance to deformation

Solids
Bladder
108 Skeletal tissues Adipose tissue Skin
Brain Adipose
107 Liver Muscle
Lung Liver
106 Marrow
Skin Fracture haematoma
Soft tissues Cornea Coagulated marrow
105 Kidney Breast tumour
Clot 0.5 Brain
104 Vocal fold
Reconstituted
103 ECMs
Reconstituted ECMs
Type-1 collagen
102 Reconstituted
basement membrane
101 matrix
Fibrin
100 Solids
0 Fluids
100 102 104 106 108 1010 0.1 1.0 10 100 1,000
Storage modulus at 1 Hz (Pa) or ‘elasticity’ Time (s)

Fig. 2 | Biological tissues and extracellular matrices are viscoelastic and Shear storage and loss moduli were converted to storage and loss moduli by
exhibit stress relaxation in response to a deformation. a, Plot of the loss assuming a Poisson ratio of 0.5, and thus multiplying by a factor of 3. b, Stress
modulus at approximately 1 Hz (which is a measure of viscosity or dissipation) relaxation tests on the indicated tissues. Data are from refs. 14,30,31,98,193. Data for
versus the storage modulus at approximately 1 Hz (which is a measure of a and b result from various modalities of measurement (shear, compression,
elasticity) for skeletal tissues, soft tissues and reconstituted ECMs. The grey tension), various measurement tools (mechanical testers, nanoindentation,
dotted line indicates a loss modulus that is 10% of the storage modulus. atomic force microscopy, shear rheometry), and tissue of different animal
Data were taken from a set of randomly selected publications28,33,35,43,162–192. origins (human, rat, mouse, bovine, sheep, porcine, canine).

538 | Nature | Vol 584 | 27 August 2020

 Box 2

Biomaterials with tunable viscoelasticity

To reproduce both the elastic and dissipative properties of tissues between the polymers. Viscoelastic PEG hydrogels have been
in the simplified bioengineered materials used for cell culture, formed using dynamic covalent hydrazone bonds, boronate bonds
several approaches based on the principles of polymer physics or thioester exchange31,195–197. In alginate gels, weak ionic crosslinking
have recently been reported. Polymers that are inert to cell binding leads to viscoelastic gels198. Viscoelastic hyaluronic-acid-based
and not susceptible to degradation by mammalian proteases hydrogels can be formed by using hydrazone bonds or guest–host
are typically used, with cell-adhesion peptide motifs or protein crosslinking97,138. Weak crosslinks can also be programmed into
coupling to the polymer serving as tunable design parameters. A peptide-based hydrogels199. In these networks with weak bonds,
purely elastic hydrogel involves the formation of an ideal covalent viscoelasticity can be modulated independently of the initial elastic
polymer network, as uncrosslinked polymers and loose ends lead to modulus by some combination of varying the following parameters:
energy dissipation194. By contrast, non-ideally crosslinked polymer the molecular weight of the constituent polymer; the coupling of
networks, such as polyacrylamide crosslinked to just beyond the gel inert molecules to the constituent polymer as spacers; the affinity
point, form materials with incomplete crosslinking that allow for loss of the weak bonds; the ratio between weak and covalent bonds;
and creep76. Varying the concentrations of acrylamide (monomer) and the total number of bonds30,96,97,138,153,199,200. Networks formed
and bisacrylamide (crosslinker), or the inclusion of non-crosslinked exclusively from weak crosslinks are expected to be viscoplastic,
linear acrylamide polymers into crosslinked polyacrylamide whereas single or double networks formed with a combination of
gel79, enables the formation of a set of gels with the same storage covalent and weak crosslinks may or may not exhibit viscoplasticity
modulus, but varying loss moduli. at the bulk scale, depending on the molecular architecture.
Other approaches are based on hydrogel materials that are
formed, at least in part, with weak (dynamic or physical) crosslinks

Elastic Viscoelastic; not viscoplastic Viscoelastic and viscoplastic

Covalent Covalent network with Covalent and weak Covalent network and Weakly crosslinked Weakly crosslinked
network entangled polymer network in parallel weak crosslinks in series network network; low connectivity

Polymer Covalent Entangled polymer Polymer, Weak crosslink;

crosslink or loose ends second network affinity may be tunable

Box 2 Figure | Strategies for forming hydrogels that are elastic, viscoelastic but not viscoplastic, or viscoelastic and viscoplastic.

of energy dissipation and viscoelasticity53,54, and has been reported can lead to intracellular poroelastic effects and transient pressure
in fibrin55, spectrin56,57 and intermediate filament58 networks in vitro. gradients that persist for biologically relevant times64. The second
The relative importance of these distinct mechanisms of dissipation distinction is that covalent links between filaments of the cytoskeleton
is expected to vary substantially in their relevance to the viscoelastic are very rare or non-existent. In addition, motor proteins apply random
spectrum displayed by different tissues. non-thermal forces to cytoskeletal filaments65, moving them faster
As tissues largely consist of water, the flow of water within the ECM than they would under thermal agitation alone, with the result that the
can cause substantial viscous dissipation and what are termed poroe- active cytoskeleton is more fluidized than one without motors66. Cel-
lastic effects, depending on the mesh size or porosity of the tissue and lular viscoelasticity can also manifest at the tissue scale. For example,
the rate of loading. Dissipation due to poroelasticity occurs under ten- rigor mortis, the stiffening and solidification of muscle that occurs
sion or compression, and results from volume changes due to water after death, happens in part because the links between actin protein
flow into or out of the network59. Variations in cell number or density filaments and myosin motor proteins in muscle fibres become both
and in the ECM composition, density and conformation in a tissue ena- more numerous and permanent, whereas living muscle hydrolyses ATP
bles fluid to be differentially held by or released from the matrix when so that the actin–myosin links rapidly form and dissociate.
under an externally imposed load or strain, resulting in variations in Finally, many tissues exhibit nonlinear elasticity and do not display
response. By contrast, shear deformations change the shape but not the the simple linear relationship between stress and strain that character-
volume of the sample, and dissipation due to water movement within izes most conventional Hookean solid materials used in engineering,
the matrix is much lower. As a result, the time- or frequency-dependent such as concrete, aluminium or steel. Analogous to a nonlinear elastic
viscoelastic modulus measured in uniaxial strain for the ECM is much material, a coiled bungee cord or rope, an exercise band or an accordion
greater than it is for shear strain60. Poroelastic effects superpose with is easy to straighten out initially, but becomes increasingly difficult to
other mechanical behaviours of tissues and ECM, including nonlinear stretch as it becomes fully extended. In addition to their role in medi-
elasticity, viscoelasticity and viscoplasticity. ating tissue viscoelasticity, networks of crosslinked collagen fibres
Similar mechanisms apply to viscoelasticity of the cytoskeleton of are thought to govern nonlinear elasticity. For both shear and tensile
cells61–63, with two important distinctions. The relatively imperme- deformations, collagen networks behave like linear elastic materi-
able cell membrane tends to prevent or retard poroelastic effects due als up to a threshold level of strain, beyond which they strain-stiffen
to global cell deformation, but local contraction of the cytoskeleton as the fibres align in the direction of maximum tensile strain25,67–71.

Nature | Vol 584 | 27 August 2020 | 539

Review
a Myosin
b
motors
Ws << Wb Ws}Wb Ws >>Wb
1
Clutch binding timescale Wb = r
on

G-actin Polymerization speed

F-actin

Cell spreading
bundle Spreading
Retrograde flow speed

I II III
Signalling feedback K
(Rho, Rac1, and so on)
Molecular Viscoelastic
ka 0.01 0.1 1 10 100 1,000
clutches substrate
kl Ws/Wb
K
Substrate relaxation timescale Ws =
ka

Fig. 3 | The molecular clutch model of mechanotransduction explains the model simulations predict optimal cell spreading when the timescale for stress
effect of matrix viscoelasticity on cell spreading in two dimensions. relaxation (τs) is similar to the clutch binding timescale, τb, which occurs in
a, Schematic of the molecular clutch model of mechanotransduction as region II of the graph. kl and ka are spring constants; η is the coefficient of
applied to viscoelastic substrates. Adapted from ref. 85. b, Molecular clutch viscosity; ron is the on-rate.

