? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
Regulation of chondrogenesis and chondrocyte
differentiation by stress
Michael J. Zuscik, Matthew J. Hilton, Xinping Zhang, Di Chen, and Regis J. O’Keefe
Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, USA.
Introduction to cartilage
Cartilage is a connective tissue that is comprised primarily of
matrix (mainly collagens and proteoglycans) containing relatively
sparse populations of chondrocytes, which perform matrix-gen-
eration and maintenance functions. During the development and
growth of vertebrates, chondrogenesis is the dynamic cellular pro-
cess that leads to the establishment of various types of cartilage,
including hyaline, fibrous, and elastic cartilage. Hyaline cartilage
is found in craniofacial structures, the trachea and bronchial
tubes, the articular surfaces of diarthrodial joints, and the growth
plate (GP) of long bones. GPs are responsible for driving the pro-
cess of limb lengthening and bone growth during development
pre- and postnatally. This type of bone growth involves the pro-
cess of endochondral ossification (otherwise known as bone for-
mation). The type of cartilage that is most prominent and most
susceptible to both normal and pathologic forms of stress is the
hyaline cartilage of the limb and trunk skeleton, which originates
from the differentiation of condensed mesenchymal cells into
clusters of cartilage cells known as chondrocytes. These cartilage
anlagen preform the skeleton and provide a framework for endo-
chondral bone development, a process that involves chondrocyte
maturation and matrix mineralization in the GPs. The cells of
each skeletal element proceed through a multi-step differentia-
tion process generating both the mature GP cartilage, which con-
trols skeletal growth during early and adolescent development,
and the permanent articular cartilage (AC) found at the joint sur-
face of all long bones.
The processes of chondrogenesis and endochondral bone forma-
tion are not restricted to the developing skeletal system. In fact,
following stress-related injuries, such as fractures of endochon-
dral bone, the developmental programs of chondrogenesis and
chondrocyte proliferation, maturation, hypertrophy, and termi-
nal differentiation are reinitiated at the site of injury. Addition-
ally, stress-related cartilage diseases such as osteoarthritis (OA)
also have marked effects on the differentiation and maintenance
of AC during adult life. This is why much attention has been paid
to studying the cellular and molecular mechanisms that regulate
chondrogenesis and chondrocyte differentiation. At the begin-
ning of this Review, we describe the processes of chondrogenesis
and chondrocyte differentiation in the hope of highlighting some
of the critical molecular regulators of these processes. With this
information as a foundation, we provide a synopsis of how several
normal and pathologic stressors, including normal mechanical
loading, hypoxia, fracture healing, heterotopic ossification, fibro-
dysplasia ossificans progressiva (FOP), OA, and a number of key
environmental factors such as heavy metals and cigarette smoke,
impinge upon the progression of these processes. The list of stress-
ors discussed in this Review is not all inclusive, but it represents
the most well understood modalities by which stress can have an
impact on the chondrogenic and cartilage maturation programs.
Overall, understanding the molecular and signaling basis underly-
ing the influence of stress on cartilage will provide the foundation
for the development of therapeutic paradigms for ameliorating
the impact of pathologic stress in particular.
Cellular events and molecular markers
of chondrogenesis and chondrocyte differentiation
during endochondral ossification
Chondrogenesis is a process that is important for the creation
of chondrocytes both during embryogenesis as well as in adult
life (e.g., during skeletal tissue repair). The process begins with
the aggregation and condensation of loose mesenchyme. Factors
such as the bone morphogenetic proteins (BMPs) are known to
play critical roles in the compaction of mesenchymal cells and
the shaping of the condensations (1). During this early step in
chondrogenesis, the condensing mesenchyme expresses various
ECM and cell adhesion molecules, including the IIa splice form of
type II collagen [Col2a1(IIa)] (2, 3), N-cadherin (Ncad) (4), N-cam
(Ncam1) (5), and tenascin C (Tnc) (6), while also broadly express-
ing an important transcription factor, SRY-box 9 (Sox9) (Figure
1A, i, and Figure 1B). The Sox family of transcription factors has
various roles during chondrogenesis and chondrocyte differentia-
tion, although Sox9 is the primary determinant during the early
stages of chondrogenesis (7, 8). As the mesenchyme differenti-
ates into chondrocytes, the cells begin to produce an ECM rich in
the IIb splice form of type II collagen [Col2a1(IIb)] and aggrecan
(Agc). During development, following early chondrocyte differen-
Nonstandard?abbreviations?used: AC, articular cartilage; Agc, aggrecan; AP, alkaline
phosphatase; BMP, bone morphogenetic protein; Col2a1(IIa), the IIa splice form of
type II collagen; FOP, fibrodysplasia ossificans progressiva; GP, growth plate; MSC,
mesenchymal stem cell; OA, osteoarthritis; SOX9, SRY-box 9; SZ, superficial zone;
Tnc, tenascin C.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 118:429–438 (2008). doi:10.1172/JCI34174.
430? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
tiation, the cells rapidly proliferate, enlarging the cartilage tem-
plates that preform individual skeletal elements (Figure 1A, i and
ii, and Figure 1C). Cells near the center of each growing element
eventually withdraw from the cell cycle, initiating the process of
hypertrophic differentiation (Figure 1A, iii). During the process
of hypertrophic differentiation (i.e., maturation), chondrocytes
enlarge, terminally differentiate, mineralize, and ultimately
undergo apoptosis. As the chondrocytes die and degrade, their
residual cartilage matrix serves as a scaffold for further mineral
deposition and bone formation and turnover by invading osteo-
blasts and osteoclasts. In parallel, the degraded cartilage becomes
vascularized by surrounding blood vessels to establish the bone
marrow cavity (Figure 1A, iv).
The cartilage GPs located at each end of a developing long bone
are generated through a continual process of chondrocyte prolif-
eration, differentiation, and removal. During this process, distinct
zones of cells are both morphologically and molecularly identifi-
able. In the embryo, cells near the distal ends of cartilage elements,
periarticular chondrocytes, appear round in shape and express
early chondrocyte lineage markers such as Sox9, Col2a1(IIb), Agc1,
and low levels of FGF receptor 3 (Fgfr3) (9), as well as specific
downstream targets of the indian hedgehog (Ihh) signaling path-
way (10–13) (Figure 1A, ii, and Figure 1C). As these cells prolifer-
ate and undergo the early steps of maturation, they flatten and
form columns parallel to the axis of longitudinal growth. The flat
columnar chondrocytes, known to be the most proliferative cells in
the cartilage element, express low levels of Runx2 and Osterix (Osx)
and high levels of Fgfr3, Nkx3.2, and Ptc1. Eventually, these colum-
nar cells begin the process of hypertrophy and withdraw from
the cell cycle. The prehypertrophic chondrocytes enlarge slightly
and initiate expression of Ihh, parathyroid hormone–related pro-
tein receptor (PTHrP-R), as well as increase expression of alkaline
phosphatase (AP), and the important regulatory transcription fac-
tors Runx2 and Osx, which aid in chondrocyte differentiation as
well as being required for mineralization of the cartilage (14–16).
As hypertrophy proceeds, the cells continue to enlarge, generate a
mineralized matrix, and further enhance their expression of type X
collagen (Col10a1), Runx2, and several growth factors that coordi-
nate chondrocyte proliferation and differentiation. These factors
are critical for signaling to the surrounding perichondrial cells to
Cellular events and molecular markers of chon-
drogenesis, chondrocyte differentiation, and AC
development and maintenance. (A) Model of
endochondral bone development beginning with
mesenchymal cell condensation (i); chondrocyte
differentiation and development of the cartilage
template (ii); chondrocyte maturation and hyper-
trophy (iii); separation of cartilage growth regions,
vascular invasion, and initiation of both cortical and
trabecular bone (iv); and finally generation of the
secondary center of ossification that separates AC
and GP cartilage during postnatal bone develop-
ment (v). MC, marrow cavity; 2°, secondary center
of ossification. Red lines mark the vasculature, and
yellow coloration marks mineralized bone. Black
box outlines AC region magnified in B. (B) Graphi-
cal representation of the distinct cellular zones in
postnatal AC. IZ, intermediate zone; RZ, radial
zone; TM, tide mark; ZCC, zone of calcified carti-
lage; SB, subchondral bone; M, marrow. Vertical
lines indicate zones of gene expression. (C) Model
outlining the process of chondrogenesis and chon-
drocyte differentiation. Important markers at each
stage of chondrocyte differentiation are listed below
the stage at which the genes are expressed. Super-
scripts indicate the level of gene expression.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
induce these cells to differentiate into osteoblast lineage cells that
further mineralize the matrix after endochondral ossification is
complete. Both hypertrophic chondrocytes and the more termi-
nally differentiated hypertrophic chondrocytes located in the cen-
ter of the cartilage produce high levels of Vegfa, which is thought
to aid in vascularization of the dying cartilage (17). Only the most
terminally differentiated hypertrophic chondrocytes express the
matrix degrading enzyme Mmp13 (18). MMP13 is an enzyme that
controls degradation of the cartilage matrix, a process that pre-
cedes mineralization by osteoblasts, that is required for creation
of the bone marrow space, and that supports vascular invasion,
which provides the cells that will populate the bone marrow (Fig-
ure 1A, iii and iv, and Figure 1C). As a point of reference, Figure 1C
depicts a summary of the genetic program in chondrocytes under-
going hypertrophic differentiation.
