973240

research-article2020
CARXXX10.1177/1947603520973240CARTILAGEKabir et al.

Basic Research Article

Cartilage

Assessment of Native Human Articular

1­–11
© The Author(s) 2020
Article reuse guidelines:
Cartilage: A Biomechanical Protocol sagepub.com/journals-permissions
DOI: 10.1177/1947603520973240
https://doi.org/10.1177/1947603520973240
journals.sagepub.com/home/CAR

Wassif Kabir1,2 , Claudia Di Bella2,3,4, Peter F.M. Choong2,3,4,

and Cathal D. O’Connell2,5

Abstract
Objectives. Recapitulating the mechanical properties of articular cartilage (AC) is vital to facilitate the clinical translation
of cartilage tissue engineering. Prior to evaluation of tissue-engineered constructs, it is fundamental to investigate the
biomechanical properties of native AC under sudden, prolonged, and cyclic loads in a practical manner. However,
previous studies have typically reported only the response of native AC to one or other of these loading regimes. We
therefore developed a streamlined testing protocol to characterize the elastic and viscoelastic properties of human
knee AC, generating values for several important parameters from the same sample. Design. Human AC was harvested
from macroscopically normal regions of distal femoral condyles of patients (n = 3) undergoing total knee arthroplasty.
Indentation and unconfined compression tests were conducted under physiological conditions (temperature 37 °C and
pH 7.4) and testing parameters (strain rates and loading frequency) to assess elastic and viscoelastic parameters. Results.
The biomechanical properties obtained were as follows: Poisson ratio (0.4 ± 0.1), instantaneous modulus (52.14 ±
9.47 MPa) at a loading rate of 1 mm/s, Young’s modulus (1.03 ± 0.48 MPa), equilibrium modulus (7.48 ± 4.42 MPa),
compressive modulus (10.60 ± 3.62 MPa), dynamic modulus (7.71 ± 4.62 MPa) at 1 Hz and loss factor (0.11 ± 0.02).
Conclusions. The measurements fell within the range of reported values for human knee AC biomechanics. To the
authors’ knowledge this study is the first to report such a range of biomechanical properties for human distal femoral
AC. This protocol may facilitate the assessment of tissue-engineered composites for their functionality and biomechanical
similarity to native AC prior to clinical trials.

Keywords
articular cartilage, knee, biomechanics, mechanical testing

Introduction native human articular cartilage.4-6 Furthermore, the vast

Loss of mechanical integrity is an irreversible consequence
of damage to articular cartilage (AC). One of the most preva-
lent examples of damaged AC is knee osteoarthritis, which is
the main reason for knee replacement surgery and a global
health burden.1 The pathophysiology of knee osteoarthritis is
complex. Altered joint mechanics in AC engenders an abnor-
mal pattern of stress distribution which modifies the stress
and strain fields experienced by cells embedded in cartilage
matrix. These trigger signaling pathways that inhibit chon-
drogenesis, reduce cell viability and effect biochemical alter-
ations in the matrix.2 The net result is the cartilage softening,
fibrillation and degeneration observed in osteoarthritic knees.
Current therapeutic strategies to repair AC such as matrix-
induced autologous chondrocyte implantation (MACI),
osteochondral grafting, and microfracture have generally
produced biomechanically poor fibrocartilage.3 Although
promising results have been obtained in tissue engineering
approaches for cartilage repair in animal models, most tissue-
engineered constructs lack the biomechanical qualities of
majority of preclinical studies fail to report the biomechani-
cal properties of neocartilage as a key outcome parameter.7,8
1
Faculty of Medicine, Dentistry and Health Sciences, University of
Melbourne, Parkville, Victoria, Australia
2
BioFab3D, Aikenhead Centre for Medical Discovery, St. Vincent’s
Hospital, Fitzroy, Victoria, Australia
3
Department of Orthopaedics, St. Vincent’s Hospital, Fitzroy, Victoria,
Australia
4
Department of Surgery, University of Melbourne, Parkville, Victoria,
Australia
5
Discipline of Electrical and Biomedical Engineering, School of
Engineering, RMIT University, Melbourne, Victoria, Australia
Supplementary material for this article is available on the Cartilage
website at https://journals.sagepub.com/home/car.
Corresponding Author:
Cathal D. O’Connell, BioFab3D, Aikenhead Centre for Medical
Discovery, St. Vincent’s Hospital, BioFab3D, 1st Floor, Clinical Sciences
Building, 29 Regent Street, Fitzroy, Victoria 3065, Australia.
Email: cathal.oconnell@svha.org.au
2 Cartilage 00(0)

Figure 1.  Schematic representation of the biomechanical testing sequence consisting of initial indentation, dynamic compression,
cyclic loading, and repeat indentation tests. Four separate sites on the chondral surface (depicted by small arrows diverging from the
indenter tip to the cartilage) of each sample were indented for each calculation of the instantaneous or Young’s modulus. The dynamic
compression step and cyclic compressive loading steps were repeated sequentially 4 times before moving onto the final indentation
step. AC, articular cartilage; SB, subchondral bone.