The alignment of fibres can enable force transmission over hundreds of nuclear localization of the myocardin-related transcription factor A
micrometres, facilitating long-range communication between cells70,72. (MRTF-A) protein on viscoelastic compared to elastic substrates80.
A theoretical fibre network model of collagen showed that strong cou- Interestingly, normal human hepatocytes also spread less and had
pling between modes of deformation can give rise to much higher lower motility on viscoelastic substrates, but the reverse was true for
strain-stiffening of the networks in triaxial and biaxial tensile loading hepatocellular carcinoma cells81.
compared to uniaxial loading73. Nonlinear elasticity is also observed To explain these seemingly disparate results, computational model-
in cytoskeletal filament networks—including actin, vimentin and neu- ling was applied. The primary sensing apparatus of substrate stiffness
rofilaments—but the origins of nonlinear elasticity in these networks for cells in two-dimensional culture is thought to be the myosin–actin–
may have a stronger contribution of entropic elasticity, owing to the adhesion system, also known as the motor clutch module (Fig. 3),
semiflexible nature of the filaments25,74,75. whose dynamics have successfully explained stiffness sensing of cells
on elastic substrates82–84. To study the effect of ECM viscoelasticity
on cell spreading, a generalized motor clutch model that explicitly
Two-dimensional culture and the molecular clutch accounts for dissipative processes both in the ECM and in the cell has
The effect of substrate viscoelasticity on cells has been demonstrated recently been developed85. In this model, myosin motors pull acto-
powerfully through a set of two-dimensional culture studies that uti- myosin networks at the leading edge of the cell towards the nucleus,
lize biomaterials with independently tunable viscoelastic properties generating actin retrograde flow. The retrograde flow is resisted by
(Box 2). In an early study, human mesenchymal stem cells (MSCs) were adhesion molecules that can randomly bind and unbind between actin
cultured on collagen-coated polyacrylamide gels that had similar stor- bundles and ECM. At the cell leading edge, the polymerization of actin
age moduli, but varying loss moduli and creep responses76. Increased filaments, countered by retrograde flow, pushes the cell membrane
loss, or creep, in the substrates promoted cell spreading, focal adhesion forward, further resulting in the spreading of the cell. To account for
formation, proliferation and differentiation towards adipogenic, osteo- processes that reinforce the adhesion (for example, talin unfolding in
genic and smooth muscle cell lineages. Studies in which myosin and Rho the FA complex, which triggers recruitment of integrins86), the clutch
GTPase, a signalling molecule, were inhibited demonstrated the role of binding rate is assumed to increase beyond a threshold level of force.
cytoskeletal tension in mediating the response to increased mechanical Interestingly, the model shows that, for soft substrates, maximum
loss. In a follow-up study, increased activation of the GTPase Rac1 and cell spreading is achieved at an optimal level of viscosity in which the
increases in motility and lamellipodial protrusions were found in human substrate relaxation time falls between the timescale for clutch bind-
MSCs on substrates with higher loss and creep77. Another study com- ing and its characteristic binding lifetime. That is, viscosity serves to
pared fibroblasts and cancer cells cultured on covalently crosslinked stiffen soft substrates on a timescale faster than the clutch off-rate,
(or elastic) versus ionically crosslinked (or viscoelastic and viscoplastic) which enhances cell−ECM adhesion and cell spreading. On the other
alginate gels that presented the RGD (arginine-glycine-aspartate) cell hand, for substrates that are stiff, the model predicts that viscosity will
adhesion peptide motif. Although cells were unable to spread on soft not influence cell spreading, since the bound clutches are saturated
elastic gels, they were able to spread on soft viscoelastic gels through by the elevated stiffness. The model was tested and validated using
the β1 integrin receptor protein, myosin and Rho, exhibiting robust experimental measurements on three different material systems and
focal adhesions and stress fibres and enhanced activation of the tran- explained the different observed effects of viscosity on each substrate85.
scriptional regulator protein YAP, similar to their behaviour on stiff The clutch model has also been applied to describing myoblast interac-
and elastic substrates78. Increased spreading was associated with plas- tions with purely viscous lipid bilayers87.
tic deformation. To distinguish the effects of viscoelasticity from the
effects of viscoplasticity, viscoelastic (but not viscoplastic) substrates
were formed using elastic polyacrylamide gels with linear acrylamide Three-dimensional culture and mechanical confinement
chains trapped inside them79. An increased loss modulus, or faster stress The role of matrix viscoelasticity has also been investigated in
relaxation, diminished fibroblast stiffness and cell spreading area, three-dimensional culture. Culture dimensionality is known to
in contrast to results with viscoplastic alginate substrates. Similarly, affect cell structure, adhesions, signalling and nutrient transport88.
hepatic stellate cells exhibited reduced spreading, stress fibres and Three-dimensional culture supports various behaviours, including

540 | Nature | Vol 584 | 27 August 2020

 a Cellular process restricted by confinement b
Matrix pore size
Volume change Morphological Combination
Cell growth Cell spreading Cell migration

Matrix deposition Mitosis Organoid formation Confinement

XXX
XX
Matrix degradability
X XXX

XX
XX
X

X
Matrix viscoplasticity
XX X X

Fig. 4 | Matrix viscoplasticity mediates mechanical confinement in combination of matrix pore size, matrix degradability, and matrix
three-dimensional culture. a, In confining three-dimensional matrices, viscoplasticity. A sufficiently large value for any one of these properties
processes that involve volume change, morphological changes, or a releases confinement.
combination of both are restricted. b, Confinement is governed by a

epithelial morphogenesis, maintenance of pluripotency in human The effect of hydrogel viscoelasticity and viscoplasticity on
embryonic stem cells and the differentiated state in chondrocytes89–91. cell spreading, proliferation, matrix deposition and migration in
Culture dimensionality has also been specifically implicated in medi- three-dimensional culture indicates a link to the concept of mechanical
ating mechanotransduction. For example, whereas two-dimensional confinement. Many cellular processes involve changes in cell volume,
culture studies have implicated the YAP transcriptional regulator as shape or movement (Fig. 4a). When these processes are physically
a universal mechanotransducer, mediating the response of cells to restricted in three dimensions by the surrounding ECM or cells, the
stiffness in all two-dimensional culture contexts92, YAP-independent cells are considered to be mechanically confined107,108. The established
mechanotransduction is found in a three-dimensional culture model view has been that pore size and matrix degradability are key regu-
of stiffness-induced breast cancer, which is consistent with an analy- lators of mechanical confinement107. For example, in the context of
sis of samples from patients with breast cancer93. Similarly, culture cancer cell migration, it has been shown that rigid pore sizes below
dimensionality affects YAP/TAZ signalling in human MSCs94. YAP has approximately 3 μm block migration, with cells unable to squeeze their
been shown to mediate mechanotransduction in some in vivo con- stiff nuclei through smaller pores109–111. We note that PEG, alginate and
texts, such as pancreatic cancer95, highlighting that the importance hyaluronic-acid-based hydrogels typically have nanometre-scale pores.
of using three-dimensional culture models depends on the specific With rigid or elastic pores, matrix degradation was required for the
biological process. cells to overcome confinement and migrate. However, given sufficient
Various studies have explored the effect of matrix viscoelasticity viscoelasticity or viscoplasticity, cells can overcome confinement to
on cells in three-dimensional culture. Increased stress relaxation, grow in size, deposit matrix, change their morphology as they spread or
enhanced creep or a higher loss modulus in RGD-coupled polyethylene undergo mitosis, and migrate. This provides the new perspective that
glycol (PEG) gels31, RGD-coupled alginate gels30,96, and interpenetrat- in addition to pore size and degradability, matrix mechanical viscoplas-
ing networks of hyaluronic acid and collagen97 promotes spreading ticity governs confinement (Fig. 4b). During cell–matrix remodelling,
of adherent cells such as myoblasts, fibroblasts and MSCs. Faster these properties are coupled: cell remodelling of viscoplastic matrices
stress relaxation and increased loss also promote cell cycle progres- alters pore size106, degradation of the matrix changes its viscoelastic
sion and completion of mitosis in single cancer cells and fibroblasts, properties112, and changes in the matrix architecture probably affect
as well as osteogenic differentiation of MSCs30,98,99. Transcriptional both viscoplasticity and degradability.
responses are cell-type-specific, with human cortical progenitors In viscoelastic and viscoplastic three-dimensional matrices, various
and MSCs being sensitive to different ranges of stress relaxation and mechanisms of mechanotransduction have been reported. As with
initial elastic moduli100. Maintenance of neural progenitor stemness two-dimensional culture, actomyosin-based contractility coupled to
is also facilitated by hydrogels with fast stress relaxation, while being the matrix through integrin-mediated adhesions, and integrin-ligand
inhibited in covalently crosslinked hydrogels101. In addition, chondro- clustering, are implicated30,113. Although in principle some of these
cytes and osteogenically differentiated MSCs can form wide volumes effects could probably be explained using molecular-clutch-based mod-
of interconnected cartilage-like or bone-like matrix, respectively, in els, these models have not yet been extended to three-dimensional con-
viscoelastic hydrogels that exhibit fast stress relaxation30,102. Notably, texts involving mechanical confinement. Another mechanism involves
the viscoelastic hydrogels used in these three-dimensional culture cell volume expansion. Chondrocytes, MSCs and cancer cells expand
studies are all viscoplastic. their volume, or grow as part of the cell cycle, in matrices with fast
Matrix viscoplasticity has been implicated in enabling mechanical stress relaxation, but the volume expansion is restricted in matrices
remodelling of the matrix structure for cells cultured in three dimen- that exhibit slow stress relaxation, or are more elastic98,102,114. In MSCs,
sions in collagen gels both locally20,22,103,104 and in microtissues105. The volume expansion activates the stretch-activated ion channel protein
effect of viscoplasticity on cancer cell migration was explicitly tested TRPV4, and the signalling cascade induced by the resulting calcium
in interpenetrating networks of reconstituted basement membrane influx drives nuclear localization of the transcription factor protein
matrix and alginate106. Cancer cells were found to be able to migrate RUNX2, but not YAP, to promote osteogenic differentiation114. Similarly,
through the nanoporous matrices in a protease-independent manner growth during the G1 phase of the cell cycle activates a TRPV4–PI3K/
when the matrices exhibited sufficient mechanical plasticity. Cells Akt–p27Kip1 signalling axis to promote cell cycle progression in cancer
mechanically opened up channels in the matrix using invadopodial cells98. Restriction of cell volume expansion promotes Il-1β signalling
protrusions, independent of proteases, and then migrated through in chondrocytes, resulting in an osteoarthritic phenotype102. Finally,
the channels. as matrix remodelling and deposition are often enhanced in matrices

