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The effect of finite compressive strain on chondrocyte viability in statically

loaded bovine articular cartilage

Article in Biomechanics and Modeling in Mechanobiology · February 2007

DOI: 10.1007/s10237-006-0041-2 · Source: PubMed

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Biomechan Model Mechanobiol (2007) 6: 103–111
DOI 10.1007/s10237-006-0041-2
O R I G I NA L PA P E R
N. O. Chahine · G. A. Ateshian · C. T. Hung
The effect of finite compressive strain on chondrocyte viability
in statically loaded bovine articular cartilage
Received: 24 September 2005 / Accepted: 6 January 2006 / Published online: 4 July 2006
© Springer-Verlag 2006
Abstract Recent studies have reported that certain regimes
of compressive loading of articular cartilage result in in-
creased cell death in the superficial tangential zone (STZ).
The objectives of this study were (1) to test the prevalent
hypothesis that preferential cell death in the STZ results from
excessive compressive strain in that zone, relative to the mid-
dle and deep zones, by determining whether cell death corre-
lates with the magnitude of compressive strain and (2) to test
the corollary hypothesis that the viability response of cells
is uniform through the thickness of the articular layer when
exposed to the same loading environment. Live cartilage ex-
plants were statically compressed by approximately 65% of
their original thickness, either normal to the articular sur-
face (axial loading) or parallel to it (transverse loading). Cell
viability after 12 h was compared to the local strain distribu-
tion measured by digital image correlation. Results showed
that the strain distribution in the axially loaded samples was
highest in the STZ (77%) and lowest in the deep zone (55%),
whereas the strain was uniformly distributed in the trans-
versely loaded samples (64%). In contrast, axially and trans-
versely loaded samples exhibited very similar profiles of cell
death through the depth, with a preferential distribution in
the STZ. Unloaded control samples showed negligible cell
death. Thus, under prolonged static loading, depth-depen-
dent variations in chondrocyte death did not correlate with
the local depth-dependent compressive strain, and the preva-
lent hypothesis must be rejected. An alternative hypothesis,
suggested by these results, is that superficial zone chondro-
N. O. Chahine · G. A. Ateshian
Musculoskeletal Biomechanics Laboratory,
Department of Biomedical Engineering, Columbia University,
New York, NY 10027, USA
N. O. Chahine · C. T. Hung (B)
Cellular Engineering Laboratory,
Department of Biomedical Engineering, Columbia University,
1210 Amsterdam Avenue, 351 Engineering Terrace,
MC 8904, New York, NY 10027, USA
E-mail: cth6@columbia.edu
Tel.: +1-212-8546542
cytes are more vulnerable to prolonged static loading than
chondrocytes in the middle and deep zones.
1 Introduction
Cyclic loading is necessary for the normal maintenance of
articular cartilage function in diarthrodial joints, whereas
excessive mechanical loading can lead to matrix damage and
chondrocyte death. Recent studies have reported that static
and cyclic compressive loading of varying magnitudes, as
well as blunt impacts, result in increased cell death in artic-
ular cartilage (Chen et al. 1999, 2001, 2003; Torzilli et al.
1999; Ewers et al. 2001; Quinn et al. 2001; Lucchinetti et al.
2002; Krueger et al. 2003). Examination of the distribution of
cell death has been shown to be preferentially concentrated
in the superficial tangential zone (STZ) after cyclic loading
at moderate magnitudes (peak stress of 1 MPa) (Lucchinetti
et al. 2002; Chen et al. 2003).
Several studies have examined the nonuniform strain dis-
tributions and local stiffness arising in the differing zones
of articular cartilage under static confined and unconfined
compression and indentation (Schinagl et al. 1996; Wang
et al. 2002, 2003 Bae et al. 2003; Canal et al. 2003). Results
show that the axial normal strain is greatest near the articu-
lar surface, whereas the deep layer experienced axial strains
that were an order of magnitude smaller than in the STZ.
For example, for 5% applied platen-to-platen strain, the STZ
experienced ∼10% axial strain and the deep zone (DZ) expe-
rienced ∼1.5% axial strain (Wang et al. 2002, 2003; Chahine
et al. 2004). Thus cartilage exhibits its lowest compressive
stiffness in the STZ, and this stiffness increases nonlinearly
with increasing depth away from the articular surface.
