Annals of Biomedical Engineering, Vol. 31, pp. 1–11, 2003
Printed in the USA. All rights reserved.
0090-6964/2003/31共1兲/1/11/$20.00
Copyright © 2003 Biomedical Engineering Society
Principles of Cell Mechanics for Cartilage Tissue Engineering
ADRIAN C. SHIEH and KYRIACOS A. ATHANASIOU
Department of Bioengineering, Rice University, Houston, TX
(Received 28 September 2002; accepted 12 November 2002)
lage extracellular matrix 共ECM兲, and may even enhance
the mechanical properties of the developing tissue. While
these investigations hint at the great promise in cartilage
tissue engineering, they also reveal a fundamental lack of
understanding of what mechanical stimuli are necessary
to promote regeneration and how those mechanical signals are interpreted at the cellular level. There is still no
consensus as to the type of mechanical signals that are
most crucial or effective in modulating cell function, or
even what levels of force are appropriate. A concrete
grasp of these issues is critical to creating a cogent approach to engineering functional cartilage. At the heart of
these issues is the need to paint a clearer picture of the
biomechanical environment of the single chondrocyte. To
attain this goal, a better grasp of the mechanical properties of the cell, and their relationship to the local microenvironment and mechanisms of mechanotransduction, is crucial.
The objectives of this paper are to 共1兲 provide a brief
overview of studies that illustrate the keen importance of
mechanical signals to cartilage regeneration, 共2兲 review
salient results concerning the mechanical and mechanotransductive behaviors of chondrocytes, and 共3兲 establish a framework to integrate relevant aspects of cell
mechanics into a strategy for cartilage tissue engineering.
Abstract—The critical importance of mechanical signals to the
health and maintenance of articular cartilage has been well
demonstrated. Tissue engineers have taken a cue from normal
cartilage physiology and incorporated the use of mechanical
stimulation into their attempts to engineer functional cartilage.
However, the specific types of mechanical stimulation that are
most beneficial, and the mechanisms that allow a chondrocyte
to perceive and respond to those forces, have yet to be elucidated. To develop a better understanding of these processes, it
is necessary to examine the mechanical behavior of the single
chondrocyte. This paper reviews salient topics related to chondrocyte biomechanics and mechanotransduction, and attempts
to put this information into a context both appropriate and
useful to cartilage tissue engineering. It also describes the directions this exciting field is taking, and lays out a vision for
future studies that could have a significant impact on our understanding of cartilage health and disease. © 2003 Biomedical Engineering Society. 关DOI: 10.1114/1.1535415兴
Keywords—Biomechanics, Chondrocytes, Functional tissue
engineering, Mechanobiology, Mechanotransduction.
INTRODUCTION
Regeneration of functional articular cartilage is simultaneously one of the most tantalizing prospects and intimidating challenges facing bioengineers today. The potential benefits of cartilage regeneration and replacement
therapies are enormous. Tens of millions of Americans
suffer from acute trauma to musculoskeletal tissues as
well as various degenerative cartilage conditions. Currently patients have little recourse beyond surgical techniques that ameliorate the symptoms of disease or restore
some function to damaged tissue, but cannot otherwise
address the issue of healing articular cartilage. It is the
lack of normal healing that makes engineering cartilage
such a difficult task. The dearth of intrinsic regenerative
capacity means that bioengineers must find alternative
ways of stimulating cartilage growth.
Among the most potent modulators of cartilage regeneration are mechanical signals. Many studies have shown
that mechanical forces stimulate the synthesis of carti-
ROLE OF MECHANICAL FORCES IN
CARTILAGE REGENERATION
Overview
There is ample evidence for the importance of mechanical forces in facilitating articular cartilage regeneration. Studies employing cartilage tissue explants, chondrocytes grown in monolayers, and cells seeded on
scaffolds or in hydrogels have shown that, in general,
low to moderate magnitude loads applied at frequencies
on the order of 1 Hz substantially enhance the expression
and synthesis of matrix proteins, though there remains no
widespread agreement on these conditions. A variety of
mechanical forces has been examined, but the majority
of the work centers around compression and hydrostatic
pressure.
Address correspondence to Dr. Kyriacos Athanasiou, Department of
Bioengineering, Rice University, MS-142, P. O. Box 1892, Houston,
TX 77521-1892. Electronic mail: [email protected]
1
2
A. C. SHIEH and K. A. ATHANASIOU
Explant Studies
Cartilage explant studies have provided a wealth of
information relating to the mechanical regulation of cartilage metabolism.3,9,29,33,48 –50 These studies are invaluable because they provide a window through which the
behavior of chondrocytes in their native ECM can be
studied. However, explant studies carry the problem that
the mechanical environment of the cell is exceedingly
difficult to characterize, and numerous other factors are
brought into play by the presence of an intact cartilage
ECM. Nonetheless, much of what is known about the
biomechanical regulation of cartilage and chondrocyte
metabolism comes from observing cartilage explants under load.
