Regulation of the Friction Coefficient of Articular Cartilage
by TGF-b1 and IL-1b
Grayson DuRaine,1 Corey P. Neu,1 Stephanie M.T. Chan,1 Kyriakos Komvopoulos,2 Ronald K. June,1 A. Hari Reddi1
1
Center for Tissue Regeneration and Repair, Department of Orthopaedic Surgery, University of California, Davis, Medical Center, 4635 Second Ave.,
Sacramento, CA 95817, 2Department of Mechanical Engineering, University of California, Berkeley, CA 94720
Received 19 April 2007; accepted 24 April 2008
Published online 6 August 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20713
ABSTRACT: Articular cartilage functions to provide a low-friction surface for joint movement for many decades of life. Superficial zone
protein (SZP) is a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage that contributes to effective boundary
lubrication. In both cell and explant cultures, TGF-b1 and IL-1b have been demonstrated to, respectively, upregulate and downregulate SZP
protein levels. It was hypothesized that the friction coefficient of articular cartilage could also be modulated by these cytokines through SZP
regulation. The friction coefficient between cartilage explants (both untreated and treated with TGF-b1 or IL-1b) and a smooth glass surface
due to sliding in the boundary lubrication regime was measured with a pin-on-disk tribometer. SZP was quantified using an enzyme-linked
immunosorbant assay and localized by immunohistochemistry. Both TGF-b1 and IL-1b treatments resulted in the decrease of the friction
coefficient of articular cartilage in a location- and time-dependent manner. Changes in the friction coefficient due to the TGF-b1 treatment
corresponded to increased depth of SZP staining within the superficial zone, while friction coefficient changes due to the IL-1b treatment were
independent of SZP depth of staining. However, the changes induced by the IL-1b treatment corresponded to changes in surface roughness,
determined from the analysis of surface images obtained with an atomic force microscope. These findings demonstrate that the low friction of
articular cartilage can be modified by TGF-b1 and IL-1b treatment and that the friction coefficient depends on multiple factors, including
SZP localization and surface roughness. ß 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:249–
256, 2009
Keywords: cartilage; cytokine; growth factors; friction coefficient; SZP/lubricin/PRG4
Articular cartilage is an avascular tissue with limited
innate potential for repair and regeneration that
provides a low-friction surface for joint movement.1
Lubrication of these surfaces is critical to normal joint
function. A boundary lubricant of articular cartilage,
referred to as superficial zone protein (SZP), has been
identified as a product of the proteoglycan 4 gene
(PRG4).2 SZP is a glycoprotein secreted by chondrocytes
in the superficial layer of articular cartilage and is
homologous to lubricin and megakaryocyte stimulating
factor (MSF) precursor.3 The boundary lubricating
efficacy of SZP, either separately or in conjunction with
other synovial fluid components, has been studied at
latex-glass,4–6 cartilage-cartilage,7,8 and cartilageglass9 interfaces. Recent work10 revealed a correlation
between SZP expression level at the articular surface
and short-term friction coefficient at the cartilage-glass
interface. In addition to its function as a boundary
lubricant, SZP inhibits integrative cartilage repair
and synovial cell overgrowth.11,12 SZP is a significant
protein that plays a key role in the normal function of
synovial joints, and human and mouse mutants of the
PRG4 gene display precocious arthropathy.12,13
Changes in cartilage homeostasis are thought to
precede the initiation of osteoarthritis.14 A model of
cartilage homeostasis involves the steady-state maintenance of the tissue and the interplay between mechanical and biochemical environmental signals, such as
cytokines and their interactions with cells and surrounding extracellular matrix.1 With reference to cytokines,
cartilage homeostasis has been modeled as the balance
Correspondence to: Corey P. Neu (T: 916-734-3311; F: 916-7345750; E-mail: [email protected])
ß 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
between the actions of morphogens and growth factors,
such as transforming growth factors (TGF-b), bone
morphogenetic proteins, and insulin-like growth factor,
and the actions of catabolic cytokines, such as interleukins (IL-1b) and tumor necrosis factor alpha. Introduction of inflammatory (catabolic) cytokines, such as IL-1b,
to the articular joint reduces the production of matrix
molecules and leads to changes in matrix constitution
that result in reduced mechanical strength and ability
to maintain normal cartilage function.15,16 It is well
established that cytokines play an important role in
cartilage homeostasis and that SZP is regulated in
part by the cytokines of the joint involved in cartilage
homeostasis.17,18 Previous research in cell culture
demonstrated that the level of SZP secreted into
the media can be controlled by applying different
cytokines.19 Anabolic cytokines and growth factors
(e.g., TGF-b family members) increase the expression of
SZP, while catabolic cytokines (e.g., IL-1b) decrease the
expression and accumulation of SZP.17,19
Differences in the friction coefficient at regions of the
articular cartilage surface with distinct SZP protein
levels motivated the investigation of the effect of changes
in the SZP expression level due to cytokine treatment on
the coefficient of friction. The main objectives of this
investigation were to evaluate the friction coefficient of
cartilage explants treated with TGF-b1 and IL-1b and
examine changes in the friction coefficient in terms of
SZP accumulation and explant surface roughness. An
unexpected decrease in the friction coefficient was
observed after IL-1b treatment, independent of SZP
accumulation. Since IL-1b has been previously identified
to cause damage of the articular surface,16 this finding
was interpreted in the context of articular surface
topography (roughness) characteristics.
JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2009
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DURAINE ET AL.
EXPERIMENTAL PROCEDURES
Materials
Articular cartilage was harvested from 1–3-month-old bovine
stifle (tibiofemoral) joints obtained from a local abattoir within
6 h of sacrifice. DMEM/F12 and antibiotic solution (both from
Invitrogen, Grand Island, NY), cell culture components and
reagents (from Sigma, St. Louis, MO or Fisher Scientific,
Tustin, CA), VECTASTAIN elite ABC Reagent (PK-6105) and
DAB Substrate Kit (both from Vector Laboratories, Burlingame, CA), and human recombinant TGF-b1 or IL-1b (both
from R&D Systems, Minneapolis, MN) were used in this study.
Tissue Acquisition and Culture
Bovine calf stifle joints were opened using an aseptic
technique, and two side-by-side osteochondral explants were
harvested from M1 (anterior and load bearing) and M4
(posterior and non-load bearing) regions of the femoral medial
condyles10 (Fig. 1). Hereafter, these samples will be referred to
as M1 and M4 explants. Four total explants were collected
from each joint, specifically two explants from location M1 and
two from location M4. Only one joint per animal was used in an
experimental group. Explants were matched per joint during
treatment (e.g., control and treated explants from region M1 of
the same joint). The designation as control or treated was
assigned randomly per pair. A 5-mm coring reamer was used to
extract the osteochondral explants, whereas an adjustable
custom-made jig was used to remove subchondral bone and
trim the explants to lengths of 4 mm. The explants were
allowed to submerse and equilibrate in 2 mL of serum- and
cytokine-free culture medium consisting of DMEM/F12, 0.2%
bovine serum albumin (BSA), 1% penicillin/streptomycin,
50 mg/mL ascorbic acid 2-phosphate, and 5% CO2 at 378C for
24 h. The medium was replaced after the equilibration period.
Subsequently, the explants were incubated in a medium
supplemented with 10 ng/mL of TGF-b1 or 10 ng/mL of IL-1b
for 2 or 5 days. Control explants were treated with vehicle
alone, i.e., 5 mM HCl and 0.1% BSA for the TGF-b1 group and
phosphate-buffered saline (PBS) and 0.1% BSA for the IL-1b
group (1 mL per mL of media). The media were changed at day
2 for the 5-day experimental groups, and fresh cytokine or
control vehicle was added. Concentrations of TGF-b1 and
IL-1b were chosen based on previous changes in SZP protein
expression in culture.19,20 A decrease in SZP protein expression has been observed for IL-1a treatment of articular
cartilage7,21,22 and for IL-1b in mandibular condyle chondrocytes23 at similar concentrations.