Whether tissue-engineered cartilage is able to withstand
mechanical loading in the same way as native human articu-
lar cartilage is therefore an important question that must be
answered using biomechanical testing methods prior to clini-
cal translation.
Articular cartilage is frequently modelled as a single-
phase, incompressible, elastic solid with an instantaneous
elastic response under instantaneous loading, while under
sustained deformation, AC exhibits viscoelastic qualities
with a time-dependent, asymptotic approach to an equilib-
rium stress response.9,10 Various biomechanical models
have been developed for AC, such as the simple linear mono-
phasic elastic model11 and the more complex linear bipha-
sic,12,13 poroviscoelastic,14 fibril-reinforced poroelastic,15 and
transversely isotropic biphasic,16 models. Despite the con-
sideration of mechanical nonlinearity in sophisticated mod-
els, modeling cartilage as a homogenous, isotropic material
is practically convenient in the laboratory and it allows the
measurement of equilibrium and instantaneous elastic
responses of a cartilage matrix to applied loads in uncon-
fined compression, confined compression, and indentation
geometries.1 The dynamic viscoelastic behavior of native
AC may be assessed under cyclic compression to yield the
storage moduli, loss moduli, and thereby the loss factor
using sinusoidal wave analysis.17 This system of mechani-
cal testing can be used to assess mechanical properties of
one native AC sample relative to another or to tissue-engi-
neered cartilage.
The material properties of articular cartilage have been
extensively studied using indentation and unconfined com-
pression testing. In such studies, the influence of various fac-
tors on the derivation of cartilage mechanical properties has
been thoroughly analyzed, including the effect of patient fac-
tors like aging,18,19 tissue factors such as cartilage thickness20
and mucopolysaccharide or water content of the matrix,19,21
biomechanical factors such as topographical variation of
joint contact stresses,9,22,23 and instrumental factors such as
indenter geometry,24 indentation depth,21,25 and the type of
elastic or viscoelastic model applied.10,26,27 However, the
majority of biomechanical studies of AC have utilized ani-
mal samples, particularly bovine AC.21,24,27 Those that have
used human cartilage have evaluated either the equilibrium,
instantaneous or dynamic response to loading rather than all
of these on a single sample.13,28,29
The functional mechanical testing protocol established
herein aimed to measure the elastic properties under instan-
taneous and equilibrium loading conditions, as well as vis-
coelastic properties under dynamic loading conditions
through a sequence of tests in indentation and unconfined
compression geometry (Fig. 1). In doing so, this protocol
aims to facilitate an efficient systematic approach to mechan-
ical testing of native human knee AC and tissue-engineered
Kabir et al. 3
cartilage such that the functionality of the latter may be ade-
quately assessed in tissue engineering studies.
Methods
For the purposes of protocol validation, a preliminary set
of test samples were obtained, following institutional
approval (HREC-A 143/16) and informed consent from
patients (n = 3; 77-year-old female, 71-year-old male, and
71-year-old male) undergoing total knee arthroplasty at St.
Vincent’s Hospital, Victoria, Australia. In particular, the
“distal femoral cuts” of femoral condyles were acquired
during each operation.
Biomechanical Testing Protocol
Osteochondral Sample Preparation.  The distal femoral con-
dyles were carefully inspected for macroscopically normal
looking cartilage with no signs of degeneration or fibrilla-
tion. Cylindrical osteochondral (OC) plugs were obtained
from condyles using a core extruder of 8 mm diameter.
Three plugs each were obtained from 2 patients and 2 plugs
obtained from one patient, resulting in a total of 8 samples.
Following extrusion, the bottom bony surface of each OC
plug was immediately shaped via cutting and sanding to be
orthogonal to the cartilage surface, using a Dremel 3000-
2/30 Rotary Tool at 35,000 rpm. The OC plugs were stored
in phosphate-buffered saline (PBS) containing 1% penicil-
lin/streptomycin and protease inhibitor cocktail (Roche
Complete Mini EDTA-free) at 4 °C for 24 to 48 hours prior
to testing. These steps are outlined in the supporting infor-
mation (Supplemental Figure S1).
Cartilage Thickness and Area Measurement.  Cartilage thick-
ness was measured using stereomicroscopy as per Burgin
et al.30 The stereomicroscope was oriented perpendicular to
the articular surface and used to capture images at four loca-
tions around the perimeter. The cartilage was identified as
the white layer between articulating surface and the tide-
mark. The thickness of the white cartilage layer was mea-
sured using image analysis software (FIJI/Image J).