A seminal study by Sah and associates50 identified
high- and low-frequency regimens with different stimulatory effects on explants. Low amplitude compression at
frequencies from 0.01 to 1 Hz increased collagen and
proteoglycan synthesis, while much larger amplitudes
were necessary to stimulate biosynthesis in response to
lower frequency compression.50 These results are significant, because they established a frequency window between 0.01 and 1 Hz that many subsequent studies have
focused on to examine the effects of dynamic compression. Kim and associates33 performed similar dynamic
compression experiments on cartilage explants, and concluded that fluid flow and changes in cell shape are
likely at the root of changes in chondrocyte biosynthesis,
up to stresses of approximately 0.5 MPa. Parkkinen and
associates49 similarly found that frequencies on the order
of 0.1 Hz had stimulatory effects. Investigations by Bonassar and associates3 found that dynamic compression
of 2%–3% at 0.1 Hz, in combination with IGF-I, synergistically stimulated a 180% increase in protein synthesis, and a 290% increase in proteoglycan production. The
reasons for this synergism are not known, but some suggestion has been made that transport and accessibility of
growth factors is improved by cyclic compression.3,4
The effects of other mechanical stimuli on cartilage
explants have been pursued, though to a lesser extent
than compression.29,48 Parkkinen and associates48 applied
cyclic hydrostatic pressure to cartilage explants, and
found that sulfate incorporation increased 17% above
control levels in tissue samples exposed to 5 MPa of
pressure at 0.5 Hz. Frequencies of 0.0167–0.25 Hz, in
contrast, had no measurable effect.48 Another loading
regimen, tissue shear, was studied by Jin and
associates.29 Explants exposed to 1%–3% shear strain at
frequencies of 0.01–1 Hz exhibited increases in protein
and proteoglycans synthesis of 50% and 25%,
respectively.29
Monolayer Studies
While cartilage explant studies are integral to understanding normal cartilage homeostasis, they do not nec-
essarily reflect the environments experienced by cells in
in vitro culture. Most cartilage engineering strategies are
ex vivo approaches, using cells removed from their native ECM. Thus, studying cells in monolayer culture can
be a useful approach, because the conditions experienced
by chondrocytes are better defined and can be controlled
directly.
Smith and associates55 placed articular chondrocytes
in culture and applied fluid shear using a cone viscometer. The synthesis of glycosaminoglycans 共GAGs兲 increased by 100%; however, this increase was concomitant with a 10–20-fold increase in prostanglandin E2, a
known inflammatory mediator.55 This suggests that fluid
flow-induced shear may have both positive and negative
effects, necessitating further study. Hydrostatic pressure,
on the other hand, seems to provide a positive mechanical stimulus for chondrocytes.27,48,56,57 Parkkinen and
associates48 observed that 5 MPa of pressure applied at a
frequency of 0.25–0.5 Hz for 20 h increased sulfate
incorporation, while lower frequencies or shorter durations 共1.5 h兲 inhibited incorporation. Smith and
associates56,57 have demonstrated in two separate studies
that intermittent hydrostatic pressure can have significant
effects on aggrecan and type II collagen message.
Changes in message were very time dependent, and varied considerably with different loading regimens.56 Intermittent pressure also seemed to stimulate glycosaminoglycan synthesis, and in general was more beneficial
than constant pressure.57
Three-Dimensional Culture Studies
Situated between explant studies and cells cultured in
monolayers are investigations of mechanically stimulated
three-dimensional 共3D兲 constructs. These studies are perhaps the most relevant, because they reflect how chondrocytes seeded into or on various scaffolds respond to
mechanical forces. A variety of scaffolds have been used
to culture chondrocytes, but the majority of the work
involving mechanical stimulation of seeded matrices involves natural hydrogels, i.e., agarose and alginate, and
poly共glycolic acid兲 共PGA兲.
Buschmann and associates7 seeded chondrocytes in
agarose disks and subjected the constructs to varying
levels of compression, over a range of frequencies. Dynamic strain of 3% at frequencies of 0.01–1 Hz stimulated biosynthetic activity, resulting in the deposition of
matrix in the neighborhood of cells.7 Lee and
associates42 examined the responses of superficial and
deep zone chondrocytes seeded in agarose to dynamic
compression. Interestingly, 1 Hz dynamic compression
stimulated GAG synthesis in deep cells, whereas dynamic strain had an inhibitory effect on superficial cells.