Consequently, the presence of cell death in the STZ has
been attributed to excessive compressive strain occurring in
this softer region, as compared to the stiffer middle or deep
zones (Lucchinetti et al. 2002; Chen et al. 2003; Milentij-
evic et al. 2003) – a hypothesis inferred from the general
knowledge of the depth-dependent compressive properties
of cartilage. However, no study to date has directly measured
104 N. O. Chahine et al.

Fig. 1 a Sample preparation of specimens used in Study 1 (Top) and Study 2 (Bottom). b The two testing configurations utilized during the
injurious static compressive loading (Study 2). Axial direction: loading through the depth of the tissue (i.e. perpendicular to the articular surface);
transverse direction: loading perpendicular to the depth direction (i.e. parallel to the plane of the articular surface) (AS articular surface; DZ deep
zone). c Custom static compression device used for injurious loading that is housed in a tissue culture incubator. d Microscopy-based testing
device used to measure the depth-dependent deformation. e Schematic of samples tested in the microscopy device shown in d

the relationship between the depth-dependent strain and cell
death on the same tissue type or under the same loading con-
figuration. Moreover, injury studies have generally examined
the behavior of cell death under impact and/or cyclic load-
ing with load control that results in large compressive strain
magnitude, typically up to 50–60% strain (Ewers et al. 2001;
Flachsmann et al. 2001; Krueger et al. 2003), whereas the
literature studies on the depth-dependent mechanical prop-
erties of cartilage have generally been tested under a smaller
strain range, typically 20–30% strain.
The objective of this study was to directly measure the
relationship between cell death and local compressive strain,
under comparable loading protocols, in order to address the
hypothesis that cell death through the thickness of the
articular layer is proportional to the magnitude of the local
compressive strain. Cellular viability of immature bovine car-
tilage was examined under finite static loading and the equi-
librium compressive strain through the depth of the cartilage
layer was measured using digital image correlation. Carti-
lage explants in this study were compressed in either (a) the
axial direction, where the full thickness cartilage was loaded
through the depth of the tissue, or in (b) the transverse direc-
tion, where explants were loaded in a direction perpendicular
to the depth direction. Axial loading takes advantage of the
inherent inhomogeneity of the tissue, thereby exposing cells
in the different zones to different levels of strain. However,
since chondrocytes may exhibit different viability responses
in the various zones, axial loading alone would be insufficient
to rigorously test the hypothesis that cell viability corre-
lates with extracellular strain in light of this potential con-
founding factor. The use of transverse loading configuration
allows all three zones of cartilage to be exposed to an equal,
homogenous, finite state of strain. Thus, transverse loading
can be used to test the hypothesis that cell viability responses
are uniform through the depth, when the cells are exposed to
the same loading environment.
2 Materials and Methods
2.1 Cartilage explant culture
Full thickness osteochondral plugs (Ø 4 and 5 mm) were har-
vested under sterile conditions from carpometacarpal joints
of 3- to 4-month-old freshly slaughtered calves (N = 3 for
Study 1; N = 4 for Study 2). Explants were cultured in high
glucose Dulbecco’s Modified Essential Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), amino
acids (0.5X minimal essential amino acids, 1X nonessen-
tial amino acids), buffering agents (10 mM HEPES, 10 mM
sodium bicarbonate, 10 mM TES, 10 mM BES), and antibi-
otics (penicillin, streptomycin). These disks were cultured
in a humidified incubator at 37◦ C and 5% CO2 with daily
supplementation of 50 µg/ml of ascorbic acid. All explants
were allowed to acclimate to the in vitro culture environ-
ment for 2 days prior to experimentation; a time deemed suffi-
cient for chondrocytes to revert to full biosynthetic/metabolic
activity in an in vitro system (Dumont et al. 1999). On the
day of testing, full thickness cartilage layers were prepared
by removal of the subchondral bone under sterile conditions
using a custom cutting device, therefore limiting the time
allowed for significant changes in sample dimensions prior
to loading.
