Engineering lubrication in articular cartilage.
ABSTRACT Despite continuous progress toward tissue engineering of functional articular cartilage, significant challenges still remain. Advances in morphogens, stem cells, and scaffolds have resulted in enhancement of the bulk mechanical properties of engineered constructs, but little attention has been paid to the surface mechanical properties. In the near future, engineered tissues will be able to withstand and support the physiological compressive and tensile forces in weight-bearing synovial joints such as the knee. However, there is an increasing realization that these tissue-engineered cartilage constructs will fail without the optimal frictional and wear properties present in native articular cartilage. These characteristics are critical to smooth, pain-free joint articulation and a long-lasting, durable cartilage surface. To achieve optimal tribological properties, engineered cartilage therapies will need to incorporate approaches and methods for functional lubrication. Steady progress in cartilage lubrication in native tissues has pushed the pendulum and warranted a shift in the articular cartilage tissue-engineering paradigm. Engineered tissues should be designed and developed to possess both tribological and mechanical properties mirroring natural cartilage. In this article, an overview of the biology and engineering of articular cartilage structure and cartilage lubrication will be presented. Salient progress in lubrication treatments such as tribosupplementation, pharmacological, and cell-based therapies will be covered. Finally, frictional assays such as the pin-on-disk tribometer will be addressed. Knowledge related to the elements of cartilage lubrication has progressed and, thus, an opportune moment is provided to leverage these advances at a critical step in the development of mechanically and tribologically robust, biomimetic tissue-engineered cartilage. This article is intended to serve as the first stepping stone toward future studies in functional tissue engineering of articular cartilage that begins to explore and incorporate methods of lubrication.
- [Show abstract] [Hide abstract]
ABSTRACT: The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation, and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. While dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP; with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and TGFβ1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFβ1. A decrease in TGFβ1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified via paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFβ-induced SZP accumulation. TGFβ-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1), and Y27632 (Rho effector Rho Kinase). On the other hand lysophosphatidic acid, an upstream activator of Rho, increased SZP synthesis in response to TGFβ1. These results suggest that SZP production is dependent on the functional cytoskeleton, and Rho GTPases contribute to SZP accumulation by modulating the actions of TGFβ.Tissue Engineering Part A 10/2013; · 4.64 Impact Factor
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ABSTRACT: Effective lubrication of synovial joints is important to prevent cartilage degeneration and to keep the joints healthy. This paper sets out the basics of engineering lubrication with respect to the composition and properties of synovial fluid constituents. Two basic types of boundary lubrication are discussed: the presence of highly hydrophilic proteoglycans that provide a water liquid film, and the existence of multilamellar phospholipids lubricating layers at the surface ofarticular cartilage. Based on current knowledge, we may conclude that no single mechanism of boundary lubrication exists, and that effective boundary lubrication of synovial joints is maintained by the synergic effect of all synovial fluid constituents.Wiener Medizinische Wochenschrift 10/2013;
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ABSTRACT: Costochondral cells may be isolated with minimal donor site morbidity and are unaffected by pathologies of the diarthrodial joints. Identification of optimal exogenous stimuli will allow abundant and robust hyaline articular cartilage to be formed from this cell source. In a three factor, two level full factorial design, the effects of hydrostatic pressure (HP), transforming growth factor beta1 (TGF-beta1), and chondroitinase ABC (C-ABC), and all resulting combinations, were assessed in third passage expanded, redifferentiated costochondral cells. After 4 wks, the new cartilage was assessed for matrix content, superficial zone protein (SZP), and mechanical properties. Hyaline articular cartilage was generated, demonstrating the presence of type II collagen and SZP, and the absence of type I collagen. TGF-beta1 upregulated collagen synthesis by 175% and glycosaminoglycan synthesis by 75%, resulting in a nearly 200% increase in tensile and compressive moduli. C-ABC significantly increased collagen content, and fibril density and diameter, leading to a 125% increase in tensile modulus. Hydrostatic pressure increased fibril diameter by 30% and tensile modulus by 45%. Combining TGF-beta1 with C-ABC synergistically increased collagen content by 300% and tensile strength by 320%, over control. No significant differences were observed between C-ABC/TGF-beta1 dual treatment and HP/C-ABC/TGF-beta1. Employing biochemical, biophysical, and mechanical stimuli generated robust hyaline articular cartilage with a tensile modulus of 2 MPa and a compressive instantaneous modulus of 650 kPa. Using expanded, redifferentiated costochondral cells in the self-assembling process allows for recapitulation of robust mechanical properties, and induced SZP expression, key characteristics of functional articular cartilage.Arthritis research & therapy 12/2013; 15(6):R214. · 4.27 Impact Factor
Engineering Lubrication in Articular Cartilage
Sean M. McNary, B.S.,1Kyriacos A. Athanasiou, Ph.D., P.E.,1,2and A. Hari Reddi, Ph.D.1
Despite continuous progress toward tissue engineering of functional articular cartilage, significant challenges
still remain. Advances in morphogens, stem cells, and scaffolds have resulted in enhancement of the bulk
mechanical properties of engineered constructs, but little attention has been paid to the surface mechanical
properties. In the near future, engineered tissues will be able to withstand and support the physiological
compressive and tensile forces in weight-bearing synovial joints such as the knee. However, there is an in-
creasing realization that these tissue-engineered cartilage constructs will fail without the optimal frictional and
wear properties present in native articular cartilage. These characteristics are critical to smooth, pain-free joint
articulation and a long-lasting, durable cartilage surface. To achieve optimal tribological properties, engineered
cartilage therapies will need to incorporate approaches and methods for functional lubrication. Steady progress
in cartilage lubrication in native tissues has pushed the pendulum and warranted a shift in the articular cartilage
tissue-engineering paradigm. Engineered tissues should be designed and developed to possess both tribological
and mechanical properties mirroring natural cartilage. In this article, an overview of the biology and engineering
of articular cartilage structure and cartilage lubrication will be presented. Salient progress in lubrication treat-
ments such as tribosupplementation, pharmacological, and cell-based therapies will be covered. Finally, fric-
tional assays such as the pin-on-disk tribometer will be addressed. Knowledge related to the elements of
cartilage lubrication has progressed and, thus, an opportune moment is provided to leverage these advances at a
critical step in the development of mechanically and tribologically robust, biomimetic tissue-engineered carti-
lage. This article is intended to serve as the first stepping stone toward future studies in functional tissue
engineering of articular cartilage that begins to explore and incorporate methods of lubrication.