However, superficial cells did respond to dynamic compression by an increase in proliferation, an effect not
Cell Mechanics for Cartilage Engineering
seen in the deep zone chondrocytes.42 In a significant
study performed by Mauck and associates,44 dynamic
compression at a frequency of 1 Hz and an amplitude of
10% caused a substantial increase in equilibrium modulus over unstimulated controls, from 15 to 100 kPa.
GAG and hydroxyproline content also increased in response to compression.44
Carver and Heath10 demonstrated that intermittent
pressure—applied by pressurization of the gas phase—
stimulated immature and adult equine chondrocytes
seeded on PGA meshes to increase GAG synthesis. Additionally, if sufficient pressure was applied, collagen
content also increased in the PGA constructs.10 Carver
and Heath11 further demonstrated the stimulatory effect
of pressurizing the constructs, and also found that an
initial culture period in spinner flasks improved the quality of the constructs, compared to cell-seeded scaffolds
exposed only to pressure or mixing in a spinner flask.
Vunjak-Novakovic and associates60 demonstrated that
chondrocytes seeded onto PGA scaffolds and cultured in
a rotating wall bioreactor exhibited mechanical properties
and biochemical compositions superior to static and
mixed flask cultures. They hypothesized that the superior
properties of the bioreactor-cultivated constructs were in
part a result of the hydrodynamic environment in the
rotating wall vessels.60 Gooth and associates15 cultured
calf articular chondrocytes on PGA scaffolds in static
and mixed flasks, and in a rotating wall vessel, with and
without IGF-I. They found that the mechanical environment and IGF-I independently modulated the morphology, composition, and mechanical properties of the cartilage constructs, and that, when used together, yielded
superior tissue.15
MECHANISMS FOR MECHANOTRANSDUCTION
IN CHONDROCYTES
3
pression appear to be connected to changes in interstitial
pH.16 Osmotic stress has been shown to change the mechanical properties of chondrocytes,19 which in turn alters how much the cell deforms under a given load.
Electrical potentials have also been identified as possible
regulators of chondrocytes function.32,38 For the most
part, these physicochemical changes act as transducers,
changing a mechanical event, e.g., compression, into
stimuli that the cell can interpret directly, e.g., osmotic
pressure.
Ion Pumps and Channels
The flow of ions into and out of the cell is a common
upstream event in signal transduction. Stretch-activated
Ca2⫹ channels in the plasma membrane have been implicated
in
several
studies
of
chondrocyte
mechanotransduction.12,66 Calcium is a ubiquitious second messenger in cells, so its possible role in chondrocyte mechanosensing is not surprising. In these cases,
mechanical stimulation resulted in intracellular Ca2⫹
waves, which were inhibited by gadolinium, a known
blocker of stretch-activated channels.12,66
Pressure also seems to have an effect on various ion
transport pathways. Wright and associates63 showed that
pressurization of the gas phase above cells in culture to
16 kPa induced membrane depolarization in chondrocytes, a response that can be abolished by tetrodotoxin, a
Na⫹ channel blocker. Browning and associates6 showed
that H⫹ efflux mediated by the Na⫹/H⫹ exchanger increased in response to 30 MPa of hydrostatic pressure,
and that the change in exchanger activity may have involved phosphorylation of the transport protein. A study
by Hall26 demonstrated that hydrostatic pressure can inhibit the Na⫹/K⫹ pump, the Na⫹/K⫹/Cl⫺ cotransporter,
and even basal membrane K⫹ permeability.
Overview
Integrins
While the influence of mechanical forces on cartilage
and chondrocyte metabolism has been extensively documented, the machinery by which cells sense these mechanical signals and effect change remains poorly understood. A number of mechanisms have been proposed, and
it is likely that most of these are involved, in some way
or another, in the transduction of mechanical forces to
biochemical signals in chondrocytes.
Integrins are widely recognized as important mediators of cell-matrix interactions. As the primary bridge
between the ECM and the actin cytoskeleton, integrins
are uniquely positioned to act as cellular mechanosensors. Work by Wright and associates62 implicated the
␣ 5 ␤ 1 integrin, as well as associated downstream components such as phosphalipase C, the actin cytoskeleton,
and various kinases, in the hyperpolarization response of
chondrocytes to mechanical strain. To explain this phenomenon, a novel integrin-regulated interleukin-4 共IL-4兲
secretion pathway has been proposed as means for chondrocyte mechanotransduction.43,45,46 A thorough treatment of this research and putative pathway can be found
in a review by Salter and associates.51 Briefly, mechanical stimulation results in Ca2⫹ influx through stretchactivated channels and a cascade of signal transduction
events via integrin receptors. These two pathways both
Physicochemical Effects
Deformation of cartilage precipitates a host of
changes in the matrix environment around cells. Interstitial fluid flow, electrical potentials, increased osmotic
pressure, and decreased pH are some of the effects generated during cartilage loading.16,19 Changes in proteoglycan and collagen metabolism caused by static com-
4
A. C. SHIEH and K. A. ATHANASIOU
precipitate secretion of IL-4. IL-4 then acts, either in an
autocrine or paracrine manner, by binding to the IL-4
receptor. Binding of the IL-4 receptor causes a series of
intracellular events that lead to the opening of specific
potassium channels, resulting in hyperpolarization. The
IL-4 receptor may also be connected to pathways that
lead to changes at the gene expression level.51
forces can induce deformations resulting in changes in
cell behavior, the biomechanical characteristics of the
cell must be accurately quantified. A number of studies
have endeavored to qualitatively and quantitatively
determine the mechanical properties of single
chondrocytes.