The effect of finite compressive strain on chondrocyte viability
2.2 Study 1: Dose response of chondrocyte viability
to static axial compression
In a preliminary study, cylindrical cartilage explants were
diametrically cut in half (Fig. 1a, top panel), and compressed
to determine the dose-dependent response of chondrocyte
viability to varying magnitude and duration of finite static
compressive strain (n = 23). One half of each explant was
compressed axially, through the depth, to either 50 or 80% of
the original thickness for a duration of either 6 or 12 h. The
other half of each sample served as the unloaded control.
Axial static compression was applied using a custom com-
pression device housed in a tissue culture incubator (Fig. 1c).
Samples were loaded in unconfined compression between a
frictionless impermeable platen and a supported petri dish
containing the explants. Displacement was manually applied
using a micrometer (MRO Industrial Supplies, Manville, NJ,
USA) to the nominal strain at a loading rate of ∼2.5 µm/s.
Upon completion of the loading protocol, samples were un-
loaded and allowed to equilibrate in fresh media for 30 min.
Chondrocyte viability was assessed in the loaded and control
explants using the cellular viability protocol described below.
2.3 Study 2: Comparison of injurious static axial
and transverse compressive loading
Rectangular cartilage specimens (1.5 mm × 4 mm × thick-
ness) were prepared from the center of each cylindrical disk,
resulting in an average articular layer thickness of 1.2 ±
0.3 mm (Fig. 1a, bottom panel). The cut bony surface was
marked with sterile trypan blue to indicate specimen orienta-
tion during subsequent testing. Twenty-one specimens were
divided into three equal groups: (1) free swelling control, (2)
axial loading and (3) transverse loading. Loaded specimens
were compressed in unconfined static compression, applied
to 80% of original tissue thickness (applied at ∼2.5 µm/s) for
duration of 12 h, in a custom loading device (Fig. 1c). This de-
vice was housed in a tissue culture incubator where samples
were loaded between a frictionless impermeable platen and
a supported petri dish containing the explants. Samples were
loaded in either the (1) axial direction, through the depth of
the tissue (i.e. perpendicular to the articular surface), or in the
(2) transverse direction, perpendicular to the depth direction
(i.e. parallel to the plane of the articular surface) (Fig. 1b).
Upon completion of the loading protocol, samples were un-
loaded and allowed to equilibrate in fresh media for 30 min.
Chondrocyte viability was assessed in the loaded and con-
trol explants using the cellular viability protocol described
below. The remaining tissue from each disk was stored at
−20◦ C and later tested in the microscopy-based loading de-
vice to determine the depth-dependent strain (Fig. 1d, e), and
its relationship to cellular viability.
2.4 Cellular viability
Cellular viability of specimens was assessed using a Live/
Dead Assay Kit (Molecular Probes, Eugene, OR, USA),
105
where Live cells fluoresce green due to activation of Calcein
AM by intracellular esterase activity, and dead cells fluoresce
red due to permeation and binding of Ethidium homodimer
to nucleic acids of damaged cells. Full thickness fluorescence
images of each explant were captured using an inverted fluo-
rescence microscope (Olympus IX-70; excitation: ∼495 nm,
emission: Calcein ∼515 nm, EthD-1 ∼ 635 nm) near the cut
edges of the rectangular specimens; thus assessing the via-
bility of the cells that are a few cell layers inward from the
edge. Intensity analysis was performed on each full-thickness
image using NIH Image software to assess the distribution
of dead cells (red nuclei) through the entire thickness. The
average intensity profile of cell death in the loaded samples is
normalized to the average cell death in the control group, in
order to provide a comparable measure of cell death between
loading configurations.
2.5 Axial and transverse finite strain measurements
In order to determine the depth-dependent strain in these sam-
ples, cartilage specimens were tested in unconfined compres-
sion using a custom microscopy-loading device mounted on
the stage of an inverted microscope (Fig. 1d), as previously
described (Wang et al. 2002). Uniaxial compressive defor-
mation was manually applied using a micrometer with 1 µm
resolution (MRO Industrial Supplies). Each sample was posi-
tioned between two parallel glass-loading platens (Fig. 1e)
and surrounded by 0.15 M NaCl solution in an immersion
chamber. The device was positioned on a motorized stage
(ProScan H128 Series; Prior Scientific, Rockland, MA, USA)
of an inverted microscope (Olympus IX70; Olympus) and
aligned so that the platens are perpendicular to the moving
axis of the stage. The optical path of the microscope was
adjusted through the immersion chamber, and the face of the
specimen between the platens was visualized.