Experimental Setup. Mechanical testing was conducted
using the ElectroForce Biodynamic 5500 instrument (Bose,
Eden Prairie, MN) controlled with WinTest 7 software
(Bose). The mover arm of the instrument was fitted with an
indenter or 25 mm circular metal platen for tests under
indentation or unconfined compression geometry, respec-
tively. OC plugs were attached to the surface of a glass petri
dish with glue (bonded to residual bone) and immersed in
PBS. Temperature inside the dish was maintained at 37 ± 1
°C using a heated circulation system (Lauda-Brinkmann,
Deltran, NJ). A 25-mm metal platen was attached to either a
250 g load cell or a 50 lb (200 N) load cell for indentation
and unconfined compression respectively. This platen
served as the platform for the petri dish. A photograph of the
test setup is provided in the supporting information (Sup-
plemental Figure S2).
Poisson Ratio Calculation.  In unconfined compression geom-
etry, the mover arm fitted with a 25 mm metal platen was
lowered until it came into contact with the cartilage surface
and the load measured in real time using a 50 lb load-cell.
The minimum load required to achieve even contact with
the chondral surface was set as the zero or baseline value.
Then 20% axial compression was applied at 0.01 mm/s and
the compression was observed in real time using video
microscopy. The sample was allowed to stress-relax until
equilibrium, at which point photos of the samples were
taken horizontally. The axial strain was measured between
the top platen and bottom of the OC plug and the lateral
strain was measured from the horizontal expansion of the
cartilage layer at equilibrium. Using image processing
(ImageJ software), the ratio of the average lateral strain to
average axial strain was calculated to optically determine
the Poisson ratio, similar to previous studies.22,31
Mechanical Testing Sequence
Indentation.  First, stepwise indentation tests were per-
formed at 1 mm/s for strains of 0% to 5%, 5% to 8%, and
8% to 10%, with 300 seconds relaxation to equilibrium
between each step. Indentations were performed either with
a diameter flat-ended cylindrical indenter of diameter 0.36
mm or 0.5 mm. The indenters had polished edges and were
cleaned with paraffin oil before and after each experiment.
After the indenter (see Supplemental Figure S2) was lifted
off the cartilage at the end of each indentation step, the por-
table stereomicroscope (affixed to a clamp stand) was used
to zoom in to the area of indentation to ensure that the
indenter had not penetrated or caused fissures in the carti-
lage surface. Examination of the osteochondral unit for
potential damage is an important part of the protocol as a
damaged sample cannot be used to accurately conduct sub-
sequent biomechanical tests. The gradient of the instanta-
neous stress-strain curve at 5% strain was calculated to
derive the instantaneous modulus Ei. The gradient of the
equilibrium stress-strain curve from all 3 indentation steps
was calculated to derive the equilibrium modulus Eeq.
Under the assumption of a monophasic, isotropic elastic
material indented using a flat-ended cylindrical indenter,
we used the mathematical solution used by Korhonen and
colleagues27 to determine the Young’s modulus Es at equi-
librium, from the original solution of Hayes et al.32:
Es =
(1 − v ) πa E
2
eq
2kh
where v is the Poisson ratio measured optically, a is indenter
radius, h is cartilage thickness, and k is a theoretical scaling
4 Cartilage 00(0)
factor that depends on the aspect ratio (a/h). Values for k
were obtained for finite indentation according to calcula-
tions by Zhang et al.25
Viscoelasticity.  Following indentation, the sample was set up
in an unconfined compression arrangement, and put through
a sequence involving dynamic mechanical testing, com-
pressive modulus measurement, and cyclic compressive
loading. Viscoelastic measurements were performed using
sinusoidal compression with a strain amplitude of 1.25%
for 50 cycles at frequencies of 0.1 Hz, 1 Hz, and 10 Hz. The
storage modulus (E′) and loss modulus (E″) were calculated
using viscoelastic theory. The loss factor was obtained from
the ratio E″/E′.
Compressive Modulus.  Compressive modulus was measured
in unconfined compression where the cartilage was axially
compressed between 0% and 20% strain at 0.01 mm/s. The
compressive modulus was calculated as the average stress-
strain gradient between 10% and 15% strain, in a similar
fashion to previous studies.17
Cyclic Loading.  Cyclic compressive loading constituted axial
compression between 0% and 20% strain with a frequency
of 4 Hz for 2000 cycles. The three stages of dynamic test-
ing, compressive modulus measurement and cyclic loading
were conducted sequentially and repeated 4 times per sam-
ple. At the end of 4 blocks of testing, the AC had undergone
8,000 cycles of high-frequency compressive loading. Inden-