In Situ Experiments
Cytoskeleton
As one might expect, the cytoskeleton is a potential
force transducer in cells.13,28,31,39,41 Because of its structural role in the cell, the cytoskeleton can undergo numerous changes under load, including deformation, reorganization, assembly, and disassembly. The levels of
actin, vimentin, and tubulin vary from zone to zone in
cartilage, which may be correlated to the different load
environments in each zone.28,39 Another study found that
vimentin filaments changed in response to swelling pressure in explant culture.13 Chondrocytes seeded in agarose
exhibited temporal changes in the organization of their
cytoskeletons, which seemed to affect how the nucleus
deformed under load.41 Treatment of chondrocytes with
taxol, a stabilizer of microtubules, or nocodazole, a microtubule disrupter, significantly inhibited the stimulation
of proteoglycan synthesis resulting from cyclic hydrostatic pressure.31 The cytoskeleton may also be involved
in signal transduction pathways via its connection to
integrin receptors, as discussed earlier.
Nuclear Deformation
One of the most direct mechanisms for transducing
mechanical signals into changes in gene expression may
involve deformation of nucleus. Deformation of the
nucleus may physically alter the nuclear pore complex,
or result in changes in the accessibility of genomic DNA
for transcription. Changes in nuclear dimensions and volume have been previously documented in deformed cartilage explants.17 It has also been suggested that changes
in aggrecan biosynthesis are correlated to changes in
nuclear structure.8 Guilak and associates24 used micropipette aspiration to demonstrate that the nucleus is viscoelastic in nature, and is substantially stiffer than the
cell as a whole. These results point to a possible role for
nuclear strain in chondrocyte mechanotransduction.
MECHANICAL BEHAVIOR OF CHONDROCYTES
Overview
If one considers the mechanisms described above, it
becomes apparent that cellular deformation is an essential component for at least three putative mechanosensors: stretch-activated ion channels, the cytoskeleton, and
nuclear deformation. To properly understand the stress–
strain environment of a chondrocyte, and how certain
The logical place to start when considering the mechanical properties of chondrocytes is in the tissue itself.
To this end, confocal laser scanning microscopy has been
used to measure the deformation of chondrocytes within
cartilage explants.17,22 With this technique, threedimensional reconstructed images of chondrocytes can be
produced both before and during compression of the
tissue explant. The resultant cell deformation can provide
qualitative descriptors of chondrocyte mechanical behavior and insight into some of the mechanisms involved in
mechanotransduction. Guilak17 applied 15% surface-tosurface strain to canine cartilage explants, and measured
changes in cell and nuclear height and volume. Of particular interest is that, while the height of the chondrocytes decreased 14.7%, the nuclei of the cells only deformed 8.8%. When the explants were treated with
cytochalasin D to disrupt microfilaments, very little
nuclear deformation 共2.2%兲 was observed. These results
showed that deformation of the cell is transmitted into
intracellular compartments, and suggest that the cytoskeleton, in particular the actin filaments, are responsible
for transferring force across the membrane to the
nucleus. Another study using confocal microscopy by
Guilak and associates22 showed that changes in cell
height resulting from 15% tissue compression varied
from 26% in the superficial zone to 19% and 20% in the
middle and deep zones, respectively. The implication of
these findings is that tissue mechanical properties vary
with depth, which is known to be true,53 or that the
mechanical properties of the cells themselves may differ,
or both.