Samples were compressed in both the axial and trans-
verse directions, corresponding to the same configuration as
the injurious loading (Fig. 1b). These smaller rectangular
samples (∼ 2 mm × 1 mm × thickness) were prepared from
the remaining semi-cylinder of tissue from each cartilage
disk (Fig. 1a, bottom panel). Under axial loading, these sam-
ples were compressed and visualized through the rectangu-
lar cross-section representing the depth of the cartilage layer.
Under transverse loading, the samples were compressed while
imaging the cross-section of this transverse face. Images of
the specimen cross-sections were acquired before deforma-
tion using a digital camera (MicroMax 5 MHz; Princeton
Instruments, Trenton, NJ USA). Compression was applied
in nominal 10% increments from 0 to 80%, and images were
acquired at equilibrium after each increment. Strain analy-
sis was performed using optimized digital image correlation
(DIC) (Wang et al. 2002), by automatically tracking the tex-
ture on the specimen surface across the entire cross-section
of tissue, yielding depth-dependent strain along either the
axial or transverse direction. For this loading protocol, im-
ages were acquired in the reference, undeformed state and
106
Fig. 2 Representative live/dead images of samples compressed axially (through depth) to 50 or 80% strain for 6 or 12 h; AS indicates articular
surface
at eight increasing compressive increments. The incremental
Lagrangian strain was calculated with DIC, through the depth
of the articular layer, from each incremental pair of images,
and converted to depth-dependent incremental stretch ra-
tios (= current length/original length). The cumulative stretch
ratio, λ, relative to the undeformed configuration, was
calculated from the product of the incremental stretches,
according to standard finite deformation strain analysis. The
stretch ratio is related to the axial compressive engineering
strain, ε, by ε = λ − 1 (where ε = change in length/original
length). In principle, the nominal platen-to-platen strain
(−80% or −0.8) represents the average value of ε through
the depth; the corresponding average value of λ should thus
be 0.2.
In order to assess the dependence of the deformation re-
sponse on the applied nominal strain, the degree of inhomoge-
neity was calculated at each applied compressive increment
(10 through 80%). The degree of strain inhomogeneity was
calculated as the ratio of strain in the STZ (top 20% of tis-
sue thickness) to the strain in the DZ (bottom 30% of tissue
thickness).
2.6 Statistical analysis
A two-way analysis of variance was performed examining the
effect of loading configuration and depth-dependent zone on
cell death. LSD post hoc test was performed with
p < 0.05 considered statistically significant. Statistical test-
ing was conducted using STATISTICA (StatSoft Inc., Tulsa,
OK, USA).
N. O. Chahine et al.
3 Results
All sample preparation and tissue incubation induced mini-
mally detectable and uniformly distributed cell death in the
cartilage explants (Fig. 2a, d), indicating that the co-culture
of osteochondral explants did not result in significant release
of chemical factors that mediate cell injury, such as cytokines
or free radicals.
3.1 Study 1
Results of the preliminary study indicated that uniaxial com-
pression to 50 or 80% strain applied for 6 h resulted in non-
continuous (scattered) cell death in the superficial zone
(Fig. 2b, e), with no major cell death occurring in the other
zones of the tissue. Once compression duration was increased
to 12 h, continuous cell death was seen across the entire cross-
section of the superficial zone for both 50 and 80% strain.
Based on these results, all subsequent injurious loading was
carried out at 80% strain for 12 h.
3.2 Study 2
Control samples showed minimal and constant cell death
intensity across all depth-dependent layers of the tissue
(Fig. 3). Static compression of 80% strain for 12 h resulted in
an increase in cell death in both the axially and transversely
loaded explants, compared to unloaded controls (Fig. 3).