During early postnatal development, epiphyseal chondrocytes
(immature chondrocytes located in the center of the epiphyses
of long bones) undergo maturation similar to the chondro-
cyte differentiation process described above that occurs during
embryonic skeletogenesis. These cells differentiate, hypertrophy,
undergo apoptosis, and are replaced by invading vasculature
and osteoblasts, creating the secondary center of ossification
(labeled 2° in Figure 1). The secondary center of ossification
separates the only two areas of remaining cartilage within indi-
vidual long bones of the adult skeleton: the AC and the mature
GP cartilage (Figure 1A, v). When chondrogenesis and chon-
drocyte maturation occur in the adult, such as during fracture
repair, no secondary centers of ossification are formed, nor is
a novel limb generated, but the processes progress essentially
the same way and hypertrophic chondrocytes are ultimately
required for analogous purposes — the initiation of mineraliza-
tion and the induction of vascular invasion.
In the mouse, the development of AC begins during embryo-
genesis at sites of synovial joint formation. These joints develop
through processes including patterning of the joint site, inter-
zone formation, cavitation, and morphogenesis (reviewed in refs.
19, 20). It has been demonstrated that articular chondrocytes
are formed from interzone cells during development. Following
embryonic joint formation and postnatal growth, the adult skel-
eton maintains the cellularity and phenotype of AC via mecha-
nisms largely unknown, whereas GP cartilage completely erodes
following adolescent growth in humans. Adult AC is maintained
as four distinct cellular zones: the superficial zone (SZ), the inter-
mediate zone (IZ), the radial zone (RZ), and the zone of calcified
cartilage (Figure 1B). The SZ consists of 1–2 layers of flattened
chondrocytes expressing proteoglycan 4 (Prg4) (also known as SZ
protein and lubricin), Sox9, Col2a1(IIb), Agc1, Tnc, and low levels
of cartilage intermediate layer protein (Cilp). Chondrocytes of the
IZ are round in appearance and express many of the same mol-
ecules as the SZ chondrocytes, although they do not express Prg4
and express higher levels of Cilp. Below the IZ reside the RZ chon-
drocytes and the zone of calcified cartilage. The RZ chondrocytes
express markers of chondrocyte differentiation and hypertrophy
such as Col10a1 and AP. Each of the AC regions is normally main-
tained throughout adulthood unless stress related injury, inflam-
mation, or a genetic defect leads to the loss of either the signals
required to maintain these cells or the signals required to inhibit
excessive differentiation of these cells. Disruption or impairment
of the signals that inhibit excessive differentiation is believed to
provide the basis for diseases such as OA.
Effects of stress on chondrogenesis and endochondral
ossification during normal skeletal development
Within the framework of the processes of chondrogenesis and chon-
drocyte differentiation described above and depicted in Figure 1,
information about the regulatory impact of normal mechanical and
physiologic stress has been published. Presented below is a summary
of the influence of normal mechanical and hypoxic stress on the
programs of chondrocyte commitment and differentiation.
Mechanical stress. Cellular perception of mechanical stress within
cartilaginous tissues is an important modulator of chondrocyte
function. This is particularly the case in AC, where the sensing of
mechanical forces by chondrocytes leads to profound changes in
the health and normal function of the joint (21, 22). Mechanical
stresses sensed by chondrocytes are often referred to as mechano-
electrochemical events (23), and these coordinate with other envi-
ronmental, hormonal, and genetic factors to regulate chondrocyte
metabolic activity and contribution to the maintenance of the ECM
(24). The mechano-electrochemical events sensed by chondrocytes
include compressive loading, hydrodynamic/osmotic pressure,
fluid flow, ion flow, and electrical current. The generation of these
stresses occurs as a coordinated response to mechanical loading
and their biophysical nature has been previously reviewed (23, 25).
The net impact of mechano-electrochemical stresses on cartilage
dynamics is an area of intensive research. Excellent reviews have
been published that provide detailed analysis of the anabolism and
catabolism that occurs under various mechanical stress situations
(see refs. 26, 27). A prevailing hypothesis predicts that the chon-
drocyte response to moderate mechanical loading is necessary for
normal cartilage homeostasis (27). Although static (i.e., constant)
loading of cartilage explants in culture leads to the suppression of
chondrocyte metabolism and a reduction in chondrocyte Agc and
collagen biosynthesis (28), dynamic (i.e., oscillatory) loading at spe-
cific frequencies has a net anabolic effect (26, 27). Correlating with
this, in vivo experiments have shown that changes in joint load-
ing without altering the stability of the tissues (i.e., without injury)
can elicit cellular responses that are either catabolic or anabolic
depending on frequency, duration, and magnitude of loading (29).