tation measurements were then repeated at 4 sites for each
plug using the initial parameters.
Rationale for the Testing Parameters and
Conditions
Strain.  According to a study of tibiofemoral cartilage strains
measured by magnetic resonance imaging (MRI), immedi-
ately following a dynamic hopping activity the maximum
compressive strain was 6% in human medial femoral carti-
lage and 3% in the lateral femoral cartilage.33 Similarly, the
average medial femoral cartilage deformation after walking
was 6.7% in another study.34 Some in vivo MRI studies
have shown that 30 minutes of running in healthy individu-
als produces significant acute medial femoral cartilage
deformation up to 5.3% and lateral femoral cartilage defor-
mation up to 4.0%, when measured within 2 minutes of
ending the activity.35
While it is not possible to measure the in vivo strains in
real time as a human joint is loaded, the immediate postac-
tivity and nonequilibrium cartilage deformation values cap-
tured in the above literature suggest that physiological
strains to estimate cartilage stiffness ex vivo are likely to be
close to or slightly greater than reported values. Hence, in
this protocol, the range of strains 5% to 7.5% in the dynamic
mechanical testing phase, instantaneous strain of 5% and
stepwise indentation axial strains of 5%, 8%, and 10% at
equilibrium are deemed appropriate.
Strain Rate or Loading Rate.  For an average cartilage thick-
ness of 2.09 ± 0.23 mm in our study, when axial strain is
applied at 1 mm/s, a 5% deformation is achieved on average
within 105 ms. These conditions are designed to mimic
physiological loading conditions during running or drop-
landing, where tibiofemoral cartilage experiences large
force and frequent instances of rapid loading.33-35
In a study of noninvasive measurements of in vivo human
tibiofemoral cartilage deformation via MRI, when a cyclic,
half-body-weight load was applied axially to a subject’s
knee, it took less than 200 ms to apply the required force
during every cycle before the cartilage tissue experienced
creep.36 Higher loads (equivalent to full body weight or
higher, e.g., when landing from a height) would further
reduce the time to deformation and hence increase the strain
rate. The chosen strain of 5% following rapid loading is
well supported by in vivo human knee MRI studies, which
have shown average deformations of 6% ± 2% in the distal
femur and 5% ± 1% in the tibial plateau immediately fol-
lowing single legged hopping.33 Thus, our chosen rate and
axial strain for instantaneous deformation falls within the
range of deformations observed in vivo using radiographic
techniques. It is however important to note that the reported
literature values of maximal axial strain due to rapid load-
ing lie somewhere between the true peak strain and the
equilibrium strain depending on the timing of measurement
following the joint-loading activity.
This protocol follows similar methods to several
published studies that have utilized the Hayes’ elastic
model to obtain the Young’s modulus for cartilage under
indentation.23,24,27 The rate of impact is less influential when
using the equilibrium stress-strain values to determine the
equilibrium modulus and subsequently the Young’s modu-
lus of the cartilage.
While we chose to probe a regime of high force and
rapid loading, researchers more interested in the response of
the cartilage to more gentle activities, such as walking, may
opt to choose a slower strain rate of 0.1 s−1.
Viscoelastic Frequency. The frequency chosen (1 Hz) to
obtain the dynamic modulus via sinusoidal compression
matches that used previously22 and approximates the aver-
age physiological loading frequency of 1.2 Hz which has
been reported for the human tibiofemoral (knee) joint dur-
ing running.37
Statistical Analysis
Due to a small number of samples, the nonparametric
Wilcoxon matched-pairs signed rank test (statistical
Kabir et al. 5
significance at α = 0.05) was used to analyze the difference
in the modulus values before and after cyclic compression
for each sample separately, as done in previous studies.27
For similar reasons, the nonparametric Spearman rank cor-
relation test (statistical significance at α =0.05) was con-
ducted to investigate correlations between the different
elastic and viscoelastic parameters both before and after
cyclic compression.
Results
The functional mechanical testing protocol was validated
through preliminary indentation studies conducted on 8
osteochondral samples from macroscopically normal
regions of distal femoral condyles from 3 patients (patient
1, patient 2, and patient 3). Calculations of dynamic and
compressive elastic properties were performed using
unconfined compression data at baseline and after 8,000
compressive loading cycles to simulate joint loading
(4 consecutive steps of 20% axial compression for 2,000
cycles at 4 Hz). Final indentation studies on each sample