The effect of finite compressive strain on chondrocyte viability
Fig. 3 Representative live/dead staining of control, axially and trans-
versely loaded samples compressed to 80% strain for 12 h. AS indicates
articular surface
Fig. 4 Mean cell death intensity profile under axial and transverse load-
ing (normalized to the average intensity of cell death in the control
samples). Articular surface is located at 0% of tissue depth
Consequently, the average distributions of cell death for the
axially and transversely loaded groups were normalized to
the average death intensity in the control samples (Fig. 4).
Loaded samples exhibited a peak in the average cell death
intensity, which is 32 times greater than cell death in the
control samples (Fig. 4). This peak occurred at 2.2% of tis-
sue thickness, closest to the articular surface (Figs. 3, 4).
Thereafter, the average cell death under axial and transverse
loading decreased to 1.6 ± 0.3 and 1.8 ± 0.6 times that in the
control samples, respectively. The decrease in cell death oc-
107
curred over a narrow region occupying the superficial layer,
though the extent of cell death throughout the STZ was sig-
nificantly greater for transversely loaded samples when com-
pared to axial loading. Under axial loading, the extent of cell
death permeated 6.8 ± 3.1% of the tissue thickness, whereas
cell death reached 11.5 ± 2.8% of the tissue thickness under
transverse loading ( p < 0.05). The intermediate and deep
zone showed a generally less intense cell death distribution
when compared to the superficial zone.
The average cell death in the superficial (0–15% of
thickness), middle (40–60% of thickness) and deep zones
(70–90% of thickness) are shown in Table 1 for both load-
ing configurations. The average cell death was significantly
greater in the STZ compared to the MZ and DZ for axial
and transverse loading ( p < 0.03). Moreover, the DZ dem-
onstrated a significantly higher cell death under transverse
loading than under axial loading (Fig. 4, p < 0.05). How-
ever, loading along the axial versus. transverse directions did
not result in significant difference in cell death in either the
STZ or MZ (Table 1).
The spatial distribution of the deformation, and conse-
quently the stretch ratio, were determined for samples
compressed in the axial and transverse directions (Fig. 5).
Samples loaded in the axial direction demonstrated a non-
linear displacement along the depth of the tissue (Fig. 5a),
resulting in an inhomogenous stretch ratio (Fig. 5b). Under
the nominal 80% compressive strain, the stretch ratio was
smallest near the superficial zone (λ = 0.23 ± 0.04), cor-
responding to a maximum compressive strain in the STZ
(ε = −0.77) (Fig. 6). Moreover, the stretch ratio increased
nonlinearly from the articular surface to the deep zone (λ =
0.45 ± 0.08 or ε = −0.55) (Fig. 6). Under transverse load-
ing, samples demonstrated a linear deformation and a cor-
responding homogeneous stretch ratio throughout the depth
(λ = 0.36 ± 0.03 or ε = −0.64) (Figs. 5, 6).
Along with spatial dependence, the strain distribution
also demonstrated a dependence on the applied nominal strain,
as indicated by the degree of strain inhomogeneity (Fig. 7).
At the lowest applied strain (10%), samples demonstrated
a high ratio of STZ to DZ compressive strain of 8.0 ± 3.3
(Fig. 7). However, this degree of inhomogeneity significantly
decreased with increasing applied compression, reaching an
average value of 1.3 ± 0.11 at 80% applied nominal strain
( p < 0.01).
Table 1 Average cell death intensity in the axially and transversely
loaded samples
Loading configuration STZ MZ DZ
Axial 8.9 ± 5.0* 1.5 ± 0.75 1.8 ± 1.4
Transverse 11.5 ± 7.9* 2.6 ± 1.9 7.0 ± 3.6#
Values represent mean ± SD
*significance relative to the other layers within the same loading con-
figuration ( p < 0.03)
# significance relative to the axial loading direction within the same
tissue layer ( p < 0.05)
108 N. O. Chahine et al.

Fig. 5 Representative plots of the (a) incremental depth-dependent displacement under axial loading, and the corresponding (b) incremental
depth-dependent stretch ratio under axial loading. Representative plots of the (c) incremental depth-dependent displacement under transverse
loading, and the corresponding (d) incremental depth-dependent stretch ratio under transverse loading. Articular surface is located nearest to the
position origin (i.e. 0 µm)

Fig. 6 Average ± standard deviation of the total deformation for sam-

ples loaded axially and transversely to 80% nominal compressive strain.