Specifically, moderate exercise in young rodents produces an ana-
bolic response in chondrocytes such that cartilage shows increased
proteoglycan content, decreased proteoglycan degradation, and
increased thickness (27, 29). Conversely, high-intensity exercise or
a sudden increase in joint loading leads to OA-like catabolism of
the cartilage matrix, characterized by decreased collagen network-
ing, proteoglycan loss, and reduced cartilage stiffness (29) (see OA
below). Severe inactivity can produce analogous catabolic effects,
including reduced cartilage thickness and proteoglycan loss (27). In
addition to these effects of mechanical stress on cartilage homeo-
stasis, normal joint loading might also be an important regulator
of developmental and postnatal growth of cartilage. Recently pub-
lished data indicate that abrogation of normal shoulder range of
motion via injection of botulinum toxin A into the supraspinatus
muscles of newborn mice leads to delayed development of tendon-
bone insertions (30). Although these results pertain to tendon
insertion and/or attachment, it seems plausible to imagine that a
similar dysregulation of AC development and/or growth can occur
in joints that experience abnormal mechanical loading.
In the context of chondrocyte sensing of mechano-electrochemi-
cal events (discussed above), substantial effort has been spent try-
ing to elucidate the key signaling mechanisms facilitating the cel-
432? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
lular effects of loading. A central signal transduction pathway that
regulates these processes involves the integrin receptors. Integrin
receptor complexes bind ECM components and transmit informa-
tion about mechanical perturbations of the ECM to the cytoplasm
of chondrocytes. Integrin receptors containing a β1 subunit are the
primary integrins responsible for binding the ECM (31). For exam-
ple, an α1β1 or α2β1 integrin complex typically binds collagen and
laminin, the α3β1 integrin complex binds fibronectin and type II
collagen, and the αVβ1 integrin complex is the classical receptor
for fibronectin (reviewed in ref. 25). Adhesion and binding of ECM
components to integrin receptors on the surface of chondrocytes
leads to the activation of proteins associated with the cytoskeleton,
such as paxillin (32), and intracellular signaling proteins, such as
focal adhesion kinase (FAK) (33) and MAPK signaling molecules
(34). In fact, MAPK signaling with downstream activation of the
MEK-Erk1 signaling pathway leads to downregulation of Agc gene
expression in bovine articular chondrocytes (35). Furthermore,
association of the integrin complex with IGF receptor I strongly
facilitates activation of this MAPK signaling pathway (34). There
is also evidence for activation of proline-rich tyrosine kinase 2
(Pyk2), leading to the upregulation of collagenase III expression
via PKC following exposure to fibronectin fragments (36). In
addition to these signaling responses, it is important to note that
abrogation of cell-ECM interactions (anoikis) that are mediated
via integrins leads to chondrocyte apoptosis (37), establishing a
true dependence of chondrocyte function on sensing mechanical
forces transmitted by the ECM.
It is widely thought that transduction of mechano-electrochemi-
cal events can also be facilitated via stress-activated ion channels
in the plasma membranes of chondrocytes. Of the numerous ion
channels that have been characterized, the most relevant in chon-
drocytes may be the N- and L-type voltage-gated calcium channels
(VGCCs) (38, 39). Since cytoskeletal elements control opening and
closing of VGCCs in neuronal cells, a similar regulatory paradigm
might exist in chondrocytes. Thus, transfer of mechanical stress
through the cytoskeleton could induce opening of these channels,
the propagation of intracellular calcium waves, and the subse-
quent induction of phenotypic effects in the cells (40). In general,
calcium transients lead to activation of signaling via both calmod-
ulin kinase and calcineurin/NFAT pathways (41, 42) among oth-
ers. Although these pathways are known to be important in the
modulation of both chondrogenesis and chondrocyte differen-
tiation (43, 44), work remains to fully characterize how calcium
signaling in chondrocytes contributes to the anabolic or catabolic
effects of mechanical stress.
Contribution of the periosteum to the early phase of fracture healing.
(A) Periosteum is a well-microvascularized tissue, consisting of an
outer fibrous layer and an inner cambium layer. Children have thicker
and better-vascularized cambium layers than adults (i versus ii). (B)
The cambium layer contains abundant stem/progenitor cells that can
differentiate into bone and cartilage (i). Following fracture or oste-
otomy, progenitor cells residing in the periosteum are activated and
enter the cell cycle (ii), followed by differentiation into osteoblastic and
chondrogenic (green) lineage cells (iii). Further differentiation of the
osteoblasts and chondrocytes leads to intramembranous bone forma-
tion (gray) and a mature cartilage template (blue) (iv). The vascular
invasion of the cartilage template (v) coupled with bone formation com-
pletes the process of endochondral bone repair (vi).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
Hypoxic stress. Oxygen is required for normal cellular metabo-
lism, and oxygen deficiency within cartilage tissues can induce
an hypoxic state that affects chondrocyte function. Cells exposed
to hypoxia respond in a number of ways, including altering their
expression of genes involved in cell cycle, differentiation, and
apoptosis (45). In mesenchymal stem cells (MSCs) and chondro-
cytes, the transcription factor HIF-1α is a survival factor that is
induced in hypoxic environments and inhibits proliferation but
increases ECM production (46, 47). Conditional deletion of the
gene encoding HIF-1α in chondrocytes results in massive apopto-
sis in hypoxic areas (46). Three-dimensional micromass cultures
of adipose-derived adult stromal cells with targeted deletion of the
gene encoding HIF-1α have substantially reduced chondrogenic
potential (48), suggesting that hypoxia-induced upregulation of
this factor supports chondrogenic commitment. Thus, hypoxia
is likely to be an important stressor that enhances the chondro-
genic potential of mesenchymal cell populations both during the
development of normal tissues that have limited blood supply as
well as under pathologic conditions (e.g., following disruption of
vascularity due to injury).