were conducted after a total of 8,000 compressive loading
cycles and compared to baseline.
The mean cartilage thickness obtained via stereomicros-
copy was 2.09 ± 0.23 mm, which is similar to that reported
elsewhere (2.0-3.9 mm,23 1.2- 2.0 mm,17 1.95 mm44). The
mean Poisson ratio (v) of 0.4 ± 0.1 demonstrated some
overlap with literature values in the range of 0.12 to 0. 38
for human articular cartilage.17,28
Table 1 compares the mechanical properties obtained
before cyclic compression with literature values of the same
properties in various animal and human joints. The values
obtained using the current protocol were more similar to
certain studies than others due to methodological similari-
ties and differences as well as intrinsic variations of “nor-
mal” cartilage within the same population.
Indentation.  The baseline instantaneous strain 5%, com-
pared with 58.26 ± 8.12 MPa following 8,000 high-fre-
quency compression cycles. Values for the fluid-independent
elastic properties equilibrium modulus and Young’s modu-
lus were 7.48 ± 4.42 MPa and 1.03 ± 0.48 MPa before
cyclic compression, respectively. Following compressive
loading, the corresponding values for equilibrium and
Young’s moduli were 7.43 ± 4.78 MPa and 1.01 ± 0.49
MPa, respectively. Wilcoxon matched-pairs signed rank
test27,44 for each osteochondral sample showed no statisti-
cally significant difference between the “before cyclic com-
pression” and “after cyclic compression” values in terms of
the instantaneous (P = 0.3), equilibrium (P > 0.9), or
Young’s moduli (P = 0.8) (Fig. 2).
Unconfined Compression. In unconfined compression, the
mean compressive modulus was 10.60 ± 3.62 MPa at
baseline and 9.47 ± 3.54 MPa after cyclic compression.
The dynamic, storage and loss moduli at baseline were 7.71
± 4.62 MPa, 7.66 ± 4.60 MPa, and 0.84 ± 0.47 MPa,
respectively. Following 8,000 cycles of compressive load-
ing, the corresponding values were 6.57 ± 3.88 MPa, 6.53
± 3.86 MPa, and 0.76 ± 0.41 MPa, respectively. From the
storage and loss moduli, the “before” and “after” values of
the loss factor were calculated as 0.11 ± 0.02 and 0.12 ±
0.02, respectively (Fig. 3). Similar to indentation results,
there was no statistically significant difference in the values
for each modulus before and after cyclic compression.
Correlations between Biomechanical Properties. According to
Spearman rank correlation (Fig. 4), the Young’s modulus at
equilibrium correlated poorly with the instantaneous stiffness
measured on rapid loading. While there was no significant
correlation between the baseline moduli measured using
indentation and unconfined compression, there was a signifi-
cant positive correlation (P < 0.05) between the instanta-
neous modulus and dynamic modulus (ρ = 0.79) as well as
between the instantaneous modulus and storage modulus
(ρ = 0.79) after cyclic compressive loading of the cartilage.
Discussion
In the present study, we developed a novel mechanical test-
ing protocol to enable the biomechanical characterization of
human articular cartilage and conduct a functional test of
cartilage durability. Using a preliminary set of human distal
femoral articular cartilage samples, indentation and uncon-
fined compression tests were conducted in physiological
conditions (37 ± 1 °C) to quantify elastic and viscoelastic
material properties of native human articular cartilage
before and after thousands of compressive loading cycles.
In general, the material properties obtained from this proto-
col, especially the Young’s modulus, compressive modulus,
dynamic modulus, and loss factor, demonstrated good
agreement with values reported using similar methodolo-
gies in the cartilage biomechanics literature (Table 1). The
cartilage samples showed no significant mechanical deteri-
oration in any of the 8 tested parameters following com-
pressive loading at 4 Hz for 8000 cycles, which revealed a
high degree of durability of native human femoral condylar
cartilage and highlighted that a tissue-engineered construct
would need to be highly durable to be clinically suitable for
chondral repair in human knees. While engineered con-
structs may be expected to mature biomechanically over
time, it is essential for the immature tissue to have a mini-
mum level of stiffness to maintain its shape, prevent disin-
tegration, and support chondrogenesis.
Comparisons of absolute values of cartilage mechanical
properties are difficult due to the natural variation of carti-
lage stiffness across a joint surface,30 between different
joints within the same individual20 and between joints of
6
Table 1.  Comparison of Various Mechanical Properties of Human and Large Animal Articular Cartilage from Different Sites, as Measured in Previous Studies and in This Study
(Expressed as Mean, Mean ± SD, or Range).
Cartilage Source Instantaneous Modulus (MPa) Young’s Modulus (MPa) Equilibrium Modulus (MPa) Compressive Modulus (MPa) Dynamic Modulus (MPa) Loss Factor