Stretch ratio (λ) is related to the engineering compressive strain (ε) by
ε = λ − 1. Articular surface is located at 0% of tissue depth
Fig. 7 Average degree of strain inhomogeneity, as indicated by the ratio
of strain in the STZ to the strain in the DZ, as a function of the applied
nominal strain (ε). * p < 0.01 indicates significance between the degree
of inhomogeneity measured at infinitesimal strain (i.e. ε ≤ 20%) and
finite strain range (ε > 20%)

4 Discussion
The objective of this study was to directly measure the rela-
tionship between chondrocyte death and local compressive
strain in statically loaded cartilage explants, under compara-
ble loading protocols, to address the hypothesis that cell death
through the thickness of the articular layer is proportional to
the magnitude of the local compressive strain. Axial loading
was used to produce a range of compressive strains through
the depth of the tissue under a single loading event. How-
ever, since chondrocytes in the varying zones may exhibit
different viability responses to varying magnitudes of strain,
axial loading alone was deemed insufficient to rigorously test
this hypothesis. Thus, transverse loading was used to test the
hypothesis that cell viability responses are uniform through
the depth, when the zones are exposed to the same loading
environment.
Under both loading configurations, results show that
chondrocyte death was most pervasive within the superficial
zone (< 12% of tissue thickness, Fig. 4), when compared to
unloaded controls. Yet, the strain distribution was distinctly
different in axial and transverse loading (Fig. 5). Under axial
loading, all the zones of the tissue were exposed to vary-
ing strain magnitudes, depending on the local compressive
modulus (Fig. 5b); however under transverse loading, the
The effect of finite compressive strain on chondrocyte viability
zones of the tissue were subjected to the same magnitude of
compressive strain (Fig. 5d). When examining the results of
axial loading alone, they strictly fail to support the hypoth-
esis that cell death is proportional to the magnitude of the
local compressive strain. Though the compressive strain at a
nominal tissue compression of 80% is indeed greatest in the
superficial zone under axial loading, it decreases smoothly to-
ward the deep zone (Fig. 6). In contrast, cell death decreases
abruptly beyond 10% of the thickness from the articular sur-
face (Fig. 4). This rejection of the hypothesis is further sup-
ported by the findings from transverse loading, which show
a very similar nonuniform pattern of cell death (Fig. 4) even
though the strain through the thickness of the articular layer
is uniform (Fig. 6). These results imply that we must also
reject the hypothesis that cell viability responses are uniform
through the thickness, when exposed to the same loading
environment.
Further support for the rejection of the first hypothesis
arises from the time-dependent cell death response (Fig. 2).
Chondrocyte death was noticeable near the surface of the
tissue at 6 h, and became more pronounced by 12 h of load-
ing. While there is a temporal progression of cell death, we
know from theoretical predictions (Mow et al. 1980) and
experimental verification (Wang et al. 2002) that the local
compressive strain reached equilibrium long before the 6 h
time point, and remained constant between 6 and 12 h. More-
over, it does not appear that the region of cell death is varying
spatially beyond the superficial zone. Such spreading of cell
death, emanating from an initial defined injury site, has been
reported by other investigators and has been attributed to the
release of soluble factors from injured cells, such as nitric
oxide or the cytokines IL-1β, TNF-α, or Fas ligand (Levin
et al. 2001).
Under axial loading, the higher compression observed
in the STZ relative to the middle and deep zones is con-
sistent with previous results in the literature (Guilak et al.
1995; Schinagl et al. 1996; Wang et al. 2002), though the
magnitude of compression is significantly greater in this fi-
nite deformation study (∼77 ± 4% compressive strain in
the superficial zone, ∼55 ± 8% in the deep zone). In con-
trast, the overwhelming majority of dead chondrocytes are
concentrated in a narrow region consisting of ∼10% of the
full thickness (Fig. 4), where the compressive strain varies
over a small range, ∼74–77% (Figs. 5, 6). This high level
of compaction in the STZ may result in exudation of most
interstitial fluid, thus potentially compromising the transport
of nutrients and waste products in this layer. However, under
transverse loading, all zones of the tissue are equally com-
pressed to the same level of excessive strain, and cell death
remains most pervasive in the STZ. Therefore, we conclude
that nutrient deficiency is not the primary factor resulting in
preferential cell death in the STZ. The alternative hypothe-
sis arising from the loading experiment along the transverse
direction is that STZ chondrocytes are more susceptible to
cell death or injury, because middle and deep zone chon-
drocytes exhibited significantly lower amounts of cell death
(Fig. 4).