tant quality-of-life issues for all healthy adults. However,
for more than 26 million adults in the United States alone,1
degenerative joint disease or osteoarthritis (OA) impairs these
daily activities and reduces their quality of life.2Regenerative
medicine, through tissue engineering, aims at producing
functional, engineered synovial tissues to replace and restore
these damaged joints. Despite its seemingly simple structure,
articular cartilage regeneration has been elusive and presents
an important clinical challenge. As the connective tissue lo-
cated on the ends of long bones, articular cartilage is able to
support and distribute large mechanical loads while provid-
ing a nearly frictionless surface for joint movement. Recently,
exciting progress has been made in engineering tissues
with mechanical properties, such as compressive and tensile
strength, approaching native tissue levels.3–5The next grand
challenge in cartilage engineering is to address perhaps the
most important functional attribute of cartilage: lubrication.
ain-free ambulation and joint movement are impor-
The lubrication mechanisms of articular cartilage impart
the tissue with its low friction properties. Maintenance of
these tribological properties is crucial to reducing wear and
ensuring healthy and functional cartilage for the lifetime of
the individual. For example, ineffective joint lubrication has
been demonstrated to play an important role in the devel-
opment of precocious joint degeneration and OA.6,7There-
fore, engineered cartilage should possess both mechanical
and frictional properties to function and endure in vivo.
Historically, the tribological aspects of cartilage tissue engi-
neering have been a secondary concern compared with
achieving the necessary mechanical properties. This ap-
proach was a practical prioritization, as the bulk of the en-
gineered tissue needed to withstand large mechanical loads
before surface characteristics became a concern. However,
with this goal closer to being realized, the state of tissue
engineering has advanced sufficiently to make a case for a
shift in this paradigm. In this age of mechanically functional
cartilage, tribological characterization and development
need to become a key component of any articular cartilage
1Department of Orthopaedic Surgery, Lawrence Ellison Center for Tissue Regeneration and Repair, School of Medicine, University of
California, Davis, Sacramento, California.
2Department of Biomedical Engineering, University of California, Davis, Davis, California.
TISSUE ENGINEERING: Part B
Volume 18, Number 2, 2012
ª Mary Ann Liebert, Inc.
tissue-engineering strategy (Fig. 1). The aims of this article
are to provide a synopsis of the biology and engineering of
lubrication in articular cartilage, and present approaches to
lubrication engineering. In addition to tissue-engineering-
based approaches, other methods of cartilage lubrication
augmentation such as tribosupplementation and pharma-
cological therapies will also be discussed. In presenting this
information, we wish to demonstrate that the foundations
have been laid for expansion into this exciting frontier of en-
gineering cartilage lubrication. The studies described in this
article should serve as a vital stepping stone in directing tissue
engineers of articular cartilage along the path toward me-
chanically and tribologically robust, engineered cartilage.
Structure-function relationships in articular cartilage
Articular cartilage distributes forces and lubricates the
contact surfaces between articulating long bones, as in the
knee.8,9Cartilage is an avascular and aneural tissue com-
posed of water (60%–85%), rich in extracellular matrix
(ECM), and a relatively small volume of cells, the chon-
drocytes.9,10The major ECM constituents consist of type II
collagen and the proteoglycan aggrecan.11Other ECM
components include cartilage oligomeric protein (COMP),
cartilage link protein, hyaluronan, biglycan, and decorin. The
maintenance and homeostasis of the ECM is regulated by
chondrocytes by enzymatic degradation and secretion of
matrix. Cells comprise only 2%–10% of cartilage by volume,
and this cell density decreases with age.12,13These cells
possess low proliferative and metabolic abilities.13Nutrient
and oxygen transport is mediated by diffusion. Transport
within the tissue is augmented by convection driven by cy-
clic compression.9Inherent deficiencies in cellularization and
vascularization prevent articular cartilage in adults from
engaging in any substantial self-repair.