Nature | Vol 584 | 27 August 2020 | 541

Review
a Damaged or b Biomaterial design c Biomaterial d Biomaterial-
diseased tissue introduction driven regeneration
Gene expression Morphogenesis

Materials

Stress
Cells
Proteins
Nucleotides
Mechanical Time
measurement

Fig. 5 | Designing viscoelastic biomaterials for regenerative medicine. interacting cells and morphogenesis. c, d, Introduction of the material, either
a, b, Advanced imaging is used to detect the mechanical properties of the alone or carrying various regeneration-promoting cargoes (for example, cells)
tissue, damaged and normal, in order to design materials with appropriate will then lead to (right panel) regeneration of the damaged tissue and
viscoelastic properties to guide the desired pattern of gene expression from reconstitution of function.

with increased viscoplasticity, the mechanical microenvironment to in various applications—including cartilage regeneration, vocal cord
which cells respond is time-dependent, and cell–matrix interaction regeneration and amelioration of pathologic remodelling of the myo-
becomes a dynamic and potentially iterative process. cardium following myocardial infarction—may also be related to their
viscoelastic properties125–128.
A key question is whether viscoelasticity has been a hidden vari-
Viscoelastic biomaterials in medicine able that could explain much past work in the biomaterials field more
A potential application for these findings lies in the design of bioma- broadly. Some of the most widely used and successful biomaterials in
terials for regenerative medicine. This field originated with the goal regenerative medicine—including collagen gels, hyaluronic acid and
of regenerating tissues and organs, or engineering replacements, supramolecular assemblies129—are physically crosslinked hydrogels.
for those damaged or lost to disease or trauma115. Biomaterials are The most widely used biomaterial for intestinal organoid formation
typically used for cell and drug delivery, to spatially organize trans- in vitro—reconstituted basement membrane matrix—is also a physi-
planted and resident cells, for regulation of gene expression, and to cally crosslinked viscoelastic hydrogel, as are others used to promote
guide tissue structure and function in various regenerative, tissue- formation of skeletal muscle, liver and neural organoids130–133. There
and immune-engineering applications116. The evident effect of matrix have been a number of studies aiming to delineate the effect of matrix
viscoelasticity on cell proliferation, gene expression, fate and migration degradation on tissue regeneration, and a provocative possibility is that
highlights its importance as a design parameter for biomaterials-based the effects might, at least in part, relate to the viscoelastic behaviour of
applications. Indeed, FDA-approved, tissue engineering products (for these biomaterials. Several early studies concluded that more rapidly
example, Apligraft engineered skin, Infuse bone-regeneration devices) degrading hydrogels led to greater tissue regeneration than more slowly
are often based on viscoelastic matrices. Advances in materials pro- degrading gels134,135. However, those studies used alterations in polymer
cessing techniques such as 3D printing, which often uses viscoelastic molecular weight to regulate gel dissolution, and these changes will also
materials117,118, have allowed tissue and organ structure and properties alter viscoelasticity. A number of studies examining three-dimensional
to be more faithfully recapitulated. The utility of engineered tissues as mechanotransduction have used covalently crosslinked hydrogels
improved models for basic studies of development and pathology, as and concluded that degradation of the gels was key to how cells inter-
test beds for toxicology analysis and as improved drug screening have preted gel cues136,137. However, the cellular activity leading to degrada-
also led to substantial interest in the development of microphysiologi- tion of these materials will probably transition the local matrix to a
cal systems (for example, tissue-on-chip) and cultured organoids119,120. more viscoelastic state. In addition, cells may be interacting with the
These can recapitulate tissue and organ biology more faithfully than matrix molecules they themselves deposit138, which might provide a
can standard, two-dimensional cell culture models, while also enabling viscoelastic substrate. Similarly, recent efforts to develop a synthetic
the study of human biology (as opposed to animal biology) of classic analogue to the naturally derived, physical hydrogels for organoid
preclinical studies. formation demonstrate that gel degradability is critical to designing
There exist both direct evidence and substantial correlative data that synthetic replacements139,140. While little is known regarding the role
viscoelasticity is an important design parameter for biomaterials used of viscoelasticity in the fate and functional state of cells of the innate
in regenerative medicine. The first demonstration that matrix stiffness and adaptive immune system, a recent study has implicated purely
regulates regeneration used the transplantation of stem cells within elastic covalently crosslinked synthetic matrices, rather than those
viscoelastic hydrogels121. Strikingly, the effect of stiffness on stem cell fabricated with naturally derived physically crosslinked viscoelastic
fate in those gels was related to the ability of cellular traction forces ECM, as leading to inflammatory instead of regeneration-promoting
to remodel the polymers comprising the hydrogels113, suggesting that immune cell responses141. Clearly, much more research will be required
in fact it was the viscoelasticity of the gels that was key to their effect to delineate the specific roles of viscoelasticity, other physical proper-
on cell fate in vivo. A subsequent study directly examined the effect ties and chemical composition in the cellular and tissue response to
of viscoelasticity by transplanting cells in hydrogels of matched ini- various biomaterials that mediate tissue repair and formation.
tial elastic moduli, but varying rates of stress relaxation. Hydrogels
with more rapid stress relaxation led to greater bone regeneration122;
the optimal relaxation rate corresponded to that of human fracture Outlook
haematomas isolated from patients122, which comprise the environ- Viscoelasticity is a near-universal feature of living tissues and ECMs,
ment in which bone regeneration naturally occurs. Similar viscoelastic and a rapidly expanding body of evidence is establishing that cells
hydrogels delivering inductive proteins were also found to promote sense and respond to the viscoelastic properties of ECMs, challeng-
extensive bone regeneration, probably owing to the ability of host ing the current stiffness-centric view of cell–matrix mechanotrans-
cells to readily invade the gels123,124. The beneficial effect of hydrogels duction. Measurements of the viscoelasticity and viscoplasticity of