Friction Testing
To determine the effect of growth factors or cytokines on the
friction coefficient of the articular surface, cartilage explants
treated with either TGF-b1 or IL-1b were tested using a
pin-on-disk tribometer operated in the boundary lubrication
regime in reciprocating sliding mode.24 Explants were harvested from locations M1 and M4 as described previously. The
number of samples (n ¼ 9–14) depended on the specific group
tested (i.e., n ¼ 9 for 2-day TGF-b1 treatment of M1 and M4
explants; n ¼ 11 and 12 for 5-day TGF-b1 treatment of M1 and
M4 explants, respectively; n ¼ 10 for 2-day IL-1b treatment of
M1 and M4 explants; and n ¼ 13 and 14 for 5-day IL-1b
treatment of M1 and M4 explants, respectively). The pin
specimens consisted of explants affixed to acrylic pins by ethyl
cyanoacrylate. The articular surface was brought into contact
with a polished glass disk and was fully immersed in PBS. The
choice of PBS for the current work was made to prevent
confounded interactions between native cartilage surface
molecules (e.g., SZP) and those in a test solution, such as
whole synovial fluid. The glass disk was repositioned or
ultrasonically cleaned after every fourth specimen to ensure
a fresh surface. In all of the tests, the sliding speed was
0.5 mm/s, the radius of the wear track was 5 mm, and the
normal load was 1.8 N, resulting in an average contact
pressure of 0.1 MPa. These load and speed conditions have
been shown to yield sliding in the boundary lubrication
regime.10
Prior to the initiation of each friction test, the sample was
allowed to equilibrate under the applied load (i.e., 0.1 MPa
average pressure) in an unconstrained test configuration for
2 min to minimize any fluid effects during testing.10 Test
parameters, such as normal load, sliding speed, and equilibrium conditions, were determined from analytical and experimental analyses to allow for sufficient interstitial fluid
depressurization and to obtain reproducible coefficient of
friction measurements in the boundary lubrication regime.10,25
The test duration of each friction experiment was fixed at 60 s.
Data were collected at 0.1 s intervals with a data acquisition
system (Labview, National Instruments, Austin, TX) and
processed using a standard software package (Microsoft,
Seattle, WA). Friction force data from each entire test were
used to compute the mean and standard deviation values of the
coefficient of friction for each femoral condyle location and
treatment. These experimental conditions have been previously used to investigate the effect of SZP expression level on
the coefficient of friction of articular cartilage.10
SZP Quantification and Immunohistochemistry
Samples from the treatment groups that resulted in significantly different changes in the friction coefficient were used for
SZP quantification and localization. SZP accumulation in the
media was quantified by enzyme-linked immunosorbent assay
(ELISA), using SZP purified from bovine synovial fluid and
cultured synovium and articular cartilage as a standard.26 The
SZP protein was quantified in media collected from a subset of
Figure 1. Dependence of immunolocalization of the SZP protein on femoral condyle
location at the articular cartilage surface. The
SZP expression at anterior harvest location M1
extended several cell layers into the tissue
compared to the posterior location M4.
JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2009
FRICTION COEFFICIENT OF ARTICULAR CARTILAGE
cartilage explants of standardized volume (n ¼ 9 for each group
tested) following treatment with TGF-b1 or IL-1b. The SZP
content was determined using a standard method from
samples that were serially diluted with PBS and reacted with
S6.79 (1:5,000) as the primary antibody and anti-mouse
horseradish peroxidase-conjugated goat (1:3,000, Vector Laboratories) as the secondary antibody.
For immunohistochemistry, tissues were fixed in Bouin’s
solution overnight, followed by paraffin embedding and
sectioning. Immunostaining was performed following a standard method with S6.79 (1:1,000) as the primary antibody
and an ABC kit (Vector Laboratories) with mouse IgG as
the secondary antibody for signal detection. Another set of
consecutive sections was stained with 1% toluidine blue for
histological examination to verify expected patterns of proteoglycan content (not shown here for brevity). Images were
obtained with an optical microscope (LSM 510, Carl Zeiss, Jena,
Germany) at 100 magnification.
Surface Roughness Measurement
Treatment groups that showed significant differences in the
friction characteristics were further examined in additional
samples using an atomic force microscope (AFM) (model MFP3D-CF, Asylum Research, Santa Barbara, CA) with an
extended z-range of 28 mm. Triangular silicon nitride tips
(model MSCT-AUNM, Veeco, Santa Barbara, CA) with a
nominal tip radius of 10 nm and spring constant of 0.01 N/m
were used in all of the AFM scans. AFM images of 60 60 mm2
scan areas were obtained in contact mode using 128 128
pixels, 1 Hz scan rate, and 2.5 V set point. Force calibration
performed with a glass substrate, using Igor Pro software
(version 5, Wavemetrics, Lake Oswego, OR) and based on
the thermal calibration method, showed that this set
point corresponded to an applied normal contact force of
2.39 0.02 nN.