Articular cartilage is an anisotropic and heterogeneous
tissue at multiple levels. The ECM structure, chondrocyte
phenotype, and cell shape varies between each zone.14,15
Macroscopically, cartilage is divided into three zones: su-
perficial, middle, and deep.8The surface layer, or superficial
zone, possesses type II collagen fibrils aligned parallel with
the articular surface.16Collagen fibrils are, thus, oriented in
the superficial zone to resist shearing forces at the articular
surface generated by joint articulation. Cell density is the
greatest in the superficial zone,12where chondrocytes as-
sume a flattened shape.17The central 40% to 60% of cartilage
that comprises the middle zone contains randomly aligned
type II collagen fibrils and rounded cells. The remaining 30%
makes up the deep zone, which is mineralized in the depths
of this zone. The so called ‘‘tide mark’’ is the interface be-
tween the unmineralized and mineralized ECM. In the deep
zone of articular cartilage, collagen fibrils are aligned per-
pendicularly to the articular surface. Chondrocytes are
aligned similarly in columns of ellipsoidal cells. Proteoglycan
content increases from the superficial zone to the deep zone,
whereas hydration decreases through each zone.17
Since cartilage is mechanically loaded, the forces are
supported by a combination of ECM and water. Water is
retained in the ECM by aggrecan, a post-translationally
modified proteoglycan with negatively charged glycosyla-
minoglycans (GAG), such as chondroitin sulfate and keratan
sulfate, which attract cations into the ECM. The resulting
osmotic pressures hydrate articular cartilage and provide
resistance to compression. This support is complemented by
type II collagen fibrils, which provide the tensile strength.18
Stress relaxation in human cartilage due to fluid exudation
and ECM interactions occurs over 10,800s.19Articular car-
tilage is a mechanically robust material, as it must endure
localized loading of nearly 18MPa in the hip.20Over two
decades ago, Mow et al.10,21developed a biphasic theory to
tionally follows a paradigm of utilizing primary chon-
drocytes or stem cells, growth factors and morphogens, and
scaffolding material to develop constructs that aim to replace
natural cartilage. Current functional approaches rely on ex-
perimental methods driven by biomechanical properties.
Advancement toward artificial, biomimetic cartilage will
require future developments in the tribological properties of
The tissue engineering of articular cartilage tradi-
ENGINEERING LUBRICATION IN ARTICULAR CARTILAGE89
mathematically characterize the material properties of this
complex, viscoelastic tissue. Using this biphasic model, the
aggregate compressive modulus of cartilage has been found
to vary between 0.53 and 1.82MPa. Tensile modulus mea-
surements have varied between 1 and 20MPa, depending on
the cartilage zone measured.13
Articular Cartilage Lubrication
The functional lifetime of articular cartilage is dependent
on minimizing friction and wear. Friction (F) is the opposing
force generated from the relative movement of two contact-
ing surfaces. The magnitude of friction is linearly propor-
tional to the applied normal load (W) by the coefficient of
friction (l), and is the product of multiplying the two vari-
ables: F=m$W.22Hyaline cartilage possesses the lowest
measured friction coefficient of any material, ranging from
0.005 to 0.02.10Wear occurs when asperities (microscopic
surface roughness) from opposing surfaces come into contact
and deform, resulting in removal of material.23Frictional
forces can be moderated by fluid films or lubricants that
separate the interfacing materials, preventing solid–solid
contact and reducing material deformation. Lubrication of
articular surfaces can be divided into two categories: fluid
film and boundary lubrication.
Fluid film-lubricated interfaces are separated by a viscous
fluid with a thickness greater than the surface roughness to
prevent contact.24The fluid forces that support the applied
loads from the interacting surfaces are generated through
hydrostatic and hydrodynamic mechanisms. Under hydro-
static lubrication, the opposing surfaces are separated by an
externally pressurized fluid film.24,25For hydrodynamic lubri-
cation, fluid film forces are generated by the entraining or
sliding speed of the articulating surfaces. The thickness of a
hydrodynamic film is a function of multiple factors such as
fluid viscosity, geometry and roughness of the articulating
surfaces, applied normal load, and sliding speed.23In the
elastohydrodynamic lubrication regime, the fluid film thickness
approaches the order of asperity heights.26The applied loads
and fluid pressures result in elastic deformation of the ar-
Under conditions not conducive for fluid film lubrication
such as low sliding speeds, high loads, and low fluid vis-
cosity, articulating surfaces are separated by a molecular film
or boundary lubrication.10,27,28Asperities are separated by this
one- to two-molecule thick layer of boundary lubricant.29
According to one model, surfaces are protected by a sacrifi-
cial layer of strongly adsorbed boundary lubricant that is
sheared away during sliding.30Boundary lubricants should,
therefore, be quickly and continuously replenished to pre-
vent wear from the next round of solid–solid contact.24
During the walking cycle, the articulating surfaces of the
knee bear a wide variety of contact stresses and sliding
speeds. For example, the sliding speed becomes zero during
each directional change in the leg swing.31Thus, biological
joints operate under a mixed lubrication regime where ar-
ticulating surfaces are subjected to both fluid film and
boundary lubrication.10,24,27,28,31Within mixed lubrication,
weeping and boosted lubrication may occur. Weeping lubri-
cation is generated by the release of interstitial fluid from
asperity-asperity contacts compress the cartilage matrix,
pressurizing the ECM and inducing interstitial fluid exuda-
tion. Boosted lubrication occurs under joint loading condi-
tions that force fluid back into the ECM, effectively
increasing (or ‘‘boosting’’) the concentration of lubricant
confined at the articular surface.34For detailed information
on lubrication mechanisms and wear of articular joints, the
reader is directed to a review on biotribology by Neu et al.24
Boundary Mode Lubricants
Fluid film lubrication relies on fluid forces and rheo-
logical phenomena generated from joint motion. However,
after finding the lubricity of synovial fluid was not entirely
dependent on viscosity, investigators began searching for
the lubricating component of synovial fluid. This research
led to the important discoveries on boundary lubrication,
which is unique in that it relies on molecular interactions
and biochemical properties to provide the last layer of
defense in preventing solid–solid contact and degradation
of the articular surface.28Researchers have focused on
three molecules as putative boundary lubricants: hya-
luronic acid (HA), surface active phospholipids (SAPLs),
and superficial zone protein (SZP)/lubricin/PRG4 (Fig. 2).