The influence of pathologic stress on chondrogenesis
and endochondral ossification
More is known about how cartilage biology is influenced by stress-
ors that arise from pathologic situations than is known about how
cartilage biology is affected by normal physiologic stressors. Below
is a compilation of key information regarding the influence on
chondrogenesis and chondrocyte differentiation of pathologic
stress induced by fracture healing, OA, heterotopic ossification,
FOP, heavy metal toxicity, and cigarette smoke.
Fracture healing. Skeletal fracture is an important pathologic stress
that has a critical impact on the chondrogenesis and endochondral
ossification that occurs during the fracture healing response. The
mechanisms underlying the initiation of chondrocyte progenitor
proliferation and differentiation at the time of fracture injury are
not well understood, although it has been suggested that mechani-
cal stability and the early inflammatory response play key roles
(49). Following cortical bone fracture or osteotomy, local pro-
genitor cells residing in either the periosteum or bone marrow are
sensitized, enabling them to respond to biological or biophysical
stimuli produced within the local injury milieu. During the first
few days following fracture, a hematoma is formed and this is fol-
lowed by infiltration of inflammatory cells and release of growth
factors, which direct the recruitment and proliferation of progeni-
tor cells (Figure 2). The further differentiation of the recruited
progenitor cells into chondrocyte and/or osteoblast lineage cells
determines the fate of the cells — whether they enter the endo-
chondral bone formation pathway or the intramembranous bone
formation pathway. It is evident that the environment at the site of
the injury, for example the presence of hypoxia and inflammation,
as well as the mechanical stability of the bone ends affects the fate
decision of the progenitor cells. In particular, endochondral bone
formation, which is the focus of this Review, always takes place
close to where the junction between the broken bone ends will
develop. This is where the oxygen tension is low and vascularity is
severely damaged. In terms of mechanical stability, stabilized frac-
tures heal with virtually no evidence of cartilage (intramembra-
nous bone formation), whereas nonstabilized fractures produce
abundant cartilage at the fracture site (endochondral bone forma-
tion). As mentioned earlier, the schematic in Figure 2 shows the
unique morphogenesis of reparative tissue during the early phases
of bone fracture healing via endochondral bone formation, and
this has been detailed in excellent review articles (see refs. 50, 51).
The progenitor cells that contribute to the fracture healing
response are found in both the bone marrow and, to a greater
extent, in the periosteum. Periosteum consists of microvascular-
ized connective tissue that covers the outer surface of cortical
bone. It can be separated into two distinct layers: an outer layer
that contains fibroblasts and distinct connections between the
periosteum and bone, which are known as Sharpey fibers; and an
inner layer known as cambium, which contains multipotent MSCs
and osteoprogenitor cells that contribute to normal bone growth,
healing, and regeneration (52–54). It is known that the cambium
layer in children is much thicker and better vascularized than in
adults, a possible underlying cause for faster and more complete
healing of fractures in children.
Due to the lack of cellular markers of pluripotent MSCs, the iden-
tity of the multipotent stem/progenitor cells residing in the peri-
osteum are largely unknown. This substantially hampers efforts to
track the fate of these cells in repair and further understand the
cellular and molecular mechanism(s) pertaining to the activation
of this important progenitor cell pool. A murine segmental bone
graft transplantation model that includes periosteum (55, 56) has
permitted examination of the signals and stem cell populations
involved in bone repair in transgenic and knockout mice. When
live bone grafts derived from mice constitutively expressing β-galac-
tosidase in all tissues were transplanted to wild-type mice, it was
observed that about 70% of the early bone and cartilage formation
overlying the bone graft was attributable to the proliferation and
differentiation of donor periosteal progenitors (55). These data
strongly suggest that live cortical bone healing is initiated and driv-
en by pluripotent local MSCs. Using the live bone transplantation
approach in transgenic and knockout mice should provide an in
vivo model to examine the influence of injury stress on progenitor
cell activation, proliferation, and differentiation.
The molecular signaling pathways involved in the initiation and
morphogenesis of fracture repair are only superficially under-
stood. Experimental data suggest that periosteum-initiated bone
repair might be analogous to fetal limb bud development (57, 58).