Animal  
  Bovine humeral — 0.677 ± 0.223 (Jurvelin et al. 1997) (unconfined — — 8.1 ± 6.4a (Nieminen et al. ~0.25 (Lawless
head compression) 2004)22 et al. 2017)38
0.80 ± 0.33 (Korhonen et al. 2002) (unconfined
compression)
1.15 ± 0.44 (Korhonen et al. 2002) (indentation)
0.10-1.60 (Lyyra-Laitinen et al. 1999) (unconfined
compression)
  Bovine patella 20-65b (Brown et al. 2007) 0.57 ± 0.17 (Korhonen et al. 2002) (unconfined — — 8.1 ± 6.4 (Nieminen et al. —
(indentation) compression)27 200422)a
4.0-16 (Brown et al. 2007)21 0.56 ± 0.19 (Korhonen et al. 2002) (unconfined
(indentation) compression)27
0.72 ± 0.19 (Korhonen et al. 2002) (indentation)
0.83 ± 0.21 (Korhonen et al. 2002)
(indentation)
  Bovine femoral — 0.31 ± 0.18 (unconfined compression)27 0.2-0.4 (Laasanen et al 2003)16 ~1.0 (Martin et al. 2000)39 ~15-17 (Martin et al. —
condyles 0.55 ± 0.19 (indentation)27 (unconfined compression) (confined compression) 2000)39
~4-7 (Laasanen et al. 2003)16
  Bovine femoral — — — — — ~0.20 (Lawless
head et al. 2017)38
  Sheep femoral 0.6 ± 0.4 (Di Bella et al. 2018) — 4.5 ± 4 (Di Bella et al. 2018)40 — — —
condyles (indentation) (indentation)
Human  
 Tibial plateau 11.1 ± 2.61d (Stok et al. 3.49 ± 1.06 (Stok et al. 2010)41 (indentation) — — — —
2010)41 (indentation)
 Patella — 0.64 ± 0.30 (Kiviranta et al. 2008)29 (indentation) — 3.1-10 (Shepherd et al. 1999)9 4.67 ± 2.24 (Kiviranta et al. —
2008)29
 Ankle (talar and — — — 10.0-18.6 (Shepherd et al. — —
tibial surfaces) 1999)9
  Femoral head 51.9-89.8e (Burgin et al. 2014)30 — 0.250.7 (Boschetti et al. 2004)28 ~4.89 (Taylor et al. 2012)42 — 0.14-0.17 (Temple
(unconfined compression) (unconfined compression) et al. 2016)43
  Femoral condyles — ~0.70 (Lyyra et al. 1999)23 (indentation) 0.45 ± 0.1 (Bas et al. 2017)17 1.63 ± 0.26 (Bas et al. 2017)17 — 0.12 ± 0.02 (Bas
0.85 ± 0.35 (Jurvelin et al. 2003)44 (unconfined (unconfined compression) (unconfined compression) et al. 2017)17
compression) 0.2-0.4 (Bartnikowski et al. 2015)45 1.70-2.75 (Bartnikowski 0.16-0.17
(unconfined compression) et al. 2015)45 (unconfined (Bartnikowski
0.2-0.58 (Jurvelin et al. 2003)44 compression)45 et al.)45
(unconfined compression) 4.3-13.0 (Shepherd et al. 1999)9
Femoral condyles 52.14 ± 9.47 1.03 ± 0.48 7.48 ± 4.42 10.60 ± 3.62 7.71 ± 4.62 0.11 ± 0.02
(present study)