There is a debate in the literature regarding the importance
and relevance of these putative lubricants and boundary
mode lubrication in general.24,35–37Despite these disagree-
ments, compelling results from many meticulous studies
merit the consideration of these physiologically important
HA, or hyaluronan (Fig. 2B), is a nonsulfated GAG
composed of the repeating sugars glucuronic acid and
N-acetylglucosamine.38Lacking a protein core, HA is syn-
thesized at the plasma membrane into the extracellular
space. HA is a major constituent of the ECM and is also
involved with cell signaling by binding with the cell surface
receptor CD44.38The compressive and viscoelastic properties
of cartilage tissue are due in part to the aggregating com-
plexes resulting from HA association with aggrecan and link
protein.38HA plays a major role in fluid film lubrication
by providing viscosity to synovial fluid through its high
molecular weight (0.5–3.8·106Da) and concentration (0.1–
5mg/mL).10,39However, the role of HA in boundary lubri-
cation is under debate.
Radin et al.40,41found that digestion or separation of HA
from bovine synovial fluid reduced the viscosity, but did
not affect the boundary lubrication of the treated fluid.
These results have been confirmed most recently by fric-
tion measurements of hyaluronidase-treated cartilage with
an atomic force microscope.28In addition, HA has not been
found to bind or adsorb to the cartilage surface, a re-
quirement for boundary lubrication.42,43
contrast, HA decreased friction in a cartilage–cartilage in-
terface. In combination with PRG4, the reduction was ad-
ditive.44Experiments using the surface force apparatus
suggest that HA serves a chondroprotective role by pre-
venting wear of the articular surface, rather than reducing
the coefficient of friction.45,46The utility of HA varies by
the frictional test system employed. Additional work is
needed to pinpoint the exact role of HA in the boundary
90McNARY ET AL.
Surface active phospholipids
Phospholipids such as phosphatidylcholine (Fig. 2C),
phosphatidylethanolamine, and sphingomyelin have been
identified as constituents of synovial fluid and bound to the
articular surface.47,48Inspired by their role in reducing sur-
face tension (surface active or surfactant) in the lungs, SAPLs
have been proposed as a boundary lubricant for articular
cartilage.47A strongly adsorbed layer of SAPLs could pro-
vide hydrophobicity to the articular surface and shield as-
perities from solid–solid contact. The literature contains
conflicting reports on the boundary lubricity of SAPLs in
synovial joints. Enzymatic digestion of SAPLs with phos-
pholipase was shown to eliminate the lubricating ability of
synovial fluid.49However, these results were disputed in a
later report, suggesting phospholipase may contain traces of
protease activity due to trypsin.35Phospholipase supple-
mented with trypsin inhibitors was found to have a minimal
effect on the lubricity of synovial fluid. Other studies that
examined the effects of SAPL degradation on the cartilage
surface found no effect on the frictional coefficient.28While
lacking universal agreement,37the current consensus is that
SAPLs do not contribute to articular cartilage boundary
A hyaluronate-free protein fraction of bovine synovial
fluid was purified by Radin et al. in 1970.40This protein
possessed similar boundary lubricating properties to that of
whole synovial fluid. The name lubricin was christened after
its purification a decade later.50Lubricin (227kDa) is one
of the several proteins encoded by the gene prg4. Other
homologous proteins include SZP, PRG4, megakaryocyte-
stimulating factor precursor, and hemangiopoietin.51–54Dif-
ferences between these homologous proteins appear to result
from post-translational O-linked glycosylation.53Lubricin
is typically expressed by synovial fibroblasts, whereas SZP
is produced by articular chondrocytes in the superficial
zone. Much work has been performed in characterizing the
biochemical properties, and biological and mechanical
mechanisms of these lubricating proteins. SZP is a heavily
glycosylated protein with an apparent molecular weight of
345kDa (Fig. 2A). Middle- and deep-zone chondrocytes
express little to no SZP.55,56SZP has also been found to be
expressed in the infrapatellar fat pad, meniscus, tendon,
and ligaments.57–60It is hypothesized that SZP forms a
nanofilm that reduces and smoothens asperities on artic-
ular cartilage, reducing ‘‘stick-slip motion’’ and subse-
SZP: Critical Boundary Lubricant
There is a general consensus in the cartilage lubrication
field that SZP is a critical lubricant for articular cartilage. This
hypothesis is based on an abundant number of studies in-
volving a human genetic disorder,6,62rodent gene knockout
models,63,64animal models of arthritis,65–68and functional
tribological assays.61,69In addition to serving as a boundary
lubricant, SZP has been found to prevent hyper-proliferation
of synovial cells and fouling of the articular surface.64These
functions are particularly important. Patients with campto-
dactyly-arthropathy-coxa vara-pericarditis syndrome (CACP),
a genetic autosomal recessive disease, endure excessive sy-
novial growth, fouling of the articular surface, and precocious
joint failure.6,64,70A study by Sah and coworkers71also sug-
gested that SZP expression inhibits integration of apposed,
articular cartilage surfaces, and, by extension, cartilage repair.