Theoretical Models
Attempts have been made to develop models that can
accurately describe the deformation behavior of chondrocytes in cartilage.1,21,64,65 Bachrach and associates1 modeled chondrocytes as solid–fluid mixture inclusions
within a biphasic matrix. One of the important observations made in this study is that differences in the material
properties of the cell and the neighboring ECM have a
profound influence on the mechanical environment in
and around the cell.1 Wu and associates65 added an additional level of complexity, by taking into account the
contribution of the cells to the overall mechanical behavior of cartilage. The system was modeled using a cell
surrounded by matrix, which is, in turn, surrounded by a
Cell Mechanics for Cartilage Engineering
macroscopically homogenized matrix representing the remaining cartilage matrix, which includes other cells.64 A
later model by Wu and Herzog,64 based upon the study
by Wu and associates,65 showed that the position of a
given chondrocyte within cartilage can substantially affect its loading environment during unconfined compression. Interestingly, this model predicts that the deformation of individual chondrocytes may be three to four
times greater than the deformation of the macroscopic
matrix, and that the cells do not reach equilibrium for as
long as 20 min after the onset of compression.64 Guilak
and Mow21 used a multiscale finite element approach,
modeling the cell, its pericellular matrix 共PCM兲, and the
cartilage ECM as biphasic materials, all with distinct
material properties. The inclusion of the PCM layer, distinct from the ECM and separating the cell from the rest
of the cartilage matrix, was an important addition in this
model. The relative properties of the cell and PCM had a
strong effect on the peak stresses and strains experienced
by the modeled cell.21 Wang and associates61 used a
sophisticated triphasic model of cartilage, coupled with
depth-dependent parameters for the tissue stiffness and
fixed charge density, to predict the stress–strain environment, electrical potential, and fluid and osmotic pressures
throughout the cartilage matrix. This model could ostensibly be used to describe the local environment of the
chondrocyte.61 More recently, a model was developed by
Breuls and associates5 to predict cell deformations within
engineered tissues, using approaches that differ from the
previously discussed models. In all cases, computational
tools have been developed to predict the stress–strain
environment of chondrocytes embedded in a matrix.
However, in order for these models to have true predictive value, concrete knowledge of the material properties
of the matrix and the cells is required.
In Vitro Experiments
A number of studies have employed chondrocytes embedded in agarose as a system to study deformation of
cells within a matrix.34,35,41 Lee and associates41 examined both cellular and nuclear deformations in chondrocytes seeded in agarose. Their results corroborated the
findings from the previously discussed confocal microscopy studies, where compression induced deformation of
both the chondrocyte and the nucleus, suggesting a possible mechanism for signal transduction. Knight and
associates,34 using the same chondrocyte–agarose system, investigated the effects of an elaborated PCM on
cell deformation. As expected, the presence of a PCM
significantly reduced cell deformation as more matrix
material was deposited.34 More recently, Knight and
associates35 compared the deformation of mechanically
isolated chondrons 共chondrocyte surrounded by its PCM兲
to isolated chondrocytes and enzymatically released
5
FIGURE 1. Illustration of the three direct techniques currently used to characterize the biomechanical characteristics
of chondrocytes: „A… micropipette aspiration; „B… cytoindentation; and „C… atomic force microscopy.
chondrons. Compression of the isolated cells and enzymatically isolated chondrons in agarose resulted in cell
deformation, whereas cells in mechanically isolated
chondrons did not deform.35 While these studies do provide valuable insight into the deformation of chondrocytes seeded in gels, they provide only qualitative
descriptors.
Quantitative mechanical assessments of chondrocytes
in vitro have been previously performed, but remain limited in number and in scope.14,19,20,30,36,37,59 As shown by
Guilak and Mow,21 the properties of the cell and its
associated PCM can profoundly influence the loads and
deformations experienced at the microscopic level, such
that they can differ greatly from the macroscopic loading
environment. Therefore, accurately quantifying the material properties of chondrocytes is of paramount importance both in expanding the current understanding of
cartilage biomechanics as well as providing new avenues
to manipulate and enhance cell function to promote tissue regeneration. It is important to note, however, that
the testing methodology used and the models applied to
derive the material properties of chondrocytes can have a
substantial effect on the results. This is exemplified in a
comparison made by Bader and associates,2 where three
different techniques yielded results that could have substantially different interpretations. To date, the testing
methods used include compression of cell-seeded gels,
micropipette aspiration, cytoindentation, and atomic
force microscopy 共Fig. 1兲.
Measurements of cell deformation within compressed
gels have been used to indirectly determine the material
properties of chondrocytes.2,14,36 Freeman and
associates14 used a linear elastic finite element model to
determine the properties of rat Swarm chondrosarcoma
cells embedded in a statically compressed 2% agarose
6
A. C. SHIEH and K. A. ATHANASIOU
gel. By comparing the deformation of individual cells
with the bulk gel, they estimated the Young’s modulus
and Poisson’s ratio of the cells to be 4.0 kPa and 0.4,
respectively.14 In a similar study by Knight and
associates,36 the Young’s modulus of chondrocytes from
the metacarpal–phalangeal joint of 18 month old steers
seeded in alginate gels was found to be 3.2⫾0.5 kPa.
Bader and associates2 also tested bovine chondrocytes in
agarose gels, and indirectly calculated a cell Young’s
modulus of 2.7 kPa.