542 | Nature | Vol 584 | 27 August 2020

tissues during development, and of adult and pathologic tissues, are that matrix stiffness affects genome accessibility in a three-dimensional
needed. The change in viscoelasticity and viscoplasticity associated culture model of breast cancer, which mediated induction of malig-
with diseases will be of particular interest, especially at the microscale, nancy by enhanced stiffness13. The connection between matrix
relevant to cells. Given that both two- and three-dimensional culture viscoelasticity and cell signalling, transcription factor activation, and
studies have shown that changes in matrix viscoelasticity drive broad the epigenome is an area ripe for study.
changes in gene expression, proliferation, migration and differentia- Biomaterials design has historically operated without taking
tion, it is likely that changes in tissue viscoelasticity will play a part in into account the importance of viscoelasticity, but moving forward,
disease progression, suggesting a potent target for therapeutic viscoelasticity is likely to be a key technical specification in many appli-
approaches. More work is needed to explore the relationships between cations (Fig. 5). Success will probably involve mimicking the mechanical
viscoelasticity and viscoplasticity and higher-order behaviours in devel- characteristics of developing tissues, because mimicking the develop-
opment, tissue genesis and repair and disease aetiology. mental context is often a potent strategy for promoting regeneration.
Although the effect of substrate viscoelasticity on cell spreading The role of viscoelasticity in regulating the biology of the various cell
in two-dimensional culture is increasingly understood, the effect of types regulating regeneration—possibly including pluripotent stem
viscoelasticity must also be considered in the context of other physical cells, tissue-resident stem and differentiated cells, and immune cells—
cues of the matrix. Architectural features—including geometry, poros- will also need to be delineated in order to rationally design materials that
ity, and topology (for example, nanoscale roughness)—have all been can enhance tissue regeneration. Biomaterial design may also require
demonstrated to affect various aspects of cell behaviour142–146. However, decoupling of the local viscoelastic properties that cells sense from the
these have typically been studied in the context of high-moduli, purely larger, tissue-scale properties required to achieve mechanical stability of
elastic matrices. It is unclear how cells will interpret these cues in the the regenerating or engineered tissue. Thus, the advent of biomaterials
context of viscoelastic matrices. Cells generate forces and deforma- with controlled viscoelasticity may be transformative in improving the
tions on substrates in a highly dynamic manner, leading to a complex success of biomaterials applications in regenerative medicine.
time-dependent mechanical response of the substrates, which may
greatly alter the original architecture and feature sizes to which cells 1. Katzberg, A. A. Distance as a factor in the development of attraction fields between
respond. Although externally applied stresses (for example, compres- growing tissues in culture. Science 114, 431–432 (1951).
sive and shear forces) conveyed to cells from their matrices also regu- 2. Keese, C. R. & Giaever, I. Substrate mechanics and cell spreading. Exp. Cell Res. 195,
528–532 (1991).
late cellular gene expression and tissue structure and function147,148, 3. Pelham, R. J. Jr & Wang, Y. Cell locomotion and focal adhesions are regulated by
their effects have often been studied in the context of purely elastic substrate flexibility. Proc. Natl Acad. Sci. USA 94, 13661–13665 (1997).
4. Discher, D. E., Janmey, P. & Wang, Y. L. Tissue cells feel and respond to the stiffness of
substrates. The dissipation of externally applied forces by viscoelastic
their substrate. Science 310, 1139–1143 (2005).
matrices is likely to diminish the magnitude and distance of action of 5. DuFort, C. C., Paszek, M. J. & Weaver, V. M. Balancing forces: architectural control of
these cues and may alter the mechanotransduction pathways they mechanotransduction. Nat. Rev. Mol. Cell Biol. 12, 308–319 (2011).
6. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev.
trigger.
Mol. Cell Biol. 7, 265–275 (2006).
Our mechanistic understanding of mechanotransduction in 7. Humphrey, J. D., Dufresne, E. R. & Schwartz, M. A. Mechanotransduction and extracellular
viscoelastic and viscoplastic matrices in three dimensions is still lim- matrix homeostasis. Nat. Rev. Mol. Cell Biol. 15, 802–812 (2014).
8. Kechagia, J. Z., Ivaska, J. & Roca-Cusachs, P. Integrins as biomechanical sensors of the
ited. Tools and approaches that enable cell–matrix interactions to
microenvironment. Nat. Rev. Mol. Cell Biol. 20, 457–473 (2019).
be deciphered with greater spatiotemporal resolution are needed. 9. Janmey, P. A., Fletcher, D. & Reinhart-King, C. A. Stiffness sensing in cells and tissues.
This is particularly important in viscoplastic matrices, because cell Physiol. Rev. 100, 695–724 (2020).
10. Bellin, R. M. et al. Defining the role of syndecan-4 in mechanotransduction using
interactions with the matrix would be expected to dynamically alter
surface-modification approaches. Proc. Natl Acad. Sci. USA 106, 22102–22107 (2009).
the local matrix architecture, ligand density and viscoelasticity. 11. Yang, C., Tibbitt, M. W., Basta, L. & Anseth, K. S. Mechanical memory and dosing influence
Super-resolution imaging in three dimensions, molecular force sen- stem cell fate. Nat. Mater. 13, 645–652 (2014).
12. Balestrini, J. L., Chaudhry, S., Sarrazy, V., Koehler, A. & Hinz, B. The mechanical memory of
sors and materials with dynamically tunable mechanical properties
lung myofibroblasts. Integr. Biol. 4, 410–421 (2012).
are emerging technologies that may address this need and provide 13. Stowers, R. S. et al. Matrix stiffness induces a tumorigenic phenotype in mammary
a detailed readout of the dynamic molecular-scale interactions and epithelium through changes in chromatin accessibility. Nat. Biomed. Eng. 3, 1009–
1019 (2019).
forces that occur between cells and viscoplastic matrices149–152, helping 14. Levental, I., Georges, P. C. & Janmey, P. A. Soft biological materials and their impact on
to develop a more holistic view of cell–matrix signalling. In addition, cell function. Soft Matter 3, 299–306 (2007).
most synthetic hydrogel systems used in this field are nanoporous and 15. Swift, J. et al. Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed
differentiation. Science 341, 1240104 (2013).
do not capture the fibrillarity and ligand presentation of native ECMs. 16. Wozniak, M. A. & Chen, C. S. Mechanotransduction in development: a growing role for
Incorporation of collagen fibres into synthetic hydrogels97,153, or the contractility. Nat. Rev. Mol. Cell Biol. 10, 34–43 (2009).
use of synthetic approaches to generating collagen-like fibres154, may 17. Jaalouk, D. E. & Lammerding, J. Mechanotransduction gone awry. Nat. Rev. Mol. Cell Biol.
10, 63–73 (2009).
help to address this important limitation. Furthermore, the integration 18. Wang, J. H. Mechanobiology of tendon. J. Biomech. 39, 1563–1582 (2006).
of advances in chemical synthesis routes that permit explicit control 19. Mazza, E., Papes, O., Rubin, M. B., Bodner, S. R. & Binur, N. S. Nonlinear elastic-viscoplastic
over composition, architecture and precise positioning of functional constitutive equations for aging facial tissues. Biomech. Model. Mechanobiol. 4, 178–189
(2005).
groups155,156—such as reversible addition-fragmentation chain transfer 20. Malandrino, A., Trepat, X., Kamm, R. D. & Mak, M. Dynamic filopodial forces induce
polymerization (RAFT) and DNA origami—and real-time, non-invasive accumulation, damage, and plastic remodeling of 3D extracellular matrices. PLoS
tuning of properties151, with adaptive manufacturing processes that Comput. Biol. 15, e1006684 (2019).
21. Ban, E. et al. Mechanisms of plastic deformation in collagen networks induced by cellular
can program material composition and architecture across varying forces. Biophys. J. 114, 450–461 (2018).
length scales118 is likely to produce novel material systems with which This study used computational modelling to show that the observed plasticity of
to explore the effects of viscoelasticity and viscoplasticity both in vitro collagen networks is caused by the formation of new crosslinks if moderate strains are
applied at small rates or due to permanent fibre elongation if large strains are applied
and in vivo. Synthetic semiflexible filament networks made by electro- over short periods, matching experimental findings.
spinning or from self-assembling helix-forming monomers represent 22. Nam, S., Lee, J., Brownfield, D. G. & Chaudhuri, O. Viscoplasticity enables mechanical
a novel class of materials that can more closely mimic the elastic prop- remodeling of matrix by cells. Biophys. J. 111, 2296–2308 (2016).
23. Clement, R., Dehapiot, B., Collinet, C., Lecuit, T. & Lenne, P. F. Viscoelastic dissipation
erties of native ECM157,158 as well as incorporate energy dissipation159 stabilizes cell shape changes during tissue morphogenesis. Curr. Biol. 27, 3132–3142 (2017).
and plastic deformation160. In addition, there are major gaps in our 24. Lardennois, A. et al. An actin-based viscoplastic lock ensures progressive body-axis
understanding of how matrix viscoelasticity affects signalling pathways elongation. Nature 573, 266–270 (2019).
25. Storm, C., Pastore, J. J., MacKintosh, F. C., Lubensky, T. C. & Janmey, P. A. Nonlinear
and regulation of transcription in three dimensions. Mechanical cues elasticity in biological gels. Nature 435, 191–194 (2005).
generally regulate genome architecture161, and a recent study has found 26. Shadwick, R. E. Mechanical design in arteries. J. Exp. Biol. 202, 3305–3313 (1999).