Samples sectioned with a razor blade to a thickness of
1.5 mm and affixed with ethyl cyanoacrylate to a custommade sample holder were immersed in PBS throughout
imaging to maintain tissue hydration. Sample groups from
each joint comprised treated and control pairs from both M1 and
M4 joint locations (Fig. 1), i.e., four cartilage explants per joint.
Five pairs of M1 explants and six pairs of M4 explants treated
with TGF-b1, and eight pairs of M1 explants and seven pairs of
M4 explants treated with IL-1b were used in the AFM analysis.
Images from five different locations of each explant surface
were used to calculate the mean and standard deviation values
of the root-mean-square surface roughness Rq given by
"
#1=2
N
1X
2
Rq ¼
z
;
ð1Þ
N i¼1 i
where zi is the deviation of the ith measured height from the
mean surface plane and N is the number of data points in each
surface scan.
Mechanical Testing
Compression experiments were performed to evaluate possible
chemical treatment and time-dependent load effects on the
bulk material properties. For this purpose, pairs of articular
cartilage explants harvested from bovine stifle joints were
used as control and treated [5-day treatment with mediasupplemented IL-1b (10 ng/mL), as described previously]
samples, for a total of n ¼ 6, to perform compression experiments. After measuring the sample dimensions with digital
251
calipers, the samples were placed in a custom-made chamber
of a materials testing system (Enduratec 3200, Eden Prairie,
MN) where they were compressed by polished and nonporous
stainless steel platens at a rate of 50 mm/s to an average
pressure of 12.5 kPa (preload). Subsequently, a 5% nominal
compression was applied at a rate of 10 mm/s, resulting in a
peak average pressure of 361.0 0.1 kPa, and data were
collected over a period of 30 min at a sampling frequency of 180
Hz. The average (engineering) stress was determined from the
initial cross-sectional area calculated from the measured
diameter. The initial sample height used to calculate the
engineering strain was measured at the equilibrium platen
position after a 10 min equilibration time. All of the samples
were incubated and tested at 378C. The bulk stiffness was
obtained as the ratio of the engineering stress to the engineering strain.
Statistical Analysis
For each treatment (TGF-b1 or IL-1b), location (M1 or M4),
and treatment time (2 or 5 days) combination, a paired t-test
was performed to determine any significant differences among
the friction coefficients of the treated and untreated control
samples using a standard software package (SAS Institute,
Cary, NC). Differences in SZP expression following cytokine
treatment were evaluated using a paired t-test. Average
surface roughness measurements were compared using a
one-way nested ANOVA for four treatment levels (M1 control,
M1 treated, M4 control, and M4 treated) and both 5-day IL-1b
treatment and 2-day TGF-b1 treatment groups. A nested
analysis was used to account for the multiple (five) measurements obtained from each explant. A significance level of
p < 0.05 was used to determine differences between groups. A
paired t-test was also used in the statistical analysis of the
bulk stiffness of the control and treated samples used in the
compression experiments. Results are presented in the form of
data points representing mean values one standard error of
the mean.
RESULTS
Coefficient of Friction
The friction coefficient demonstrated a dependence on
anatomical location, cytokine, and treatment duration.
The 2-day TGF-b1 treatment of posterior M4 explants
produced a decrease in friction coefficient compared to
the untreated explants (p ¼ 0.035; Fig. 2A). The friction
coefficients of the explants from other locations did not
change (statistically) following 2-day TGF-b1 or IL-1b
treatment compared to controls (p > 0.445). Five-day
IL-1b treatment of M1 explants resulted in lower
friction coefficient than that of the controls (p ¼ 0.015;
Fig. 2B). The friction coefficients of explants from
other locations did not change following 5-day TGF-b1
or IL-1b treatment compared to controls (p > 0.396).
SZP Quantification and Immunohistochemistry
SZP accumulation in media did not vary statistically
following either 2-day TGF-b1 treatment or 5-day IL-1b
treatment of M1 explants compared to untreated
controls (p > 0.149; Fig. 3A, B). The SZP depth of
staining in M4 explants increased after a 2-day TGFb1 treatment compared to untreated controls (Fig. 3C).
Furthermore, SZP localization after TGF-b1 treatment
JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2009
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DURAINE ET AL.