SZP expression is modulated by growth factors, mor-
phogens, and other cytokines. Transforming growth factor-b
(TGF-b) and bone morphogenetic protein (BMP) upregulate
structures of three putative
boundary lubricants, (A)
superficial zone protein
(SZP), (B) hyaluronic acid,
and (C) surface active
represented. SZP is a 345kDa
proteoglycan and a product
of the gene prg4. Hyaluronic
acid is a disaccharide
polymer consisting of repeat-
ing units of glucuronic acid
Phosphatidylcholine can be
composed of a variety of fatty
ENGINEERING LUBRICATION IN ARTICULAR CARTILAGE91
SZP production in both superficial zone chondrocytes and
synoviocytes.72–74These effects are additive in both popula-
tions.72Upregulation of SZP production in articular chon-
drocytes by insulin-like growth factor-1, a stimulator of
aggrecan expression, appears to be context dependent.74In-
terleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a), cata-
bolic cytokines, decrease SZP expression in synoviocytes
and articular chondrocytes.73–76This modulation of SZP
also mirrors tribological effects observed in cartilage. TGF-b1
treatment increases SZP deposition and decreases the coef-
ficient of friction in bovine cartilage explants.69Although
IL-1 also decreases the coefficient of friction by increasing
articular surface roughness, in the long term, this damage
accelerates wearing of the articular surface.
SZP is heterogeneously distributed across the articular
surface.61,77The topographical variation can be partially at-
tributed to in vivo mechanical loading. In the bovine stifle
joint, expression was found to be primarily localized to load
bearing, anterior regions (M1) of the femoral condyles (Fig.
3A, 3B).61Nonloaded, more posterior locations (M4) pro-
duced significantly less SZP. Increased amounts of SZP from
load-bearing regions were found to correlate with decreased
coefficients of friction. This localization within load-bearing
regions suggests that SZP production is mechanosensitive. In
addition, when cartilage explants were isolated from M1 and
M4 and subjected to reciprocal sliding, SZP protein was
upregulated in M1 explants but not M4 tissue (Fig. 3C). This
sliding shear-mediated upregulation of SZP was inhibited by
SB431542, an inhibitor of TGF-b receptor type I kinase,
suggesting a role for TGF-b signaling in SZP mechan-
otransduction.61Taken together, the functional phenotype of
articular chondrocytes differs across the geometry of condyle
in addition to depth.
The mechanical regulation of SZP has been investigated
using other loading methods. Nugent et al.78found SZP ex-
pression to be modulated by unconfined, compressive, me-
chanical forces. Patellofemoral, articular cartilage explants
were compressed for 1 day using static conditions of 6 and
100kPa, or oscillatory compressive loads (0.01Hz) of 3–
10kPa and 3–300kPa. All loading conditions induced a sig-
nificant decrease in SZP protein immediately following
treatment.78Compared with unloaded controls, only the
dynamic loading of 3–300kPa induced a significant 46% in-
crease in SZP secretion at day 1 post-treatment. In addition,
static treatment of 100kPa induced a significant decrease of
SZP production by day 3 post-treatment. All other treat-
ments were insignificant at all other time points.78In another
study, no changes were observed in prg4 gene and protein
expression when bovine articular chondrocytes seeded in
porous polyurethane scaffolds were dynamically com-
pressed at 10%–20% strain at 0.1Hz.79It is difficult to draw
firm conclusions from a limited sample set. Although SZP
synthesis is responsive to compressive loading, the lack of a
robust response suggests that compression may not act as a
major mechanical regulator of expression.
In monolayer cultures, 0.5% and 3.0% biaxial strain of
primary human chondrocytes induced a nearly twofold in-
crease in transcription of prg4.80However, identical treat-
ment of chondrocytes passaged thrice resulted in 5-fold and
2.5-fold decreases, respectively. This study corroborates
other publications demonstrating a change in chondrocyte
gene expression due to dedifferentiation of chondrocytes
during monolayer culture.81An earlier study looked at the
effects of cyclic hydrostatic pressure (5MPa) and cyclic ten-
sion at 9% strain (0.5Hz, 3h/day for 3 days) on hypertrophic
and matrix proteins in primary bovine chondrocytes seeded
in 2% alginate gel. Cyclic tension significantly upregulated
SZP mRNA, whereas hydrostatic pressure had no effect.82
Due to its anatomical location at the articular surface,
superficial zone chondrocytes experience shearing forces.
Bovine cartilage explants dynamically loaded (0.1Hz) under
3% shear strain, and 20% compressive strain produced over
three-times greater PRG4 protein than either unloaded or
compressively loaded controls.83These experimental obser-
vations utilizing tensile, shear, sliding shear, and compres-
sive mechanical stimuli suggest that SZP expression is
sensitive to multiple types of mechanical loading conditions
SZP plays a key role in the development and homeostasis
of the joint as illustrated in prg4 knockout mice and CACP
patients.6,62–64Mice lacking prg4 were born with ordinary
joints. However, during maturation, Rhee et al.64observed a
loss of superficial zone chondrocytes, fouling of the articular
surface, and synovial hyperplasia; characteristics observed in
CACP. Mechanical tests of articular cartilage from prg4
knockout mice demonstrated an increase in the coefficient of
loading (M1) in the femoral condyles of bovine stifle joints is
(B) significantly greater than in regions experiencing lower
contact stresses (M4). L, lateral condyle; M, medial condyle;
1–4, locations on condyle numbered from anterior to pos-
terior. SZP expression is (C) increased by sliding shear
loading in cartilage explants obtained from M1. SZP syn-
thesis in M4 explants is unaffected by shear. (Mean–SEM,
Expression of SZP in (A) regions of high in vivo
92McNARY ET AL.