Nature | Vol 584 | 27 August 2020 | 543

Review
27. Li, W., Shepherd, D. E. T. & Espino, D. M. Frequency dependent viscoelastic properties of 61. Mollaeian, K., Liu, Y., Bi, S. & Ren, J. Atomic force microscopy study revealed velocity-
porcine brain tissue. J. Mech. Behav. Biomed. Mater. 102, 103460 (2020). dependence and nonlinearity of nanoscale poroelasticity of eukaryotic cells. J. Mech.
28. Bilston, L. E., Liu, Z. & Phan-Thien, N. Linear viscoelastic properties of bovine brain tissue Behav. Biomed. Mater. 78, 65–73 (2018).
in shear. Biorheology 34, 377–385 (1997). 62. Hu, J. et al. Size- and speed-dependent mechanical behavior in living mammalian
29. Budday, S., Sommer, G., Holzapfel, G. A., Steinmann, P. & Kuhl, E. Viscoelastic parameter cytoplasm. Proc. Natl Acad. Sci. USA 114, 9529–9534 (2017).
identification of human brain tissue. J. Mech. Behav. Biomed. Mater. 74, 463–476 (2017). 63. Mitchison, T. J., Charras, G. T. & Mahadevan, L. Implications of a poroelastic cytoplasm for
30. Chaudhuri, O. et al. Hydrogels with tunable stress relaxation regulate stem cell fate and the dynamics of animal cell shape. Semin. Cell Dev. Biol. 19, 215–223 (2008).
activity. Nat. Mater. 15, 326–334 (2016). 64. Moeendarbary, E. et al. The cytoplasm of living cells behaves as a poroelastic material.
This study demonstrated an approach to modulating the stress relaxation or loss Nat. Mater. 12, 253–261 (2013).
modulus of alginate hydrogels independent of the initial elastic modulus, and found 65. Guo, M. et al. Probing the stochastic, motor-driven properties of the cytoplasm using
that increased stress relaxation promoted cell spreading, proliferation and osteogenic force spectrum microscopy. Cell 158, 822–832 (2014).
differentiation of mesenchymal stem cells in three-dimensional culture. 66. Humphrey, D., Duggan, C., Saha, D., Smith, D. & Kas, J. Active fluidization of polymer
31. McKinnon, D. D., Domaille, D. W., Cha, J. N. & Anseth, K. S. Biophysically defined and networks through molecular motors. Nature 416, 413–416 (2002).
cytocompatible covalently adaptable networks as viscoelastic 3D cell culture systems. 67. Vader, D., Kabla, A., Weitz, D. & Mahadevan, L. Strain-induced alignment in collagen gels.
Adv. Mater. 26, 865–872 (2014). PLoS One 4, e5902 (2009).
This study demonstrated the use of hydrozone bonds to form viscoelastic PEG gels, 68. Hall, M. S. et al. Fibrous nonlinear elasticity enables positive mechanical feedback
and found that the viscoelastic gels enabled myoblast spreading in three-dimensional between cells and ECMs. Proc. Natl Acad. Sci. USA 113, 14043–14048 (2016).
culture. 69. Steinwachs, J. et al. Three-dimensional force microscopy of cells in biopolymer networks.
32. Reihsner, R. & Menzel, E. J. Two-dimensional stress-relaxation behavior of human skin Nat. Methods 13, 171–176 (2016).
as influenced by non-enzymatic glycation and the inhibitory agent aminoguanidine. 70. Wang, H., Abhilash, A. S., Chen, C. S., Wells, R. G. & Shenoy, V. B. Long-range force
J. Biomech. 31, 985–993 (1998). transmission in fibrous matrices enabled by tension-driven alignment of fibers. Biophys.
33. Geerligs, M., Peters, G. W., Ackermans, P. A., Oomens, C. W. & Baaijens, F. P. Linear J. 107, 2592–2603 (2014).
viscoelastic behavior of subcutaneous adipose tissue. Biorheology 45, 677–688 (2008). 71. Licup, A. J. et al. Stress controls the mechanics of collagen networks. Proc. Natl Acad. Sci.
34. Qiu, S. et al. Characterizing viscoelastic properties of breast cancer tissue in a mouse USA 112, 9573–9578 (2015).
model using indentation. J. Biomech. 69, 81–89 (2018). 72. Han, Y. L. et al. Cell contraction induces long-ranged stress stiffening in the extracellular
35. Liu, Z. & Bilston, L. On the viscoelastic character of liver tissue: experiments and matrix. Proc. Natl Acad. Sci. USA 115, 4075–4080 (2018).
modelling of the linear behaviour. Biorheology 37, 191–201 (2000). 73. Ban, E. et al. Strong triaxial coupling and anomalous Poisson effect in collagen networks.
36. Perepelyuk, M. et al. Normal and fibrotic rat livers demonstrate shear strain softening and Proc. Natl Acad. Sci. USA 116, 6790–6799 (2019).
compression stiffening: a model for soft tissue mechanics. PLoS One 11, e0146588 (2016). 74. Gardel, M. L. et al. Elastic behavior of cross-linked and bundled actin networks. Science
37. Forgacs, G., Foty, R. A., Shafrir, Y. & Steinberg, M. S. Viscoelastic properties of living 304, 1301–1305 (2004).
embryonic tissues: a quantitative study. Biophys. J. 74, 2227–2234 (1998). 75. Chaudhuri, O., Parekh, S. H. & Fletcher, D. A. Reversible stress softening of actin networks.
38. Gersh, K. C., Nagaswami, C. & Weisel, J. W. Fibrin network structure and clot mechanical Nature 445, 295–298 (2007).
properties are altered by incorporation of erythrocytes. Thromb. Haemost. 102, 76. Cameron, A. R., Frith, J. E. & Cooper-White, J. J. The influence of substrate creep on
1169–1175 (2009). mesenchymal stem cell behaviour and phenotype. Biomaterials 32, 5979–5993
39. Streitberger, K. J. et al. High-resolution mechanical imaging of glioblastoma by (2011).
multifrequency magnetic resonance elastography. PLoS One 9, e110588 (2014). This study demonstrated an approach to modulating the loss modulus of PAM
40. Sack, I. et al. The impact of aging and gender on brain viscoelasticity. Neuroimage 46, hydrogels independently of the elastic modulus, thereby creating a range of
652–657 (2009). stiffness-matched substrates of varying viscoelasticity, showing that substrates that
41. Streitberger, K. J. et al. Brain viscoelasticity alteration in chronic-progressive multiple permitted increased creep under cell-generated stresses promoted increased cell
sclerosis. PLoS One 7, e29888 (2012). spreading, proliferation, and tri-lineage differentiation of mesenchymal stem cells
42. Sinkus, R. et al. MR elastography of breast lesions: understanding the solid/liquid duality in two-dimensional culture.
can improve the specificity of contrast-enhanced MR mammography. Magn. Reson. Med. 77. Cameron, A. R., Frith, J. E., Gomez, G. A., Yap, A. S. & Cooper-White, J. J. The effect of
58, 1135–1144 (2007). time-dependent deformation of viscoelastic hydrogels on myogenic induction and Rac1