Figure 2. Effects of femoral condyle
(explant) location and type and duration
of treatment on the friction coefficient of the
cartilage surface. (A) Two-day treatment
with TGF-b1 of explants from location M4
yielded a decrease in friction coefficient
over untreated controls, while 2-day treatment with IL-1b produced insignificant
differences compared to that of controls.
(B) Five-day treatment with IL-1b of
explants from location M1 yielded a
decrease in friction coefficient over
untreated controls, while 5-day treatment
with TGF-b1 or IL-1b of all the other
explants did not produce statistically different friction coefficients. (The bars with the
asterisks correspond to p < 0.035.)
of the M4 explants was similar to that of the untreated
M1 explants. The SZP staining in all of the cartilage
explants did not show discernible changes after a 5-day
IL-1b treatment (Fig. 3D).
Surface Roughness
Figure 4 shows surface roughness results and representative topography images of treated and control
explants obtained from cartilage locations M1 and M4.
The surface roughness [Equation (1)] of the M1 explants
increased as a result of the 5-day IL-1b treatment
(Rq ¼ 465.96 63.53 nm) compared to that of the
controls (Rq ¼ 271.41 22.92 nm) (p < 0.0001), while
other differences between treatments with TGF-b1
and IL-1b and explants from locations M1 and M4 were
insignificant (p > 0.05) (Fig. 4A, B). The AFM images
shown in Figure 4C and D demonstrate that the surface
topographies of the M1 explants contained fibrillated
structures and fine-scale irregularities, while those of
the M4 explants were essentially featureless. It appears
Figure 3. Dependence of SZP staining
depth in cartilage explants on location and
type of treatment: (A) 2-day treatment with
TGF-b1; (B) 5-day treatment with IL-1b.
(C) and (D) show corresponding immunolocalization SZP results.
JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2009
FRICTION COEFFICIENT OF ARTICULAR CARTILAGE
253
Figure 4. Surface roughness
of cartilage explants from locations M1 and M4 treated for
5 days with (A) TGF-b1 and
(B) IL-1b. Representative AFM
images of explant surface topographies after (C) 2-day treatment with TGF-b1 and (D) 5-day
treatment with IL-1b. Results
from corresponding untreated
(control) explants are shown
as a reference. (The bar with
the asterisk corresponds to
p < 0.0001.)
that the 5-day IL-1b treatment resulted in loss of the
fibrillated structures (especially for M1 explants) and
the development of larger wavelength surface waviness.
Mechanical Behavior
The bulk properties of the explants were not influenced
by the IL-1b treatment. In particular, insignificant
differences (p > 0.512) in the stiffness of control and
IL-1b treated explants were found after 5, 120, and
1,800 s (i.e., equilibrium) in stress relaxation experiments (Fig. 5). In addition, the stiffness was constant for
both control and treated samples after 120 s.
DISCUSSION
The presented results indicate a dependence of the
friction coefficient of articular cartilage on explant
location and cytokine (TGF-b1 or IL-1b) treatment. It
was found that the SZP staining depth in the cartilage
explants was influenced by the type and duration of the
TGF-b1 treatment. These experimental findings demonstrate that the addition of these cytokines has direct
implications on the friction coefficient of the articular
surface. The decrease in the friction coefficient following
the 2-day TGF-b1 treatment corresponded to increased
SZP staining depth in the explants. The 5-day IL-1b
treatment resulted in significant surface roughening of
the explants harvested from the anterior cartilage
location.
The decrease in the friction coefficient of the M4
explants following a 2-day TGF-b1 treatment (Fig. 2A)
was attributed to the increased depth of staining of SZP
in the near-surface region (Fig. 3A, C). SZP immunolocalization to a depth within the cartilage has been shown
previously.19,20,27 The SZP level and the decrease in the
friction coefficient of these explants were similar to those
of the untreated M1 explants. An increase in the depth of
SZP staining of the M1 explants was also detected after
a 2-day TGF-b1 treatment, although the friction coefficient did not differ significantly from that of the
untreated M1 explants (p > 0.05). The lack of further
reduction in the friction coefficient of the TGF-b1 treated
M1 explants is presumed to be a consequence of an
articular surface with saturated SZP (Fig. 3C). A
comparison of SZP accumulation in the media of TGFb1 treated and untreated M1 explants (p > 0.05, Fig. 3A)
suggests that this treatment did not result in a
significant increase in SZP released into the medium,
despite the increased staining depth indicated by the
SZP immunolocalization results (Fig. 3C). Although SZP
Figure 5. Stiffness of untreated and treated (5-day treatment
with IL-1b) cartilage explants obtained after 5, 120, and 1800 s
(equilibrium) under 5% compression. The data show insignificant
differences (p > 0.512) in stiffness between treated and untreated
samples for a given time.
JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2009
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DURAINE ET AL.
was released in the media of the 2-day culture model, the
former finding suggests that an available pool of SZP still
remained within the explant, depending on sample
location and cytokine treatment.
The 5-day TGF-b1 treatment did not produce a
decrease in the friction coefficient of the M4 explants
(Fig. 2B). It is believed that this resulted from the loss of
SZP under the longer culture conditions (even with the
TGF-b1 treatment). The SZP (PRG4) concentration is
commonly expressed in mg/cm2. Based on the data shown
in Figure 3A and the 5-mm explant diameter and 2-mL
media volume, the SZP expression levels of M1 and M4
explants are in the range of 100–200 mg/cm2. Recently
published data of SZP released into media using slightly
smaller (i.e., 3 mm diameter) explants from the patellofemoral groove have shown SZP expression levels of
30 and 40 mg/cm2 per 24 h after incubation for 1 and
2 days, respectively.27
The decrease in the friction coefficient following a
5-day IL-1b treatment of the M1 explants (Fig. 2B)
indicated that a compensatory mechanism might have
increased the SZP in the matrix similar to that observed
for the 2-day TGF-b1 treatment. An increase in SZP that
would account for the decrease of the friction coefficient
did not occur during the IL-1b treatment (Fig. 3).
However, alterations in GAG content,28 which was not
measured in this study, have been observed to change the
friction coefficient. The friction coefficients of the 2-day
IL-1b treated M1 and M4 explants were similar (Fig. 2B).
Differences in the SZP staining in the controls of the
2-day and 5-day immunohistochemistry data (Fig. 3C, D)
were attributed to culture conditions (i.e., duration of
treatment) and not to differences in the vehicle controls,
since both would have been diluted at a ratio of 1:1,000.
Although changes in the cartilage coefficient of
friction that resulted from the 2-day TGF-b1 treatment
corresponded to the increase of the SZP staining depth
in the superficial zone, in the case of the 5-day IL-1b
treatment, the friction coefficient did not correspond to
the SZP staining depth. Consequently, it was hypothesized that the IL-1b could have had a destructive effect on
the articular cartilage surface. The destruction of the
articular cartilage matrix by IL-1b is a well-known effect
that produces characteristic damage features attributable to surface erosion.15,29 IL-1b has been partly
implicated in cartilage matrix degradation by enhancing
the expression of matrix metalloproteinases (MMPs)14,30
and aggrecanases.29 Furthermore, there is evidence that
IL-1b and TNF-a colocalize with MMPs in the superficial
layer of the arthritic cartilage, revealing a key role of this
layer in the pathogenesis of arthritic diseases.15
Cartilage surface roughening was only observed with
M1 explants following 5-day IL-1b treatment (Fig. 4B).
Surface topography images (Fig. 4C, D) showed a
generally fibrous structure with feature heights as large
as 1 mm. The disruption of the distinct fibrous structure
on the surfaces of the M1 explants after 5-day IL-1b
treatment may be responsible for both the increased
surface roughness and the higher scatter in the roughJOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2009
ness measurements. Surface roughening decreases the
real contact area, resulting in lower friction.31 This effect
is bimodal since large asperities may increase friction
due to the enhancement of asperity interlocking, while
very smooth surfaces yield larger real contact areas and,
in turn, higher friction coefficients. Alternatively, early
destruction of the cartilage matrix components by IL-1b
actions may lead to a loosely bound and short-lived layer
of molecules. While the low shear strength of this layer
could contribute to a decrease in the coefficient of friction,
it would be expected to also increase the wear rate
dramatically, producing a detrimental effect on the longterm durability of the joint. Therefore, the higher surface
roughness of articular cartilage after 5-day IL-1b treatment may be attributed to the degradation of molecules
in the lamina splendens resulting from enzymatic
degradation cascades induced by the IL-1b. However, it
is not clear whether surface roughening was due to an
overall increased fibrillation of small to moderately sized
topographical maxima and minima occurring in high
frequency at the surface or the formation of large but
infrequent extremes along an otherwise smooth surface.