The mesenchymal condensation that forms at the fracture site
that is analogous to the developing limb bud (blastema) origi-
nates from the extensive proliferation of progenitor cells at the
injury site (59). Although animals and humans have only very
limited capacity to regenerate damaged tissues, it has long been
suspected that postnatal bone repair, such as fracture healing,
recapitulates some of the essential pathways in limb development,
although the fracture repair process is not capable of supporting
limb regeneration per se.
In the past decade, progress has been made in the identification
of factors and genes involved in fracture healing. The most notable
of these are BMPs, which belong to the TGF-β superfamily, hedge-
hog proteins, and Wnt proteins. For example, BMP-2 expression
was found in the early fracture callus (i.e., the tissue that forms
at the fracture site; see Figure 2) just a few days following cortical
bone fracture (60). Most recently, Tsuji et al. demonstrated that
elimination of BMP-2 in the mouse limb disrupted the initiation
of postnatal fracture healing (61), indicating an essential role for
BMP-2 in bone repair and healing. Evidence has also emerged to
show that morphogens such as hedgehog proteins and Wnt pro-
teins, which are involved in embryonic pattern formation, also
434? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
postembryonically function to initiate pathways that control self-
renewal, migration, differentiation, and cell-fate commitment of
adult stem or progenitor cells (62). However, detailed studies are
needed to determine the role of these two pathways in fracture
healing in adults.
In addition to mechanisms analogous to limb development,
genes that are involved in injury and inflammatory responses dur-
ing the repair process have been shown to play key roles in endo-
chondral bone repair (63, 64). Nonsteroidal antiinflammatory
drugs (NSAIDs) that target COX isoforms are the most commonly
used pain-relieving medications in our society. Several studies
have shown that COX activity is involved in normal bone metabo-
lism and suggested that NSAIDs have a negative impact on bone
repair (65, 66). The most compelling data implicating COX activ-
ity in bone repair come from genetic models that demonstrate a
critical role for the inducible COX isoform COX-2 — although
they develop normally, bone repair is markedly impaired in adult
Cox-2–/– mice following fracture (67). Defects occur at various
stages of healing, including during chondrogenesis and chondro-
cyte differentiation. Histology of the fracture callus, derived from
Cox-2–/– mice given a tibia fracture, shows delayed chondrogenesis
and persistent mesenchymal cells at the junction of the fracture
site. The point during the bone healing process that is impaired in
Cox-2–/– mice coincides with the early induction of COX-2 expres-
sion following bone fracture in wild-type mice, further demon-
strating the requirement of this enzyme in early chondrogenesis
during skeletal repair.
Heterotopic ossification. A well-characterized pathologic stress
response of muscle to injury involves the development of hetero-
topic ossification. Ectopic bone formation develops through a
cartilage intermediate, so the initial events of heterotopic ossi-
fication require chondrogenesis. Muscle injury results in tissue
damage, hemorrhage, and an inflammatory response. Recent data
have suggested that the development of a hypoxic environment is
critical if the early activation tissue response is to lead to chon-
drogenesis (68). Although the subsequent induction of BMPs in
the local environment has been connected with the formation of
ectopic bone in both animal models (69–72) and humans (73),
the manner in which stress signals regulate BMP expression and
enhance the BMP signaling response is not clear. However, the
finding that noggin, gremlin, and follistatin (inhibitors of BMP
signaling) can suppress the development of ectopic bone in ani-
mal models suggests that control of BMP signaling in injured tis-
sues is a critical event (74, 75).
Signals downstream of the stress response have been implicated
in regulating the BMP signaling pathway. For example, inhibi-
tion of COX-2–dependent stimulation of PGE2 production from
human bone marrow–derived MSCs using NS-398 suppresses
expression of BMP-2 (76). This suppression can be reversed by
an agonist of the EP4 receptor for PGE2 as well as by PGE2. In
addition, BMP-2 expression is also suppressed by an EP4 recep-
tor antagonist in these cells (76). Based on this, COX-2/PGE2/
EP4 receptor/BMP-2 signaling in MSCs is likely to be important
in heterotopic bone formation. Recent reports also demonstrate
that BMP-2 stimulates Osx mRNA expression and AP activity in
ST2 pluripotent stromal cells, and these effects are enhanced by
the selective EP4 receptor agonist ONO-4819 (77). In this study,
pretreatment of the ST2 cells with a PKA inhibitor abolished the
effects of ONO-4819, suggesting that the anabolic effect of activa-
tion of the EP4 receptor is mediated by the PKA pathway. Thus,
COX-2 induction of PGE2 production and signaling through PKA
in muscle stem cells is a possible link between the inflammatory
response to injury and ectopic bone formation. Although these
studies have not involved the use of muscle stem cells, recent
human data demonstrating that inhibition of COX-2 prevents het-
erotopic bone formation following hip arthroplasty are consistent
with these findings (78–80).