109
Increased vulnerability of STZ chondrocytes has been
previously reported for cartilage explants loaded under dy-
namic conditions (Chen et al. 2003). Depth-dependent differ-
ences in the behavior of chondrocytes to loading may arise
from intrinsic differences in the cell properties and/or metab-
olism of chondrocytes, as previously reported (Aydelotte et
al. 1988; Aydelotte and Kuettner 1988; Guilak et al. 1995; Lee
and Bader 1995; Lee et al. 1998). Metabolically, it has been
shown that STZ chondrocytes demonstrate a lower rate of
proliferation (Aydelotte and Kuettner 1988; Lee et al. 1998),
lower PG and fibronectin synthesis (Aydelotte et al. 1988;
Hayashi et al. 1996; Lee et al. 1998), and more rapid PG deg-
radation (Aydelotte et al. 1988), relative to chondrocytes from
the deep zone. Moreover, STZ chondrocytes show a greater
vulnerability to the harmful effects of interleukin-1 (IL-1)
and appear to be less responsive to the potential therapeutic
effect of an IL-1 receptor antagonist protein (Hauselmann
et al. 1996).
Zonal chondrocytes have also exhibited variable responses
to identical loading protocols, depending on their anatomi-
cal origin (Lee et al. 1998). When compressed in agarose,
chondrocytes freshly isolated from the deep zone deformed
to a greater extent than those from the STZ, in the absence
of matrix elaboration (Lee and Bader 1995). Moreover, a
recent study has directly measured the unconfined compres-
sion modulus of chondrocytes, and has demonstrated that
STZ chondrocytes possess a higher instantaneous and equi-
librium stiffness than deep zone cells (Shieh and Athanasiou
2005). Although isolated STZ chondrocytes appear to pos-
sess a higher compressive stiffness, these cells also undergo a
greater decrease in cellular height and volume than DZ cells
under static loading (Guilak et al. 1995), suggesting that cell
behavior in situ is dominated by the ECM properties. Despite
the mechanical and metabolic variations in cell subpopula-
tions and surrounding ECM, it is yet unknown how these
unique zonal properties result in increased vulnerability of
STZ chondrocytes to injury or death.
In transverse loading, all zones of the tissue were com-
pressed to 64% of their original sample thickness (Figs. 5,
6). The extent of cell death within the STZ appears to be in-
creased relative to axial loading conditions (from 7% in axial
loading to 13% in transverse loading), which may reflect the
anisotropy of the tissue. Earlier studies examining the aniso-
tropic response of cartilage under compression have shown
that the compressive stiffness of cartilage is greatest in the
Deep
deep zone under axial loading (denoted by E −Y 3 ) (Wang et
al. 2002, 2003; Chahine et al. 2004). Moreover, the stiffness
of the STZ when compressed axially (E −Y Surface ) was found to
3
be equal to the stiffness of cartilage when compressed trans-
versely (indicated by E −Y 1 and E −Y 2 ). Therefore, it doesn’t
appear that the collagen fiber orientation plays a dominant
role in the compressive response of STZ cartilage under infin-
itesimal deformation.
Cell death in the deep zone was found to be more appar-
ent in the transversely loaded samples, compared to axially
loaded samples (Fig. 4). This difference cannot be attributed
to compressive strain magnitudes, which exhibit little differ-
110
ence in these zones between the two loading configurations
(Fig. 6). It is possible that this cell death is a consequence
of the non-physiological loading conditions imposed during
transverse loading. Alternatively, the increased localized cell
death may be a result of higher tensile expansion strain in
these zones under transverse loading. Although the direction
representing the lateral expansion was randomized during
sample preparation in this study (no preferred direction was
chosen relative to the collagen fiber orientation in the STZ),
earlier studies have indicated that the lateral expansion of
cartilage is homogenous for samples loaded both axially and
transversely. Moreover, it was found that the lateral expan-
sion due to axial loading is significantly greater in the deep
Deep Surface )
zone (denoted by ν−31 ) than in the superficial zone (ν−31
(Wang et al. 2002, 2003; Chahine et al. 2004). An increase
in cell death near the bottom surface of transversely loaded
explants has been previously reported for explants loaded
dynamically under a 5 and 10 MPa creep load (Chen and Torz-
illi 2002). However in that study, no cell death was observed
near the STZ in the transversely loaded samples.