These studies (Streitberger et al. (2014) and Sinkus et al. (2007)) utilized magnetic activity in mesenchymal stem cells. Biomaterials 35, 1857–1868 (2014).
resonance elastography to analyse changes in tissue viscoelasticity during cancer, This study demonstrated that increasing levels of dissipation in viscoelastic substrates
and find that there were striking differences in viscoelasticity between malignant and matching skeletal muscle stiffness biased Rho-GTPase activity to drive Rac1-mediated
benign breast tumors, and between glioblastoma and healthy brain parenchyma. myogenic induction of mesenchymal stem cells in two-dimensional culture.
43. Nam, S., Hu, K. H., Butte, M. J. & Chaudhuri, O. Strain-enhanced stress relaxation impacts 78. Chaudhuri, O. et al. Substrate stress relaxation regulates cell spreading. Nat. Commun. 6,
nonlinear elasticity in collagen gels. Proc. Natl Acad. Sci. USA 113, 5492–5497 (2016). 6365 (2015).
44. Gerth, C., Roberts, W. W. & Ferry, J. D. Rheology of fibrin clots. II. Linear viscoelastic 79. Charrier, E. E., Pogoda, K., Wells, R. G. & Janmey, P. A. Control of cell morphology and
behavior in shear creep. Biophys. Chem. 2, 208–217 (1974). differentiation by substrates with independently tunable elasticity and viscous
45. Liu, W. et al. Fibrin fibers have extraordinary extensibility and elasticity. Science 313, dissipation. Nat. Commun. 9, 449 (2018).
634 (2006). This study reported a method of producing viscoelastic solid substrates with
46. Connizzo, B. K. & Grodzinsky, A. J. Multiscale poroviscoelastic compressive properties of separately tunable elastic and viscous moduli and showed that several cell types
mouse supraspinatus tendons are altered in young and aged mice. J. Biomech. Eng. 140, respond to viscoelastic substrates as though they were softer than purely elastic
051002 (2018). substrates of the same elastic modulus.
This and earlier related studies emphasize the importance of poroelastic relaxation in 80. Hui, E., Gimeno, K. I., Guan, G. & Caliari, S. R. Spatiotemporal control of viscoelasticity
the design of tissues and their changes with injury, disease and ageing. in phototunable hyaluronic acid hydrogels. Biomacromolecules 20, 4126–4134
47. Sauer, F. et al. Collagen networks determine viscoelastic properties of connective tissues (2019).
yet do not hinder diffusion of the aqueous solvent. Soft Matter 15, 3055–3064 (2019). 81. Mandal, K., Gong, Z., Rylander, A., Shenoy, V. B. & Janmey, P. A. Opposite responses of
48. Munster, S. et al. Strain history dependence of the nonlinear stress response of fibrin and normal hepatocytes and hepatocellular carcinoma cells to substrate viscoelasticity.
collagen networks. Proc. Natl Acad. Sci. USA 110, 12197–12202 (2013). Biomater. Sci. 8, 1316–1328 (2020).
49. Yang, W. et al. On the tear resistance of skin. Nat. Commun. 6, 6649 (2015). 82. Bangasser, B. L., Rosenfeld, S. S. & Odde, D. J. Determinants of maximal force transmission
50. Silver, F. H., Freeman, J. W. & Seehra, G. P. Collagen self-assembly and the development in a motor-clutch model of cell traction in a compliant microenvironment. Biophys. J.
of tendon mechanical properties. J. Biomech. 36, 1529–1553 (2003). 105, 581–592 (2013).
51. Oxlund, H., Manschot, J. & Viidik, A. The role of elastin in the mechanical properties of 83. Chan, C. E. & Odde, D. J. Traction dynamics of filopodia on compliant substrates. Science
skin. J. Biomech. 21, 213–218 (1988). 322, 1687–1691 (2008).
52. Vesely, I. The role of elastin in aortic valve mechanics. J. Biomech. 31, 115–123 (1997). 84. Bangasser, B. L. et al. Shifting the optimal stiffness for cell migration. Nat. Commun. 8,
53. DeBenedictis, E. P. & Keten, S. Mechanical unfolding of alpha- and beta-helical protein 15313 (2017).
motifs. Soft Matter 15, 1243–1252 (2019). 85. Gong, Z. et al. Matching material and cellular timescales maximizes cell spreading on
54. Zhao, X. H. Multi-scale multi-mechanism design of tough hydrogels: building dissipation viscoelastic substrates. Proc. Natl Acad. Sci. USA 115, E2686–E2695 (2018).
into stretchy networks. Soft Matter 10, 672–687 (2014). This study used analytical and Monte Carlo methods to simulate the dynamics of
55. Brown, A. E., Litvinov, R. I., Discher, D. E., Purohit, P. K. & Weisel, J. W. Multiscale mechanics motor clutches (focal adhesions) formed between the cell and a viscoelastic substrate,
of fibrin polymer: gel stretching with protein unfolding and loss of water. Science 325, and found that that intermediate viscosity maximizes cell spreading on soft substrates,
741–744 (2009). while cell spreading is independent of viscosity on stiff substrates, in agreement with
56. Paramore, S., Ayton, G. S. & Voth, G. A. Extending a spectrin repeat unit. II: rupture experiments on three different material systems.
behavior. Biophys. J. 90, 101–111 (2006). 86. Elosegui-Artola, A. et al. Mechanical regulation of a molecular clutch defines force
57. Takahashi, H., Rico, F., Chipot, C. & Scheuring, S. α-Helix unwinding as force buffer in transmission and transduction in response to matrix rigidity. Nat. Cell Biol. 18,
spectrins. ACS Nano 12, 2719–2727 (2018). 540–548 (2016).
58. Block, J. et al. Viscoelastic properties of vimentin originate from nonequilibrium 87. Bennett, M. et al. Molecular clutch drives cell response to surface viscosity. Proc. Natl
conformational changes. Sci. Adv. 4, eaat1161 (2018). Acad. Sci. USA 115, 1192–1197 (2018).
59. Oftadeh, R., Connizzo, B. K., Nia, H. T., Ortiz, C. & Grodzinsky, A. J. Biological connective 88. Baker, B. M. & Chen, C. S. Deconstructing the third dimension: how 3D culture
tissues exhibit viscoelastic and poroelastic behavior at different frequency regimes: microenvironments alter cellular cues. J. Cell Sci. 125, 3015–3024 (2012).
application to tendon and skin biophysics. Acta Biomater. 70, 249–259 (2018). 89. Petersen, O. W., Ronnov-Jessen, L., Howlett, A. R. & Bissell, M. J. Interaction with
60. van Oosten, A. S. et al. Uncoupling shear and uniaxial elastic moduli of semiflexible basement membrane serves to rapidly distinguish growth and differentiation pattern of
biopolymer networks: compression-softening and stretch-stiffening. Sci. Rep. 6, 19270 normal and malignant human breast epithelial cells. Proc. Natl Acad. Sci. USA 89,
(2016). 9064–9068 (1992).