The majority of the information available concerning
the properties of chondrocytes has been obtained using
the micropipette aspiration technique.19,20,30,59 The technique has been used to assess changes in chondron and
chondrocyte properties as a result of osteoarthirtis.20,30,59
Cells from osteoarthritic human cartilage in general exhibit substantially different volumetric properties, increased stiffness, and increased viscosity.30,59 In contrast,
enzymatically isolated chondrons from osteoarthritic cartilage have a lower Young’s modulus than normal
chondrons.20 The effects of osmotic stress on the viscoelastic properties of chondrocytes have also been studied by Guilak and associates.19 Hypoosmotic 共but not
hyperosmotic兲 conditions induced significant decreases in
the instantaneous modulus, equilibrium modulus, and
viscosity of porcine chondrocytes.19 In general, micropipette aspiration experiments have demonstrated that
chondrocytes have a mean Young’s modulus on the order
of 0.1 kPa and a mean viscosity on the order of 1 kPa s,
while enzymatically isolated chondrons have a modulus
on the order of 1 kPa. More recently, there is evidence
that mechanically isolated chondrons are substantially
stiffer than chondrocytes or enzymatically isolated chondrons, with a Young’s modulus of ⬃25 kPa 共27.1⫾16.3
kPa from the superficial zone, 23.3⫾7.5 kPa in the
middle/deep zones兲.18 Because enzymatic degradation of
the PCM likely reduces the mechanical integrity of the
chondron, the modulus of mechanically isolated chondrons is probably more indicative of the matrix properties in vivo.
An alternative method for biomechanically characterizing single adherent cells called cytoindentation has
been developed by our laboratory.37,54 Tests performed
on bovine chondrocytes yielded an instantaneous modulus 共8.00⫾4.41 kPa兲 and relaxed modulus 共1.09⫾0.54
kPa兲 that are approximately one order of magnitude
greater than values obtained from micropipette aspiration
experiments.37 A summary of the mechanical properties
of chondrocytes obtained using various methods is presented in Table 1.
Conclusions
The mechanics of chondrocytes remains a fertile area
for research. The current literature provides a glimpse
into the properties of chondrocytes, but the influence of
many different environmental factors on these properties
must still be examined. It is also abundantly clear that
the testing parameters can have a serious influence on
the properties obtained. For example, Bader and
associates2 observed that the Young’s modulus for single
chondrocytes was strongly dependent on the internal diameter of the micropipette. The results of studies employing the cell-seeded gel compression technique2,14,36
or cytoindentation37 yield moduli that are an order of
magnitude higher than studies using micropipette
aspiration.2,19,30,59 An excellent editorial by Guilak and
associates23 outlines the difficulties in comparing cell
mechanics studies, and emphasizes the need to explicitly
state every testing condition, to aid in the interpretation
of the final results.
The field of single chondrocyte mechanics has
reached a critical point that requires several questions be
answered. The most obvious question is how different
conditions, such as the addition of cytokines, using cells
from different zones, or seeding cells onto various biomaterials, affect the mechanical properties of chondrocytes. It will also be important to determine how the
single chondrocyte changes its properties in response to
different conditions, e.g., via remodeling of the cytoskeleton, or modulating the expression of cytoskeletonassociated proteins. However, before addressing these
highly significant issues, more fundamental challenges
involving the analytical or computational methods used
to obtain cell biomechanical properties must be resolved.
As Bader and associates2 have so clearly demonstrated,
the testing methodology is a major determinant of material properties, as much so as any biological or physicochemical factor. For example, the differences in the results of Koay and associates,37 compared to those
obtained via micropipette aspiration,2,19,30,59 are likely
the consequence of several differences in their respective
experimental protocols. Cytoindentation experiments
were performed on adherent chondrocytes, as opposed to
the free floating cells tested using micropipette aspiration. Cell attachment and adhesion initiates a series of
intracellular events that include the reorganization of the
cytoskeleton.40 Thus, it is reasonable to expect that the
stiffness of adherent cells would be substantially greater.
Cytoindentation and micropipette aspiration tests also apply fundamentally different types of stresses. Whereas
micropipette aspiration applies a tensile stress, cytoindentation results in primarily compressive loads. These
distinct loading regimens could result in the observed
differences in the material properties, as the mechanical
contribution of various subcellular structural elements,
e.g., the nucleus and cytoskeleton, could vary under conditions of tension and compression. Clearly, serious investigations of different testing methods, their advantages, and their caveats must be performed so that the
Cell Mechanics for Cartilage Engineering
7
TABLE 1. Summary of published values for chondrocyte mechanical properties.
Author
Cell Type/Source
Technique
Model
Properties
Freeman et al.