FOP. FOP is a rare and disabling genetic disorder characterized
by the inappropriate deposition of cartilage leading to progressive
heterotopic ossification. Throughout childhood and early adult
life, the joints of the normotopic skeleton of individuals with FOP
become progressively immobilized, rendering movement impossi-
ble. Any small injury to connective tissue (muscles, ligaments, and
tendons) typically results in the formation of hard bone around
the damaged area. FOP is an autosomal dominant condition, but
most cases are sporadic. Recently, a recurrent mutation in the gly-
cine-serine activation domain of the type IA activin receptor gene
(ACVR1) was reported in all sporadic and familial cases of classic
FOP, indicating that this activating mutation of the ACVR1 gene
is responsible for the disease (81). It has been additionally shown
that BMP-4 and type IA BMP receptor proteins are overexpressed in
cultured lymphocytes from FOP patients (82). Recent studies have
shown that stem cells isolated from the skeletal muscle of mice
exhibit long-term proliferation, high self-renewal, multipotent dif-
ferentiation, and have osteogenic potential (83–85). Overexpression
of BMP-4 induces these muscle-derived stem cells to acquire a
chondrogenic phenotype in vitro (84). One conceivable explana-
tion for FOP is that MSCs in the area surrounding a future site of
heterotopic bone are not prone to differentiate into bone-forming
osteogenic cells under normal conditions but that mutations in
ACVR1 in these tissues trigger mesenchymal cells to differentiate
into cells that support heterotopic endochondral ossification.
OA. OA, the most common form of arthritis, is a noninflam-
matory degenerative joint disease characterized by dysfunction of
articular chondrocytes, AC degradation, periarticular bone forma-
tion (i.e., osteophytes), and enhanced bone density below the AC
surface (i.e., subchondral sclerosis) (86). Although the etiology of
OA is not fully understood, it is generally held that biochemical,
genetic, and mechanical factors participate in the progression of
the AC degeneration (87). In the early stages of disease, the produc-
tion of catabolic cytokines by the membrane surrounding the joint
capsule (i.e., the synovium) induces transient AC proliferation and
increased matrix synthesis (type II collagen, Agc) in an attempt to
initiate repair (88, 89). However, the chronic production of these
cytokines, which include IL-1, TNF-α, IL-17, and IL-18 as well as
PGE2, leads to the enhanced synthesis of collagenases (MMP-1,
MMP-8, MMP-9, and MMP-13) and aggrecanases (ADAMTS4 and
ADAMTS5), which drive matrix degradation and progressively
erode the articular surface (87, 90, 91). Progressive degradation of
the matrix is associated with the dysregulation of AC, denoted by
the inappropriate expression of genetic and morphologic mark-
ers of endochondral ossification, including type X collagen, AP,
and increased apoptosis (90, 92). It has been hypothesized that
this alteration of AC phenotype, leading to disruption of normal
matrix anabolism, is an initiating step in the disease process. Of
particular interest, from the perspective of this Review, is the influ-
ence of cytokines and abnormal mechanical stress on the degen-
eration of the AC at the onset and during the progression of OA.
As mentioned in the above discussion of mechanical loading of
cartilage, high-intensity exercise, abnormally large static loading,
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
and a sudden increase in joint loading all lead to OA-like matrix
catabolism characterized by decreased collagen networking, pro-
teoglycan loss, and reduced cartilage stiffness (29). Alterations in
joint mechanics that are caused by injury to cartilage or surround-
ing structures (such as the shock-absorbing meniscus in the knee)
would presumably also lead to this type of cartilage catabolism.
This would be due to both the production of cytokines as well as
the abnormal mechanical loading of the joint caused by tissues
damaged by injury (reviewed in ref. 27). Specifically, cytokines
such as IL-1 and TNF-α are inducers of cartilage matrix degrada-
tion due to their ability to induce the expression of genes encoding
matrix catabolizing proteins such as MMPs and aggrecanases (93,
94). In fact, both IL-1 and TNF-α induce PGE2 production and NO
metabolism, which both act as strong catabolic signals in carti-
lage by promoting chondrocyte injury and enhancing chondrocyte
apoptotic potential (95).
Articular chondrocytes might also respond directly to
cytokines and abnormal mechanical loading via the generation
of intracellular ROS, which also is likely to contribute to the
pathogenesis of OA (96, 97). Generation of ROS is associated with
enhanced production of MMPs and cytokines (98) and with con-
comitant enhancement of NF-κB signaling (99). When in excess,
ROS (e.g., superoxide anion and hydrogen peroxide) induce apop-
tosis by oxidative stress and/or damage to DNA, lipids, and pro-
teins. In the context of endochondral ossification (and possibly
in chondrocytes that are undergoing inappropriate hypertrophy
during OA), ROS are increased as the cells differentiate (100). Cor-
related with this, treatment of chondrocytes with oxidants induces
hypertrophy, whereas antioxidant treatment with N-acetyl cysteine
inhibits maturation with decreased expression of the maturation
markers Mmp13, Col10a1, and Runx2 (100). Maturing hypertrophic
chondrocytes release matrix vesicles, and this event is increased fol-
lowing hydrogen peroxide treatment, supporting the hypothesis
that ROS induce hypertrophy (101). Given these findings, the use
of antioxidant therapy as a possible therapeutic paradigm in OA
has been discussed (102).