544 | Nature | Vol 584 | 27 August 2020

90. von der Mark, K., Gauss, V., von der Mark, H. & Muller, P. Relationship between cell shape 122. Darnell, M. et al. Substrate stress-relaxation regulates scaffold remodeling and bone
and type of collagen synthesised as chondrocytes lose their cartilage phenotype in formation in vivo. Adv. Healthc. Mater. 6, 1601185 (2017).
culture. Nature 267, 531–532 (1977). 123. Kolambkar, Y. M. et al. An alginate-based hybrid system for growth factor delivery in the
91. Gerecht, S. et al. Hyaluronic acid hydrogel for controlled self-renewal and differentiation functional repair of large bone defects. Biomaterials 32, 65–74 (2011).
of human embryonic stem cells. Proc. Natl Acad. Sci. USA 104, 11298–11303 (2007). 124. Kolambkar, Y. M. et al. Spatiotemporal delivery of bone morphogenetic protein enhances
92. Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011). functional repair of segmental bone defects. Bone 49, 485–492 (2011).
93. Lee, J. Y. et al. YAP-independent mechanotransduction drives breast cancer progression. 125. Lin, X. et al. A viscoelastic adhesive epicardial patch for treating myocardial infarction.
Nat. Commun. 10, 1848 (2019). Nat. Biomed. Eng. 3, 632–643 (2019).
94. Caliari, S. R., Vega, S. L., Kwon, M., Soulas, E. M. & Burdick, J. A. Dimensionality and 126. Ruvinov, E. & Cohen, S. Alginate biomaterial for the treatment of myocardial infarction:
spreading influence MSC YAP/TAZ signaling in hydrogel environments. Biomaterials 103, progress, translational strategies, and clinical outlook: from ocean algae to patient
314–323 (2016). bedside. Adv. Drug Deliv. Rev. 96, 54–76 (2016).
95. Tito Panciera, A. C. et al. Reprogramming normal cells into tumour precursors requires 127. Chhetri, D. K. & Mendelsohn, A. H. Hyaluronic acid for the treatment of vocal fold scars.
ECM stiffness and oncogenemediated changes of cell mechanical properties. Nat. Mater. Curr. Opin. Otolaryngol. Head Neck Surg. 18, 498–502 (2010).
19, 797–806 (2020). 128. Atala, A., Kim, W., Paige, K. T., Vacanti, C. A. & Retik, A. B. Endoscopic treatment of
96. Nam, S., Stowers, R., Lou, J., Xia, Y. & Chaudhuri, O. Varying PEG density to control stress vesicoureteral reflux with a chondrocyte-alginate suspension. J. Urol. 152, 641–643
relaxation in alginate-PEG hydrogels for 3D cell culture studies. Biomaterials 200, 15–24 (1994).
(2019). 129. Boekhoven, J. & Stupp, S. I. 25th anniversary article: supramolecular materials for
97. Lou, J., Stowers, R., Nam, S., Xia, Y. & Chaudhuri, O. Stress relaxing hyaluronic regenerative medicine. Adv. Mater. 26, 1642–1659 (2014).
acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion 130. Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell:
formation in 3D cell culture. Biomaterials 154, 213–222 (2018). mechanism and applications. Science 340, 1190–1194 (2013).
98. Nam, S. et al. Cell cycle progression in confining microenvironments is regulated by a 131. Shansky, J., Del Tatto, M., Chromiak, J. & Vandenburgh, H. A simplified method for tissue
growth-responsive TRPV4–PI3K/Akt-p27Kip1 signaling axis. Sci. Adv. 5, eaaw6171 (2019). engineering skeletal muscle organoids in vitro. In Vitro Cell. Dev. Biol. Anim. 33, 659–661
99. Nam, S. & Chaudhuri, O. Mitotic cells generate protrusive extracellular forces to divide in (1997).
three-dimensional microenvironments. Nat. Phys. 14, 621–628 (2018). 132. Balikov, D. A., Neal, E. H. & Lippmann, E. S. Organotypic neurovascular models: past
100. Darnell, M. et al. Material microenvironmental properties couple to induce distinct results and future directions. Trends Mol. Med. 26, 273–284 (2019).
transcriptional programs in mammalian stem cells. Proc. Natl Acad. Sci. USA 115, 133. Prior, N., Inacio, P. & Huch, M. Liver organoids: from basic research to therapeutic
E8368–E8377 (2018). applications. Gut 68, 2228–2237 (2019).
This work revealed that the transcriptional responses of cells in three-dimensional 134. Alsberg, E. et al. Regulating bone formation via controlled scaffold degradation. J. Dent.
culture to stress relaxation, matrix stiffness and adhesion ligand density exhibit Res. 82, 903–908 (2003).
substantial independent effects and coupling among these properties, demonstrating 135. Simmons, C. A., Alsberg, E., Hsiong, S., Kim, W. J. & Mooney, D. J. Dual growth factor
a clear cell type and context dependence of viscoelasticity sensing. delivery and controlled scaffold degradation enhance in vivo bone formation by
101. Madl, C. M. et al. Maintenance of neural progenitor cell stemness in 3D hydrogels transplanted bone marrow stromal cells. Bone 35, 562–569 (2004).
requires matrix remodelling. Nat. Mater. 16, 1233–1242 (2017). 136. Khetan, S. et al. Degradation-mediated cellular traction directs stem cell fate in
102. Lee, H. P., Gu, L., Mooney, D. J., Levenston, M. E. & Chaudhuri, O. Mechanical confinement covalently crosslinked three-dimensional hydrogels. Nat. Mater. 12, 458–465 (2013).
regulates cartilage matrix formation by chondrocytes. Nat. Mater. 16, 1243–1251 (2017). 137. Bryant, S. J. & Anseth, K. S. Hydrogel properties influence ECM production by
103. Mohammadi, H., Arora, P. D., Simmons, C. A., Janmey, P. A. & McCulloch, C. A. Inelastic chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. J. Biomed.
behaviour of collagen networks in cell-matrix interactions and mechanosensation. Mater. Res. 59, 63–72 (2002).
J. R. Soc. Interface 12, 20141074 (2015). 138. Loebel, C., Mauck, R. L. & Burdick, J. A. Local nascent protein deposition and remodelling
104. Kim, J. et al. Stress-induced plasticity of dynamic collagen networks. Nat. Commun. guide mesenchymal stromal cell mechanosensing and fate in three-dimensional
8, 842 (2017). hydrogels. Nat. Mater. 18, 883–891 (2019).
105. Liu, A. S. et al. Matrix viscoplasticity and its shielding by active mechanics in microtissue This study found that mesenchymal stem cells deposit matrix within a day of culture
models: experiments and mathematical modeling. Sci. Rep. 6, 33919 (2016). in proteolytically degradable covalently crosslinked or dynamically crosslinked
106. Wisdom, K. M. et al. Matrix mechanical plasticity regulates cancer cell migration through viscoelastic hyaluronic acid hydrogels, and that the deposited proteins mediated
confining microenvironments. Nat. Commun. 9, 4144 (2018). mechanotransduction.
This study demonstrated that mechanical plasticity in nanoporous matrices allows 139. Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature
protease-independent migration of cancer cells, with cells using invadopodial 539, 560–564 (2016).
protrusions to mechanically open up micrometre-size channels to migrate through. 140. Cruz-Acuna, R. et al. Synthetic hydrogels for human intestinal organoid generation and
107. Paul, C. D., Mistriotis, P. & Konstantopoulos, K. Cancer cell motility: lessons from colonic wound repair. Nat. Cell Biol. 19, 1326–1335 (2017).
migration in confined spaces. Nat. Rev. Cancer 17, 131–140 (2017). These studies (Gjorevski et al. (2016) and Cruz-Acuna et al. (2017)) demonstrated the
108. Caiazzo, M. et al. Defined three-dimensional microenvironments boost induction of use of synthetic covalently crosslinked hydrogels for organoid formation, and
pluripotency. Nat. Mater. 15, 344–352 (2016). identified gel degradability as an important design parameter.
109. Sabeh, F., Shimizu-Hirota, R. & Weiss, S. J. Protease-dependent versus -independent 141. Sadtler, K. et al. Divergent immune responses to synthetic and biological scaffolds.
cancer cell invasion programs: three-dimensional amoeboid movement revisited. Biomaterials 192, 405–415 (2019).
J. Cell Biol. 185, 11–19 (2009). 142. Ehrig, S. et al. Surface tension determines tissue shape and growth kinetics. Sci. Adv. 5,
110. Wolf, K. et al. Physical limits of cell migration: control by ECM space and nuclear eaav9394 (2019).
deformation and tuning by proteolysis and traction force. J. Cell Biol. 201, 1069–1084 143. Petersen, A. et al. A biomaterial with a channel-like pore architecture induces
(2013). endochondral healing of bone defects. Nat. Commun. 9, 4430 (2018).
111. Harada, T. et al. Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit 144. Jain, N. & Vogel, V. Spatial confinement downsizes the inflammatory response of
survival. J. Cell Biol. 204, 669–682 (2014). macrophages. Nat. Mater. 17, 1134–1144 (2018).
112. Schultz, K. M., Kyburz, K. A. & Anseth, K. S. Measuring dynamic cell-material interactions 145. Reimer, A. et al. Scalable topographies to support proliferation and Oct4 expression by
and remodeling during 3D human mesenchymal stem cell migration in hydrogels. human induced pluripotent stem cells. Sci. Rep. 6, 18948 (2016).
Proc. Natl Acad. Sci. USA 112, E3757–E3764 (2015). 146. Han, P. et al. Five piconewtons: the difference between osteogenic and adipogenic fate
This study examined how the viscoelastic properties of PEG hydrogels with degradable choice in human mesenchymal stem cells. ACS Nano 13, 11129–11143 (2019).
crosslinks were altered due to cellular degradation during migration of mesenchymal 147. Vining, K. H. & Mooney, D. J. Mechanical forces direct stem cell behaviour in development
stem cells and found that the cells converted the elastic hydrogel into a viscoelastic and regeneration. Nat. Rev. Mol. Cell Biol. 18, 728–742 (2017).
fluid. 148. Panciera, T., Azzolin, L., Cordenonsi, M. & Piccolo, S. Mechanobiology of YAP and TAZ in
113. Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface physiology and disease. Nat. Rev. Mol. Cell Biol. 18, 758–770 (2017).
to control stem-cell fate. Nat. Mater. 9, 518–526 (2010). 149. Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high
114. Lee, H. P., Stowers, R. & Chaudhuri, O. Volume expansion and TRPV4 activation regulate spatiotemporal resolution. Science 346, 1257998 (2014).
stem cell fate in three-dimensional microenvironments. Nat. Commun. 10, 529 (2019). 150. Grashoff, C. et al. Measuring mechanical tension across vinculin reveals regulation of
This study identified the role of cell volume expansion and activation of focal adhesion dynamics. Nature 466, 263–266 (2010).
mechanosensitive ion channels in mediating how mesenchymal stem cells sense 151. Rosales, A. M. & Anseth, K. S. The design of reversible hydrogels to capture extracellular
matrix viscoelasticity. matrix dynamics. Nat. Rev. Mater. 1, 15012 (2016).
115. Langer, R. & Vacanti, J. P. Tissue engineering. Science 260, 920–926 (1993). 152. Liu, A. P., Chaudhuri, O. & Parekh, S. H. New advances in probing cell-extracellular matrix
116. Huebsch, N. & Mooney, D. J. Inspiration and application in the evolution of biomaterials. interactions. Integr. Biol. 9, 383–405 (2017).
Nature 462, 426–432 (2009). 153. Vining, K. H., Stafford, A. & Mooney, D. J. Sequential modes of crosslinking tune
117. Grosskopf, A. K. et al. Viscoplastic matrix materials for embedded 3D printing. viscoelasticity of cell-instructive hydrogels. Biomaterials 188, 187–197 (2019).
ACS Appl. Mater. Interfaces 10, 23353–23361 (2018). 154. Baker, B. M. et al. Cell-mediated fibre recruitment drives extracellular matrix
118. Truby, R. L. & Lewis, J. A. Printing soft matter in three dimensions. Nature 540, 371–378 mechanosensing in engineered fibrillar microenvironments. Nat. Mater. 14, 1262–1268
(2016). (2015).
119. Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 155. Braunecker, W. A. & Matyjaszewski, K. Controlled/living radical polymerization: features,
(2016). developments, and perspectives. Prog. Polym. Sci. 32, 93–146 (2007).
120. Prantil-Baun, R. et al. Physiologically based pharmacokinetic and pharmacodynamic 156. Ong, L. L. et al. Programmable self-assembly of three-dimensional nanostructures from
analysis enabled by microfluidically linked organs-on-chips. Annu. Rev. Pharmacol. 10,000 unique components. Nature 552, 72–77 (2017).
Toxicol. 58, 37–64 (2018). 157. Liu, K. Z., Mihaila, S. M., Rowan, A., Oosterwijk, E. & Kouwer, P. H. J. Synthetic extracellular
121. Huebsch, N. et al. Matrix elasticity of void-forming hydrogels controls transplanted- matrices with nonlinear elasticity regulate cellular organization. Biomacromolecules 20,
stem-cell-mediated bone formation. Nat. Mater. 14, 1269–1277 (2015). 826–834 (2019).