Table 1. Effects of Mechanical Stimulation on Superficial Zone Protein Expression in Articular Chondrocytes
Effects on SZP expression
Nugent et al.78
1- to 3-w/o bovine
3–300kPa, 0.01Hz, for 24h
Secretion into media increased
46%, 1 day after loading
Wong et al.82
Calf (bovine) humeral
Primary MZ/DZ cells in 2%
9% strain (0.004MPa), 0.5Hz,
3h/day for 3 days
Gene expression increased
1.5-fold, 1 day after loading
Kamiya et al.116
6- to 8-m/o porcine
Confluent monolayer of
mandibular condyle cells
7% or 21% strain, 0.5Hz, for 12,
24, or 48h
7% strain increased gene and
protein expression; 21% strain
decreased protein secretion
Nugent et al.83
1- to 3-w/o bovine
3% strain, 0.1Hz (with 20% static
compression) for 1 day
Secretion into media increased
three- to fourfold during
loading, 1 day and 2 days after loading
Neu et al.61
Femoral condyle explants
0.5mm/s, 0.1MPa, for 5min
Secretion into media increased
two- to threefold, 2 days after
Wong et al.82
Calf (bovine) humeral
Primary MZ/DZ cells in 2%
5MPa, 0.5Hz, 3h/day for 3 days
No significant change in gene
expression, 1 day after loading
Grad et al.100
3- to 4-m/o bovine
Metacarpal joint cells in
Hip ball rotated 60? 0.6Hz,
applied 10%–20% comp. strain,
for 1h, 2x/day, for 3 days
Gene expression increased
sevenfold, protein secretion
detected and increased during
1- to 3-w/o bovine
Patellofemoral groove and
femoral condyle explants
10?–46? flexion, 110?/min
(0.025Hz), for 1 day
Increased % of PRG4 positive
cells in regions subjected to continuous sliding, no
significant differences insecretion
y/o, year-old; m/o, month-old; w/o, week-old; MZ/DZ, middle zone/deep zone; 2x, twice; SZP, superficial zone protein.
friction and a decrease in superficial zone cartilage stiffness
compared with wild-type mice.63These results suggest that
SZP dysfunction may play a role in OA. Indeed, numerous
animal models of OA demonstrate a downregulation in lu-
bricin synthesis and increase in friction coefficient after knee
injuries such as anterior cruciate ligament (ACL) transection
and meniscectomy.65–68,84,85These animal models were con-
firmed by human studies where lubricin expression was found
to be decreased in individuals afflicted with ACL injuries.86In
vitro models of post-traumatic OA, such as hyper-physiological
impact loading of articular cartilage, similarly display de-
creased levels of prg4 expression.87Inflammatory or rheuma-
toid arthritis animal models also follow this response.68,88
SZP protein levels were upregulated in cartilage explants
obtained from human knees with advanced OA. This study
complements other reports by suggesting an overactive re-
covery in SZP expression later in the progression of OA.89
However, this rebound could be classified as ‘‘too little, too
late’’ given the ruinous state of the cartilage. Taken together,
there is persuasive evidence linking OA disease development
with dysfunctional lubricin regulation. The downregulation
of SZP leads to increases in joint friction and wear.7,69,90
Replenishment of boundary lubricants by supplementation
may inhibit wear and prevent significant cartilage degrada-
tion from occurring.
Approaches to Enhancing Cartilage Lubrication
To successfully recapitulate cartilage lubrication, fluid film
and boundary modes need to be established. Due to complex
loads and sliding speeds, articular cartilage should function
over mixed regimes of lubrication. However, modulation of
hydrostatic and hydrodynamic lubrication is a difficult task
due to the multifactorial nature of these regimes. Small al-
terations to the joint such as fibrillations on the articular
surface can impede the formation of fluid films.10Boundary
lubrication requires only a molecular monolayer preventing
asperity-asperity contact, and as such is conceptually sim-
pler. Regeneration of functional and enduring articular car-
tilage will require the restoration of boundary lubrication.
Potential OA treatments exploiting this approach include
intra-articular injections of lubricants or tribosupplementa-
tion, pharmacological agents, and cartilage tissue engineer-
ing. Each of these therapeutic strategies has advantages and
limitations that will be discussed.
The underlying hypothesisof boundary lubricant-
centered treatments is that maintaining or increasing
boundary lubricant levels (tribosupplementation) may slow
or prevent the onset of cartilage degradation. Accordingly,
the effects of lubricin were examined in rat models of OA. In
two separate studies, purified lubricin, full-length recombi-
nant human PRG4, and modified recombinant lubricin
(LUB:1) were injected into knees 7 days after surgical in-
duction of OA.84,85Compared with controls, increased lo-
calization of lubricin at the articular surface and decreased
cartilage degradation were observed. Long-term studies are
needed to determine whether intra-articular supplementa-
tion of SZP halts or simply slows the progression of OA.