Insight into the specific roughening mechanism during
IL-1b treatment would elucidate the pattern and
distribution of the degradation of surface and matrix
molecules and their role in boundary lubrication of
articular joints. Additionally, representative images
(Fig. 4) were chosen based on the collective difference
between the average roughness of all measurements and
those of a single joint. The surface roughness of the
chosen sample was within 33 nm of respective average
roughness values. AFM images revealed variability in
the surface structure between joints, which has also been
observed in other studies.32,33
In the present work, AFM images were acquired using
an ultra-sharp (i.e., 10 nm radius) tip attached to a
flexible microcantilever. Although an ultra-sharp tip is
desirable for imaging fine surface features, it may alter
the surface topography by scratching. To minimize the
contact force exerted on the cartilage surface by the
sharp tip while maintaining the capability to detect
extreme height variations, a microcantilever of very low
stiffness was used in all of the AFM scans. Similarly
sharp probe tips have been used to image articular
cartilage, and the obtained blurred images were attributed to the high viscosity of the surface.33 However, the
probe stiffness in that study was two times higher than
that used in this investigation. Except for some evidence
of surface smearing, a microcantilever of stiffness equal
to 0.01 N/m (as opposed to 0.022 N/m) mitigated the
problem of smearing in the present AFM scans. The
surface roughness values of the control samples (typically 271 23 nm) (Fig. 4A, B) are comparable with the
roughness of articular cartilage surfaces measured with
micrometer-sized probe tips (379 83 nm)34 and blunt
probe tips (462 216 nm).32
The decrease in the friction coefficient of the articular
cartilage treated with IL-1b is likely culture condition
dependent. To avoid confounding results of other
FRICTION COEFFICIENT OF ARTICULAR CARTILAGE
cytokines on SZP expression during TGF-b1 or IL-1b
treatment, all tissues were maintained in serum-free
chemically defined media. Furthermore, the explants
remained in an unloaded free-swelling condition. This
culture technique does not exactly mimic the in vivo
situation of a rich cytokine environment and associated
mechanical loading, both of which can regulate SZP
expression.17,19,21,35,36 This may further explain the loss
of SZP in the controls of the 5-day culture media and
tissue (Fig. 3B, D).
The effects of potential confounding factors, such as
interstitial fluid pressurization and/or bulk changes in
the matrix due to chemical treatment, on the obtained
results were found to be minimal. Considering the
contact parameters used in this study, particularly the
low average contact pressure (0.1 MPa), and the stiffness
results obtained from compression experiments (Fig. 5),
fluid depressurization occurred within 2 min under load.
In addition, increasing the equilibration time up to
10 min did not produce any changes in the coefficient of
friction measured upon subsequent sliding in the
boundary lubrication regime.10 In view of the results
shown in Figure 5, it may be interpreted that the bulk
material properties were not altered significantly by the
cytokine treatment under the experimental conditions of
this study. It should be realized that time effects in the
stress-relaxation experiment do not directly resemble
those in a creep experiment (as was used in the friction
testing). Additionally, while the effects of interstitial
fluid depressurization cannot be completely ruled out
(although believed to be minimal based on analytical and
experimental results10,25), any influence of depressurization would similarly influence all samples tested.
Therefore, at a minimum, relative differences in the
friction coefficients of the control and treated samples are
relevant and important for comparison.
From a perspective of applying these results to tissue
engineering and regenerative medicine, producing a
construct that replicates the lubricating function of
articular cartilage depends on regulating the friction
coefficient of that construct, potentially by using TGF-b1
or other growth factors and morphogens. The results
of this investigation illustrate the need for multiple
functional assessments (including biochemical and tribological assays), which can ascertain the long-term
lubricating ability of a construct. To produce functional
constructs for the regeneration of articular cartilage,
a set of criteria, including the presence of boundary
lubricants (i.e., SZP) and characterization of the surface
roughness, must be satisfied to ensure a construct that
would provide a long-lasting repair.
ACKNOWLEDGMENTS
The authors are grateful to Dr. J. Zhou (University of
California, Berkeley) for technical support and Dr. T. Schmid
(Rush University, Chicago, IL) for the generous gift of the
S6.79 primary antibody. This research was funded by the
National Institutes of Health under Grant No. NIBIB 1F32
EB003371-01A1, the Lawrence J. Ellison Endowed Chair, and
255
the National Science Foundation under Grant No. CMS0528506.
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