Nature | Vol 584 | 27 August 2020 | 545

Review
158. Wang, Y. M. et al. Biomimetic strain-stiffening self-assembled hydrogels. Angew. Chem. 187. Töyräs, J., Nieminen, M. T., Kroger, H. & Jurvelin, J. S. Bone mineral density, ultrasound
132, 4860–4864 (2020). velocity, and broadband attenuation predict mechanical properties of trabecular bone
159. Wang, Y. F. et al. Architected lattices with adaptive energy absorption. Extreme Mech. differently. Bone 31, 503–507 (2002).
Lett. 33, 100557 (2019). 188. Isaksson, H. et al. Precision of nanoindentation protocols for measurement of
160. Davidson, M. D. et al. Mechanochemical adhesion and plasticity in multifiber hydrogel viscoelasticity in cortical and trabecular bone. J. Biomech. 43, 2410–2417 (2010).
networks. Adv. Mater. 32, 1905719 (2020). 189. Cowin, S. C., Van Buskirk, W. C. & Ashman, R. B. in Handbook of Bioengineering
161. Shivashankar, G. V. Mechanical regulation of genome architecture and cell-fate (McGraw-Hill, 1987).
decisions. Curr. Opin. Cell Biol. 56, 115–121 (2019). 190. Les, C. M. et al. Long-term ovariectomy decreases ovine compact bone viscoelasticity.
162. Shah, J. V. & Janmey, P. A. Strain hardening of fibrin gels and plasma clots. Rheol. Acta 36, J. Orthop. Res. 23, 869–876 (2005).
262–268 (1997). 191. Polly, B. J., Yuya, P. A., Akhter, M. P., Recker, R. R. & Turner, J. A. Intrinsic material properties
163. Chan, R. W. Measurements of vocal fold tissue viscoelasticity: approaching the male of trabecular bone by nanoindentation testing of biopsies taken from healthy women
phonatory frequency range. J. Acoust. Soc. Am. 115, 3161–3170 (2004). before and after menopause. Calcif. Tissue Int. 90, 286–293 (2012).
164. Nasseri, S., Bilston, L. E. & Phan-Thien, N. Viscoelastic properties of pig kidney in shear, 192. Abdel-Wahab, A. A., Alam, K. & Silberschmidt, V. V. Analysis of anisotropic viscoelastoplastic
experimental results and modeling. Rheol. Acta 41, 180–192 (2002). properties of cortical bone tissues. J. Mech. Behav. Biomed. Mater. 4, 807–820 (2011).
165. Hatami-Marbini, H. Viscoelastic shear properties of the corneal stroma. J. Biomech. 47, 193. Purslow, P. P., Wess, T. J. & Hukins, D. W. Collagen orientation and molecular spacing
723–728 (2014). during creep and stress-relaxation in soft connective tissues. J. Exp. Biol. 201, 135–142
166. Pereira, H. et al. Biomechanical and cellular segmental characterization of human (1998).
meniscus: building the basis for tissue engineering therapies. Osteoarthritis Cartilage 22, 194. Parada, G. A. & Zhao, X. H. Ideal reversible polymer networks. Soft Matter 14, 5186–5196
1271–1281 (2014). (2018).
167. Coluccino, L. et al. Anisotropy in the viscoelastic response of knee meniscus cartilage. 195. Tang, S. C. et al. Adaptable fast relaxing boronate-based hydrogels for probing
J. Appl. Biomater. Funct. Mater. 15, e77–e83 (2017). cell-matrix interactions. Adv. Sci. 5, 1800638 (2018).
168. Chaudhuri, O. et al. Extracellular matrix stiffness and composition jointly regulate the 196. Brown, T. E. et al. Photopolymerized dynamic hydrogels with tunable viscoelastic
induction of malignant phenotypes in mammary epithelium. Nat. Mater. 13, 970–978 (2014). properties through thioester exchange. Biomaterials 178, 496–503 (2018).
169. Jansen, L. E., Birch, N. P., Schiffman, J. D., Crosby, A. J. & Peyton, S. R. Mechanics of intact 197. Marozas, I. A., Anseth, K. S. & Cooper-White, J. J. Adaptable boronate ester hydrogels with
bone marrow. J. Mech. Behav. Biomed. Mater. 50, 299–307 (2015). tunable viscoelastic spectra to probe timescale dependent mechanotransduction.
170. Suki, B. & Lutchen, K. R. in Wiley Encyclopedia of Biomedical Engineering (Wiley, 2006). Biomaterials 223, 119430 (2019).
171. Holt, B., Tripathi, A. & Morgan, J. Viscoelastic response of human skin to low magnitude 198. Zhao, X. H., Huebsch, N., Mooney, D. J. & Suo, Z. G. Stress-relaxation behavior in gels with
physiologically relevant shear. J. Biomech. 41, 2689–2695 (2008). ionic and covalent crosslinks. J. Appl. Phys. 107, 063509 (2010).
172. Barnes, S. C. et al. Viscoelastic properties of human bladder tumours. J. Mech. Behav. 199. Dooling, L. J., Buck, M. E., Zhang, W. B. & Tirrell, D. A. Programming molecular association
Biomed. Mater. 61, 250–257 (2016). and viscoelastic behavior in protein networks. Adv. Mater. 28, 4651–4657 (2016).
173. Kiss, M. Z., Varghese, T. & Hall, T. J. Viscoelastic characterization of in vitro canine tissue. 200. Richardson, B. M., Wilcox, D. G., Randolph, M. A. & Anseth, K. S. Hydrazone covalent
Phys. Med. Biol. 49, 4207–4218 (2004). adaptable networks modulate extracellular matrix deposition for cartilage tissue
174. Klatt, D. et al. Viscoelastic properties of liver measured by oscillatory rheometry and engineering. Acta Biomater. 83, 71–82 (2019).
multifrequency magnetic resonance elastography. Biorheology 47, 133–141 (2010).
175. Nicolle, S. & Palierne, J. F. Dehydration effect on the mechanical behaviour of biological
soft tissues: observations on kidney tissues. J. Mech. Behav. Biomed. Mater. 3, 630–635 Acknowledgements O.C. acknowledges support from a National Institutes of Health National
(2010). Cancer Institute grant (R37 CA214136), a National Science Foundation CAREER award (CMMI
176. Nicolle, S., Lounis, M., Willinger, R. & Palierne, J. F. Shear linear behavior of brain tissue 1846367), and an American Cancer Society Research Scholar Grant (RSG-16-028-01). J.C.-W.
over a large frequency range. Biorheology 42, 209–223 (2005). acknowledges support from the Australian Research Council Discovery Grants Scheme
177. Hrapko, M., van Dommelen, J. A., Peters, G. W. & Wismans, J. S. The mechanical behaviour (DP190101969). P.A.J. acknowledges NIH awards EB017753, GM136259 and CA193417 and the
of brain tissue: large strain response and constitutive modelling. Biorheology 43, 623–636 Penn Materials Research Science and Engineering Center (DMR-1720530). D.J.M.
(2006). acknowledges support from the NIH (R01 DE013033, U01CA214369) and the Harvard
178. Netti, P., D’amore, A., Ronca, D., Ambrosio, L. & Nicolais, L. Structure-mechanical University Materials Research Science and Engineering Center (grant DMR-1420570). V.B.S.
properties relationship of natural tendons and ligaments. J. Mater. Sci. Mater. Med. 7, acknowledges NIH awards R01EB017753, U01CA202177, U54CA193417 and R01CA232256 and
525–530 (1996). the NSF Center for Engineering Mechanobiology (CMMI-154857).
179. Tanaka, E. et al. Dynamic shear properties of the porcine molar periodontal ligament.
J. Biomech. 40, 1477–1483 (2007). Author contributions All authors contributed to planning, writing, and editing of the
180. Tanaka, E. et al. Comparison of dynamic shear properties of the porcine molar and incisor manuscript.
periodontal ligament. Ann. Biomed. Eng. 34, 1917–1923 (2006).
181. Troyer, K. L. & Puttlitz, C. M. Human cervical spine ligaments exhibit fully nonlinear Competing interests The authors declare no competing interests.
viscoelastic behavior. Acta Biomater. 7, 700–709 (2011).
182. Fessel, G. & Snedeker, J. G. Evidence against proteoglycan mediated collagen fibril load Additional information
transmission and dynamic viscoelasticity in tendon. Matrix Biol. 28, 503–510 (2009). Correspondence and requests for materials should be addressed to O.C., J.C.-W., P.A.J., D.J.M.
183. Nagasawa, K., Noguchi, M., Ikoma, K. & Kubo, T. Static and dynamic biomechanical or V.B.S.
properties of the regenerating rabbit Achilles tendon. Clin. Biomech. 23, 832–838 (2008). Peer review information Nature thanks Sanjay Kumar, Cynthia Reinhart-King, Pere
184. Koolstra, J. H., Tanaka, E. & Van Eijden, T. M. Viscoelastic material model for the Roca-Cusachs and the other, anonymous, reviewer(s) for their contribution to the peer review
temporomandibular joint disc derived from dynamic shear tests or strain-relaxation of this work.
tests. J. Biomech. 40, 2330–2334 (2007). Reprints and permissions information is available at http://www.nature.com/reprints.
185. Tanaka, E. et al. Shear properties of the temporomandibular joint disc in relation to Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
compressive and shear strain. J. Dent. Res. 83, 476–479 (2004). published maps and institutional affiliations.
186. Tanaka, E. et al. Dynamic shear behavior of mandibular condylar cartilage is dependent
on testing direction. J. Biomech. 41, 1119–1123 (2008). © Springer Nature Limited 2020

546 | Nature | Vol 584 | 27 August 2020