In clinical practice, intra-articular injections of HA have
been approved by the FDA for the symptomatic relief of
OA.91This treatment is considered ‘‘viscosupplementation’’
due to the effect of increasing the viscosity of synovial fluid
and supporting fluid film lubrication.92However, HA may
also function as a tribosupplement according to results from
a boundary lubrication study in a cartilage-cartilage sys-
tem.44HA and SZP could form a potential combination
therapy for OA, improving both the rheological properties of
synovial fluid and boundary mode lubricity.84,93
In addition to LUB:1, other tribosupplements have been
proposed and experimentally tested in vitro. Chawla et al.94
functionalized PRG4 with an aldehyde group to increase
binding to the cartilage surface. Other GAG such as chon-
droitin sulfate have also been studied as boundary lubri-
cants.95Based on SAPLs, an Israeli group engineered
phospholipid-based liposomes to behave as boundary lu-
bricants.96Factors to be considered in designing a tribo-
supplementation therapy include cartilage surface binding
affinity, reservoir kinetics, and delivery route. Intra-articu-
lar injections would provide the easiest, direct method of
administration and minimize systemic interactions. The
tribosupplement reservoir in the synovial fluid would de-
pend on the enzymatic degradation rate as well as synovial
capsule retention, which would affect the dosage frequency.
Lubricant reservoir (synovial fluid) concentrations could
have a large effect on binding kinetics and replenishment of
the boundary lubricant. Lastly, the lubricant itself will need
to strongly bind or adsorb to the cartilage surface so that
interfacial contact is continuously prevented with a lubri-
cating, molecular monolayer.
Boundary lubrication is the final, defensive guard against
wear. SZP or other boundary lubricants have not been found
to repair the ECM or induce cell signaling for cell-mediated
regeneration. Additional lubricant (SZP) was observed to
have no beneficial effects in severely damaged cartilage.89
Thus, tribosupplementation would likely be most effective as
a preventative treatment of OA.
Another approach toward lubricant replenishment is
stimulating endogenous, cellular production. Much work has
been published on the cellular pathways controlling expres-
sion of boundary lubricants. For example, growth factors such
as TGF-b and BMP have been demonstrated to upregulate the
expression of SZP, whereas catabolic cartilage cytokines such
as IL-1b and TNF-a depress expression.72–75More impor-
tantly, modulation of SZP protein expression levels has a di-
rect functional significance, as it led to corresponding changes
in the frictional properties of cartilage explants.69,90This ex-
perimental evidence strongly suggests that a pharmacological
or bio-therapeutic agent that stimulates the production of
SZP or some other boundary lubricant could provide chon-
droprotection and serve as a bioactive method of tribosup-
plementation. Supporting this theory, Elsaid et al.97found that
administration of etanercept, a TNF-a inhibitor, increased the
synovial fluid concentration and cartilage surface localization
of lubricin in ACL-transected rats. In summary, several
pathways of SZP regulation have been identified in the liter-
ature that may serve as suitable targets for pharmacological
There are several barriers that should be surmounted to
enable the success of a pharmacological therapy. First,
94McNARY ET AL.
signaling pathways such as TGF-b are pleiotropic and uti-
lized by many other different cell phenotypes and cellular
processes. Any potential drug candidates would need to
specifically regulate SZP synthesis without disrupting other
cellular functions that could lead to deleterious side effects. In
addition, therapies would need to be successfully delivered to
the articular cartilage. Drug delivery could potentially be
achieved through oral medications, intra-articular injections,
or a hydrogel carrier for controlled release. The method of
delivery selected would depend on several factors such as
compound stability and cross-reactivity with other cellular
processes. Similar to any other drug therapy, it could take
many years and millions of dollars of research to develop a
working pharmaceutical compound to regulate SZP expression.
However, with a disease such as OA that affects a significant
percentage of the population, the possible payoff is large.
Incorporating boundary lubrication
into engineered cartilage
Boundary lubrication enhancement would in all likelihood
be unable to ameliorate the symptoms of OA due to extensive
degradation of the articular surface. Since cartilage is recalci-
trant to self-repair, the only recourse would be regeneration
of the articular surface. Tissue-engineered cartilage has the
potential to fill this need by providing replacement tissues
that enable pain-free joint articulation. Features that prevent
wear such as boundary lubrication will be key to the success
of these constructs. Since work progresses on enhancing me-
chanical characteristics such as aggregate and elastic moduli,
newer generations of engineered tissues are beginning to
address frictional properties.98–102
Since native cartilage operates under mixed modes of
lubrication, a boundary lubricant will need to be incorporated
into any cartilage engineering approach. Independent of a
scaffold or cellular self-assembly approach, the construct
should facilitate binding and concentration of the boundary
lubricant at the interfacial surface.99Lubricant localization at
the articular surface is crucial in two other respects. One, bulk
retention of the lubricant in the matrix may diminish surface
availability.98Two, the boundary lubricant may interfere with
in vivo implantation and tissue integration as PRG4 has been
observed to prevent cell- and cartilage-cartilage adhesion.71
Another design consideration is lubricant source. The
most common method will likely be a biomimetic approach
that incorporates a resident population of cells into the
construct. In accordance with the constraints just defined,
boundary lubricant secreting cells would be located along
the articular surface of the construct. Challenges of limited
cell availability are especially acute for SZP-based ap-
proaches, as only superficial zone cells possess the requisite
chondrocyte phenotype.55,56Stem cells have the potential to
provide a plentiful cell source, as mesenchymal progenitors
have been induced to express PRG4 at the protein lev-
el.58,99,103With the appropriate treatment and differentiation
protocol, mesenchymal stem cells or embryonic stem cells
could also serve as sources of SZP-secreting, articular chon-
drocytes. Middle- and deep-zone chondrocytes may have the
ability to secrete SZP at superficial zone levels.104However,
induction is not achievable with current methods, as little is
known about the mechanism behind the zone-specific ex-
pression of SZP.