Interpretation of cell death results and the severity of
cell death are dependent on the temporal nature of the ap-
plied loading (e.g., rate of loading, static or dynamic) and
the boundary conditions (e.g., confined or unconfined com-
pression). In the present study, a gradual load was applied
using displacement control to a maximum platen-to-paten
strain level. These loading conditions were chosen to avoid
cell injury due to the transient phase of loading, and are quite
different from impact conditions often studied in the litera-
ture (e.g., usually under load control, using confined com-
pression as well as unconfined compression). Impact loading
in unconfined compression can lead to fracture and fissuring
of the cartilage tissue.
Determination of the total applied strain was performed
under multiple deformation increments reaching a finite nom-
inal compressive strain of 80%. Under finite deformation
(|ε| ≥ 20%), the depth-dependent strain inhomogeneity ap-
pears to be less pronounced than under small strains (|ε| ≤
20%), as indicated by the ratio of STZ to DZ strain (Fig. 7).
At the largest compression increment, the tissue undergoes
a relatively more uniform strain distribution compared to the
first increment (Fig. 5a, note the progression from the top
curve to the bottom curve). These results suggest that the
inhomogeneous nature of the axial properties is mitigated
when the softer regions of the tissue (the superficial zone)
are consolidated by the applied load (Fig. 7). These findings
indicate that the relative magnitudes of deformation in the
three zones of cartilage are significantly altered between the
infinitesimal and finite deformation ranges. Therefore, di-
rect measurement of strain in the finite deformation regime
is necessary to accurately describe the relationship between
depth-dependent deformation and cell viability.
Another observation is that the average compressive strain
through the depth (∼65% as shown for both loading config-
urations in Fig. 6) is smaller in magnitude than the nomi-
nal applied strain of 80%. This disparity can be explained
by imperfect platen contact with the tissue specimen in the
N. O. Chahine et al.
reference (i.e. undeformed) state, and by compliance in the
loading apparatus. For the purposes of this study, this discrep-
ancy is of little consequence since the true strain distribution
(given by λ or ε) is determined from digital image correlation
(Figs. 5, 6).
The results of this study may be specific to immature
cartilage, as studies have shown that the response of chondro-
cytes to injury is dependent on the age of the tissue (Flachs-
mann et al. 2005; Levin et al. 2005). Studies of immature
cartilage provide insight to the susceptibility of young chon-
drocytes, which are more metabolically active and show a
relatively higher repair capacity, to injurious loading. They
also yield complementary knowledge regarding the physical
regulation of chondrocytes. Additionally, immature cartilage
and chondrocytes are widely used in tissue engineering and
mechanotransduction studies, which demonstrate that imma-
ture chondrocytes are more responsive to mechanical loading
than mature cells.
To our knowledge, this basic science study represents the
first attempt to measure the depth-dependent strain fields in
articular cartilage under applied finite deformation, and to
correlate it with cell death, providing insight into the fac-
tors governing cell viability. It is important to note that the
prolonged static loading applied on these explants is not
physiologic and the results of this study should not be extrap-
olated to in situ loading conditions in living joints. However,
they may serve to interpret comparable basic science stud-
ies of the biosynthetic response of immature articular carti-
lage. This study shows that, under prolonged static loading,
depth-dependent variations in chondrocyte death do not cor-
relate with the local depth-dependent compressive strain; and
superficial zone chondrocytes are more vulnerable to pro-
longed static loading than chondrocytes from the middle and
deep zones.
Acknowledgements This study was supported by the National Insti-
tute of Arthritis, Musculoskeletal and Skin Disease (NIAMS) at the
National Institutes of Health (AR46532, AR46568).
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