a
Combined population of bovine humeral head and patella samples.
b
Loaded up to 30% strain at a strain rate of 0.1 s−1.
c
Loaded up to 10% strain at a strain rate of 0.1 s−1.
d
Loaded at a strain rate of 0.08 s−1.
e
Measured instantaneously as the “peak dynamic modulus” on rapid loading (100 g load dropped from 8 cm above cartilage surface with impact velocity of 1.25 m/s).
Kabir et al. 7

Figure 2.  Mechanical properties of articular cartilage tested in and equilibrium loading conditions using indentation geometry before
(blue) and after (red) 8000 cycles of cyclic compression at a frequency of 4 Hz. Pairwise “before” and “after” comparisons for each
sample are shown for (A) instantaneous modulus, (B) Young’s modulus, and (C) equilibrium modulus. The pooled mean values for
equilibrium modulus (D), instantaneous modulus (E), and Young’s modulus (F) are illustrated before and after cyclic compression.

different individuals with similar ages.23 Cartilage stiffness
also varies with thickness,24,31 which differs enormously
between various human joints and between human and ani-
mal joints, which are generally thinner (e.g., 1.4 ± 0.3 mm
for bovine cartilage27). Furthermore, added variability in
experimental measurements arises due to differences in
testing conditions.
The rate of cartilage loading influences the instantaneous
and dynamic moduli,21 which reflect the load support
provided by the interstitial fluid pressurization within the
viscoelastic collagen matrix when cartilage is loaded
dynamically.9 The mean value of instantaneous modulus
obtained for this study was 52.14 ± 9.47 MPa under a load-
ing rate of 1 mm/s, which translates to a strain rate of 0.5 s−1
for the average cartilage thickness of ~2 mm. This rate
simulates the application of a sudden weight-bearing force
on cartilage, for example, when landing from a height.
Brown et al.21 conducted similar indentation studies on
bovine articular cartilage with a strain rate of 0.1 s−1 (20%
of the rate used in the current protocol), yielding values of
instantaneous stiffness around ~10 ± 5 MPa, which is 20%
of and in proportion to the value obtained in this study.
Similarly, another study employed a slower strain rate of
0.08 s−1 for instantaneous loading of human tibial plateau
cartilage, resulting in an instantaneous modulus of 11.1 ±
2.6 MPa, which is another example of the proportionality
observed between loading rate and instantaneous modulus
of the measured cartilage sample. Moreover, Burgin and
colleagues30 measured maximum stress-strain gradients in
the range of ~51.9 to 89.8 MPa following the instantaneous
8 Cartilage 00(0)