(1994)
Swarm rat chondrosarcoma
cells
Compression of
cell-seeded gels
Elastic finite element
E⫽4.0 kPa
␯⫽0.4
Jones et al.
(1999)
Human articular
chondrocytes, adult (37–83
yr), knees, hips, ankles, and
elbows
Micropipette
aspiration
Elastic half space
E⫽0.65 kPa
Guilak et al.
(1999)
Enzymatically isolated human
chondrons, from knees and
hips
Micropipette
aspiration
Elastic half space
E⫽1.54 kPa
Trickey et al.
(2000)
Human articular
chondrocytes, adult (26–86
yr), knees and hips
Micropipette
aspiration
Viscoelastic half space
E0⫽0.41 kPa
E⬁ ⫽0.24 kPa
␮⫽3.0 kPa s
Knight et al.
(2002)
Bovine articular
chondrocytes, 18 month old
steers, metacarpal-phalangeal
joint
Compression of
cell-seeded gels
Linear fit model
E⫽3.2 kPa
Bader et al.
(2002)
Bovine articular
chondrocytes, mature steers,
metacarpal-phalangeal joint
Compression of
cell-seeded gels
E⫽␴/⑀
E⫽2.7 kPa
Koay et al.
(2002)
Bovine articular
chondrocytes, adult, distal
portion of 1st metatarsal
Cytoindentation
Elastic half space
E⫽1.10 kPa
Viscoelastic half space
E0⫽8.00 kPa
E⬁⫽1.09 kPa
␮⫽1.50 kPa s
Layered elastic half space
E⫽27.1 kPa
(superficial)
Guilak et al.
(2002)
Mechanically isolated canine
chondrons, mature, femoral
condyles, superficial and
middle/deep zone
Micropipette
aspiration
E⫽23.3 kPa
(middle/deep)
Viscoelastic half space
influence of the mode of testing does not confound the
interpretation of otherwise significant results. The assumptions implicit in the continuum mechanics models
used also play an important role in determining the properties of chondrocytes. The models of choice have traditionally been the elastic half-space model, or ‘‘punch
problem,’’ and the standard linear 共viscoelastic兲 solid
half-space model.19,30,37,52,58,59 Clearly, the problem with
representing the cell as a semi-infinite solid is that the
influence of cell morphology is excluded. A model developed by Haider and Guilak25 included parameters
such as the curvature of the cell, the finite dimensions of
the cell, and the curvature of the edges of the micropipette. Inclusion of these parameters can result in variations as great as 20% compared to the half-space
model.25 It has also been suggested that chondrocytes
exhibit apparent biphasic behavior.59 Previously, biphasic
mixture theory has been used to model articular
E0⫽25.8 kPa
E⬁ ⫽14.0 kPa
␮⫽53.1 kPa s
cartilage,47 as well as chondrocytes embedded in
cartilage.1,21,64 Biphasic theory has also been used to
derive the properties from the indentation of MG63
cells.54 As the mechanical nature of the chondrocyte becomes better understood, models incorporating increasingly complex boundary conditions, constitutive theories,
and subcellular structures, e.g., the nucleus, will become
important and powerful tools for quantifying the biomechanical properties of the cell.
FUTURE DIRECTIONS
The evidence is indisputable that mechanical forces
act as potent modulators of cartilage metabolism and
chondrocyte function. However, the arenas where this
knowledge breaks down are twofold: 共1兲 how do chondrocytes sense and interpret these forces, and 共2兲 what
are the thresholds that separate ineffectual mechanical
8
A. C. SHIEH and K. A. ATHANASIOU
FIGURE 2. Proposed single cell approach, emphasizing the
potential use of this technique to study cellular responses to
mechanical forces indicative of regenerativeÕreparative behavior as well as pathology.
stimulation from those that provoke either beneficial or
deleterious responses? To answer these pressing questions, researchers have begun to examine the signaling
pathways that may be involved in mechanotransduction;
at the same time the mechanical nature of the chondrocyte has been probed. As this review shows, the work
accomplished thus far is substantial, but the vast potential of the field remains untapped.
Techniques in cell mechanics have progressed to the
point where characterization of the individual cell has
become a technically demanding but accepted approach
for extracting cell biomechanical parameters. On the
other hand, the current state of the art in studying chondrocyte mechanotransduction involves stimulating groups
of chondrocytes in a bulk manner, and then applying
powerful molecular biology and biochemistry approaches
to determine how different pathways are affected. The
limitation of this approach is that the application of
forces is crude compared to the sensitivity of the downstream measurements. Forces applied in a bulk manner
invariably result in cells receiving different stimuli, because of differences in position, orientation, and other
local environmental factors. A number of models have
shown how parameters such as position and orientation
can have profound implications in the local stress–strain
environment.21,64,65 These differences can potentially
confound any results obtained, because the response observed could potentially be a series of different changes
in cell behavior, averaged over an entire population. In
addition, the precise nature of the forces experienced by
each individual cell cannot be easily predicted or determined, making it difficult to correlate specific mechanical parameters, e.g., stress, strain, to the resulting effect
on cell behavior.