Methods of lubrication assessment
The functional tissue engineering of articular cartilage will
require careful analysis and modulation of characteristics
such as friction and wear to replicate the tribological prop-
erties of native tissue. Many tools exist for measuring the
friction properties of tissue engineered constructs, each of-
fering unique capabilities and limitations. Perhaps the most
widely used frictional assay is the pin-on-disk tribometer,
useful for determining macroscopic friction.61,105Numerous
configurations have been implemented106,107with the same,
basic operating principle: the sample surface is placed in
contact with an opposing surface such as glass. Normal loads
are applied to the sample from a fixed weight or actuator. As
one surface reciprocates or rotates about the other, the op-
posing frictional force or torque is measured.61,99
Macroscale coefficients of friction can also be assessed
using the surface force apparatus.46The SFA 2000 with
friction device attachment operates similarly to a pin-on-disk
tribometer: a cartilage sample is linearly reciprocated against
an opposing surface such as glass. Displacement is controlled
by either a motorized micrometer or bimorph (piezoelectric)
slide. The resistive, friction force is measured by a
tribological properties of
constructs may be developed
sequentially or in parallel
from a variety of potential
stimuli and regulatory
mechanisms involved in
articular cartilage lubrication.
ENGINEERING LUBRICATION IN ARTICULAR CARTILAGE 95
Wheatstone bridge strain gauge system. A ‘‘3D/XYZ’’ con-
figuration permits measuring independent forces in all or-
Friction forces can also be measured at the nanoscale using
atomic force microscopy (AFM).109,110An advantage of this
approach is that boundary lubrication is measured without
the confounding effects of fluid film lubrication or interstitial
fluid release.31As the AFM cantilever is scanned across the
surface, surface interactions are probed by measuring the
deflection of the cantilever.111Frictional forces and surface
topography/roughness can be obtained using this method.
Electron microscopy can also be employed to determine
nanoscale features.31Through a combination of these tests,
wear can be gauged through measured changes in friction
and roughness. In addition, wear products such as pro-
teins released into the lubricating solution (i.e., phosphate-
buffered saline) can be quantified through biochemical tests
such as the bicinchoninic acid assay.112,113
As a greater number of investigators turn their attention
toward lubrication engineering, it will be important for the
field of cartilage tissue engineering to come to an agreement
regarding issues such as assay operating parameters and a
set of success criteria. For example, it is difficult to compare
friction measurements when publications have used different
sets of normal loads and sliding speeds, as these parameters
are important for determining the mode and mechanism of
lubrication. The evaluation of different treatments could be
simplified greatly by a set of measurement standards. Fi-
nally, to determine the efficacy of lubrication, methods of
scoring for both friction and wear are needed. For instance, a
mutually accepted set of success criteria could apply equal
weights to low friction and low wear. It is imperative that
both sets of measurements be performed, as wear particles
can reduce friction and, thus, bias the tribological data.69
Perspectives in the Emerging Field
of Engineering Lubrication
It is not clear whether tribological characteristics can be
adjusted after mechanical maturation, or whether tribological
properties need to be developed alongside mechanical char-
acteristics. Since the iron is still hot with regard to bulk
properties, it is a good time to strike and address these and
other important questions (Fig. 4). For example, it remains to
be determined exactly how mechanical and biochemical
properties, such as compressive modulus and collagen II
content, will influence variables such as surface roughness
and lubricant binding/entrapment. Will a homogeneous
construct replicate these features, or is a particular ECM dis-
tribution and zonal heterogeneity essential for cartilage lu-
brication? In addition, as synthetic scaffold technology
matures and attains the mechanical characteristics of native
cartilage tissue, how will the tribological properties of the
scaffold evolve as well? Either the scaffolding will be natively
suited for low friction,102or the material may need to be
modified with lubricious molecules such as SZP. With the
recent advances in engineering increasingly stiffer constructs,
it is now time to shift the existing cartilage tissue-engineering
paradigm and begin addressing these questions in anticipa-
tion of meeting the next major challenge, cartilage lubrication.
Beyond lubrication, additional significant obstacles remain.
Outstanding questions include the integrity and stability of
engineered tissues in the demanding and complex biological
and mechanical environment of living articular joints. Work is
already being performed in this area, as synovial bioreactors
capable of simulating these conditions are currently in de-
velopment.114,115In addition, identification of suitable in vivo
animal models is needed, as it is unknown whether normal or
OA-induced animal joints are capable of replicating the con-
ditions of injured or diseased human joints. However, before
these issues become relevant, it is paramount that biomimetic,
engineered cartilage be developed in vitro that possesses both
native tissue-level mechanical and frictional properties.
The authors wish to thank Dr. Corey Neu for his help with
illustrations in Figure 3. Funds from the Lawrence J. Ellison
Endowed Chair supported the initial experimental work. The
current research is supported by the National Institute of
Arthritis and Musculoskeletal and Skin Diseases, NIH
(NIAMS 1R01 AR061496).
No competing financial interests exist.
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Address correspondence to:
A. Hari Reddi, Ph.D.
Department of Orthopaedic Surgery
School of Medicine
Lawrence Ellison Center for Tissue Regeneration and Repair
University of California, Davis
4635 Second Ave., Room 2000
Sacramento, CA 95817
Received: July 12, 2011
Accepted: September 28, 2011
Online Publication Date: January 5, 2012
100McNARY ET AL.