Figure 3.  Comparison of dynamic mechanical properties of articular cartilage before (blue) and after (red) cyclic compressive loading
for (A) dynamic modulus, (B) storage modulus, (C) loss modulus, (D) compressive modulus, and (E) loss tangent when tested at 1-Hz
sinusoidal compression (closest loading frequency of knee joint during walking and running). The “after cyclic compression” data were
measured after 4 separate steps of 2,000 cycles of cyclic compression conducted at a frequency of 4 Hz (total of 8,000 cycles).

Figure 4.  Spearman rank correlation matrix to assess the relationship between several mechanical properties of native hyaline
articular cartilage before (left) and after (right) cyclic compressive loading. Values of the correlation coefficient rho (ρ) greater than
0.7 were statistically significant positive correlations (P < 0.05).
Kabir et al. 9
application of a load onto cartilage samples obtained from
elderly human femoral heads. The impact testing in this
study better simulates high impact events such as running or
jumping from a height, similar to the instantaneous testing
in the present study, rather than low-impact activities like
walking. Nevertheless, the instantaneous values also closely
approximate those in the present study.
Evaluation of cartilage viscoelasticity involved cyclic
displacement between a strain amplitude of 1.25% at 1 Hz,
which is close to loading frequencies for walking and
running.37,46 The same conditions were set by Nieminen
et al.22 to test bovine humeral and patellar cartilage, resulting
in a dynamic modulus of 8.1 ± 6.4 MPa, which overlaps with
the values from the present protocol (Table 1). Similarly,
another study utilizing the same frequency obtained values of
4.67 ± 2.24 MPa for human patellar cartilage, which further
validates the results of the present protocol. The loss factor
was also comparable to literature values17,43 (Table 1), sug-
gesting that this protocol can provide an effective assessment
of viscoelastic damping due to sinusoidal compression.
As opposed to dynamic loading, when cartilage is sub-
ject to a constant deformation, the water in the collagen
matrix exudes out until an equilibrium stress is reached. At
this point, the resistance to the applied load reflects only the
intrinsic compressive stiffness of the solid phase of articular
cartilage, which is measured as the Young’s modulus.47 The
present study reported a Young’s modulus of 1.03 ± 0.48
MPa, which overlaps with the range of Young’s moduli
measured in equilibrium conditions (similar to the present
study) for human patella and femoral condyles29,44 (Table
1). The strong correlation between the Young’s modulus
and equilibrium modulus reflects the inherent dependence
of the Young’s modulus on the equilibrium modulus, which
is the slope of the equilibrium stress-strain curve.27 Since
the assumption of a monophasic, isotropic and homogenous
material is required to apply the indentation data to the
mathematical solution for the Young’s modulus, variations
in the values of this elastic property across studies may be
further explained by different Poisson ratio measurements
or incorporation of different scaling factors (k value). While
a value of the Poisson ratio was determined optically by
video microscopy31 in the current protocol, other studies
have calculated this intrinsic elastic parameter indirectly or
assumed a value of 0.5, which applies to incompressible
solids.24,27 Since the formula to obtain the Young’s modulus
is related to the “square” of the Poisson ratio (see Equation
1), variation in this value may explain differences in calcu-
lated Young’s moduli for articular cartilage across different
studies. Similarly, adoption of the theoretical scaling factor
k from Hayes et al.32 (infinitesimal deformation) rather than
Zhang et al.25 (finite indentation depth) in the formula may
alter such measurements.
In unconfined compression, the compressive modulus
was measured as 10.60 ± 3.62 MPa, under slow loading,
that is, a nonequilibrium state. This is very similar to the
4.3 – 13 MPa range obtained by Shepherd et al.9 for the
human femoral condyle (Table 1). Another study17 that
used similar methods to the present protocol has reported
lower values in the range of 1.6 to 2.75 MPa. However, the
sample size of this study was a limitation as the compres-
sive modulus measurements were conducted on a sample
from a single donor rather than multiple donors.17 This
could explain the difference in measurements, which may
be affected by the large topological variation in site-­
specific compressive stiffness across the distal femoral
AC of different individuals.
While numerous indentation and unconfined compres-
sion methods have been established for the mechanical test-
ing of articular cartilage, the major novelties of the current
protocol are that (1) it includes a comprehensive regime
of mechanical tests in physiological conditions using
relatively common instruments to characterize the instanta-
neous, equilibrium elastic, and dynamic viscoelastic prop-
erties of articular cartilage and (2) it is the first protocol to
incorporate cyclic compression to assess compressive dura-
bility. We did not find any significant difference in any of
the mechanical properties measured using indentation
(Fig. 2) or unconfined compression (Fig. 3) following com-
pressive loading, which emphasizes the durable nature of
native human articular cartilage. This negative finding is
particularly significant in the development of minimum
biomechanical benchmarks for cartilage tissue engineering.
However, a limitation was the small patient sample size
(n = 3), which may have impeded the determination of sta-
tistical significance in this study. Furthermore, since the
patients were in the age range of 78 to 82 years, these data
may not be truly representative of healthy, young hyaline
cartilage; this limitation is commonplace in the field of car-
tilage biomechanics due to ethical barriers to the acquisition
of articular cartilage from young patients. Furthermore,
since the distribution of degeneration on the femoral con-
dyles were slightly different for each patient, the mechani-
cal properties of AC may differ across the different samples
due to the topographical variation of cartilage stiffness in
the joint.26 Although the osteochondral samples were metic-
ulously shaped to have a flat surface, the natural curvatures
of the native cartilage surface could have led to subtle inac-
curacies in determination of the contact point (0% strain)
during compressive testing and Poisson ratio measurement,
leading to minor errors in the modulus calculations. Since
8,000 compressive cycles did not effect changes in cartilage
mechanics, the protocol may be modified to incorporate a
greater number of compressive loading cycles to conduct
mechanical failure testing of articular cartilage.
Much of the current research in cartilage mechanics
includes testing of 1 to 3 material parameters of carti-
lage.24,27,31 However, in order to assess the biomechanical
integrity and clinical translatability of an artificially
10 Cartilage 00(0)
engineered construct, a more comprehensive suite of tests
performed under physiological temperature and loading
conditions is essential. The need for simple laboratory tests
as a biomechanical evaluation tool for cartilage tissue engi-
neering formed the motivation for this project. The protocol
developed herein seeks to guide cartilage tissue engineering
colleagues to conduct in-depth mechanical characteriza-
tions of both native and tissue-engineered cartilage.
Conclusion
In the present study, we developed a functional biomechani-
cal testing protocol for native human articular cartilage
under physiologically relevant conditions. Primarily, this
protocol aimed to quantify both the elastic properties under
instantaneous and equilibrium loading conditions, and vis-
coelastic properties under dynamic loading conditions
through a sequence of tests in indentation and unconfined
compression geometry. A secondary aim was to test the
impact of cyclic compressive loading on the mechanical
properties of the cartilage. Using a preliminary set of articu-
lar cartilage samples, we successfully validated the protocol
through comparisons between the material properties calcu-
lated herein and those reported in the literature, taking into
account the heterogeneity in experimental parameters and
tissue sources. There was no significant difference in carti-
lage mechanics before and after 8,000 compression cycles,
highlighting the short-term mechanical durability of native
cartilage. Future experiments with a higher number of load-
ing cycles are required to elucidate the mechanical durabil-
ity of native cartilage. It is hoped that this protocol will aid
tissue engineers in the biomechanical comparisons between
native and tissue-engineered cartilage, to evaluate the clini-
cal suitability of the latter.
Acknowledgments and Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
work was supported by the AOA Research Grant (Grant No. 528),
Victorian Medical Research Acceleration Fund, and the Melbourne
Medical School Mid Career Seeding Grant, The University of
Melbourne.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
ORCID iD
Wassif Kabir https://orcid.org/0000-0003-1408-6370
Ethical Approval
The study was performed following institutional approval (HREC-A
143/16).
Informed Consent
All patients provided written informed consent.
Trial Registration
Not applicable.
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