A potential solution to these challenges is a methodology hereafter referred to as the ‘‘single cell approach’’
共Fig. 2兲. In this approach, mechanical forces would be
applied to a single cell, using the micromechanical techniques developed for cell mechanics studies, and the
resulting changes in cell behavior measured using sensitive biological assays, including but not limited to realtime polymerase chain reaction 共PCR兲, whole cell patch
clamping, in situ hybridization, and reporter gene constructs. By applying a well-defined force to a chondrocyte and recording the subsequent changes in the same
cell, definitive relationships between mechanical stimulation and cellular behavior would be elucidated. The effects of magnitude and frequency of load application, as
well as the type of stress applied—tension, compression,
shear, or hydrostatic pressure—could be studied unambiguously.
As with any methodology, the single cell approach
has caveats. The most obvious one is the relevance of
single cells to normal cartilage physiology and tissue
engineering strategies. The differences between an individual cell’s response in culture and the response of
chondrocytes surrounded by extracellular matrix or attached to a 3D scaffold are potentially substantial. However, it should be recognized that studying the single cell
FIGURE 3. Diagrammatic representation of the sequence of
studies leading from the ‘‘canonical problem’’ to effective
strategies for cartilage engineering incorporating mechanical
stimulation. The single cell approach would serve as a starting point for subsequent investigations into either „A… the
mechanobiology of native cartilage or „B… chondrocyte
mechanobiology in tissue engineering. Each step represents
an added level of complexity, and the more complex systems
necessarily require information from the previous steps to
provide context and allow for interpretation. It is important to
note that, in addition to the various systems illustrated here,
other factors, such as pH, oxygen tension, growth factors,
and cellular phenotype, can also affect how chondrocytes
interpret and respond to mechanical forces. These conditions would also be studied as part of these series of experiments.
Cell Mechanics for Cartilage Engineering
is only the first stage in an experimental continuum 共Fig.
3兲. By following the single cell approach, one could
identify key mechanical parameters, such as the magnitude, duration, and frequency of load, that would be used
as target conditions for subsequent studies involving
more complex systems. The next generation of experiments would fall into one of two series of hierarchical
studies. The first would seek to expand the current understanding of native cartilage mechanobiology. This
would be accomplished by applying external loads to
chondrons and microexplants 共chondrons surrounded by
cartilage ECM兲 that would result in forces at the cellular
level comparable to those identified in the initial single
cell studies. By comparing results from the single cell to
results from cell–ECM studies, a clearer picture of the
role of the ECM in modulating cell function would develop. Similarly, this type of approach could be applied
to studying chondrocyte mechanobiology in tissue engineering. The next logical step after the single cell would
be to examine the effects of cell attachment to twodimensional biomaterials on the cellular response to mechanical forces. As with the native cartilage experiments,
subsequent systems would involve external loading of
cell-seeded scaffolds, either devoid of ECM or with de
novo matrix, to achieve cellular loads comparable to
those used in the single cell approach. Simultaneously,
the effect of a variety of environmental factors, e.g., pH,
osmotic forces, oxygen tension, and cytokines, on the
cellular response to mechanical stimulation would also
be examined to gain a better appreciation of the interactions involved in chondrocyte mechanobiology. We believe that the knowledge acquired from this stepwise
approach, proceeding from the single cell to cells in
matrix or on a scaffold, can be applied to developing
optimal strategies for mechanically stimulating cellseeded scaffolds for cartilage tissue engineering.
CONCLUSION
It has become increasingly apparent that mechanical
stimulation is a key ingredient in successful ex vivo regeneration of articular cartilage. Therefore, a good working knowledge of cell mechanics is an indispensable tool
in efforts to develop rational strategies for cartilage tissue engineering. By understanding the biomechanical nature of the cell, the mechanisms by which the cell senses
and interprets mechanical signals, and how the cell responds to those signals, approaches can be designed to
incorporate optimal levels and types of mechanical
stimuli. The utility of a single cell approach, which is a
natural synthesis of current cell mechanics and mechanotransduction methodologies, is that it will allow researchers to study the effects of mechanical forces at the
cellular level, as well as the interaction between mechanical signals and other stimuli, such as cytokines and
9
bioactive materials. A clearer understanding of how mechanical forces modulate changes in cartilage at the cellular level promises to significantly advance the science
and engineering of articular cartilage regeneration.
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