Heavy metals. Lead (Pb), the heavy metal most widely studied in
biological systems, has toxic effects on various organs including
the skeleton (103–105). Bone is a major reservoir for ingested Pb
(106), and delayed skeletal development occurs in children with
prenatal exposure to Pb (107). In addition, levels of exposure to Pb
during childhood are inversely correlated with final height, weight,
and chest circumference (108–110). These observations suggest a
direct effect of Pb on skeletal development, with the possibility
that there are direct effects of this heavy metal on chondrogenesis
and endochondral ossification.
Pb stimulates chondrogenesis in micromass cultures of murine
mesenchymal limb buds and during in vivo ectopic bone forma-
tion and fracture healing. Regarding the in vitro work, Pb-exposed
micromass cultures of primary limb bud MSCs have increased Sox9
and Col2 expression as well as cartilage nodule formation (111). Pb
causes pathway-specific effects on these cells, including enhanced
phosphorylation of the TGF-β–specific Smads (Smad2 and Smad3)
and decreased phosphorylation of the BMP-2–specific Smads
(Smad1, Smad5, and Smad8) (111). The increase in cartilage for-
mation seems, in part, to be secondary to increased mesenchymal
cell proliferation. Correlating with this, mice exposed to Pb with
blood Pb levels mimicking the established childhood toxicity
range have accelerated and increased chondrogenesis in muscles
implanted with BMP-2–expressing C9 cells (111). However, during
the endochondral ossification phase of ectopic bone formation in
this model, Pb inhibits chondrocyte maturation (111). Since BMP
signaling is essential for chondrocyte maturation, it is possible
that the effect is secondary to downregulation of this signaling
pathway, as was observed in vitro. Thus, although Pb stimulates
chondrogenesis, it reduces chondrocyte maturation and thereby
results in delayed endochondral bone formation, as has been pre-
viously documented (112). Consistent with these in vitro and in
vivo data, Pb exposure has been found to inhibit fracture healing
in mice, with complex effects noted on chondrogenesis and chon-
drocyte maturation (113). The enhanced cartilage deposition and
delayed mineralization (i.e., delayed completion of endochondral
ossification) seen in this fracture experiment further support
the impact of stress related to Pb toxicity on chondrogenesis and
Only a few reports address the chondrogenic effect of exposure to
other heavy metals. Cadmium induces apoptosis of mesenchymal
populations adjacent to the apical ectodermal ridge (114), reduc-
ing the cartilage-forming potential of the tissue in the develop-
ing limb. Similarly, quantum dots comprised of a combination
of cadmium, selenium, and zinc inhibit chondrogenesis in bone
marrow progenitors (115). Another report documents the influ-
ence of zinc, manganese, and cadmium on chondrocytes during
endochondral ossification (98). Zinc was found to have a biphasic
effect on chondrocyte differentiation, with low doses inducing AP
activity and high doses inhibiting mineralization (116). Compara-
tively, manganese caused a reduction of mineralization, whereas
the effects of cadmium were acutely toxic (116). Although these
heavy metals clearly regulate chondrogenesis, little is known about
the molecular mechanisms underlying the toxicity in mesenchymal
populations that leads to impaired cartilage formation.
Cigarette smoke. In addition to effects on lung, heart, and vascular
tissue, smoking has been documented to have a negative impact
on skeletal healing (117–124). Cartilage is an important target of
cigarette smoke during fracture repair, and effects are observed on
mesenchymal cell recruitment to the chondrocyte lineage and the
later stages of endochondral bone formation. For example, expo-
sure of mice to second-hand smoke caused delayed chondrogen-
esis in a tibial fracture model, as evidenced by reduced toluidine
blue staining at day 7 after fracture and peak cartilage formation
at day 14 when control calluses are mineralized (125). Work to
identify which components of cigarette smoke are responsible for
this inhibitory effect on chondrogenesis is important if the under-
lying molecular mechanisms are to be elucidated.
Overall, normal and pathologic stress substantially affect the
differentiation of chondroprogenitors and chondrocytes, and
thus exert critical influence over cartilage tissue homeostasis. As
can be seen from this Review, the bulk of our knowledge derives
from work that has identified the influence of various pathologic
stresses, including skeletal fracture, OA, heterotopic ossification,
and heavy metal toxicity. Ongoing efforts to further address how
pathologic stress affects cartilage are evident in the literature and
driven by the strong interest in understanding the control of carti-
lage biology in the context of trauma, inflammation, and healing.
Regarding the influence of normal stresses on cartilage (in par-
ticular, mechanical stress), clear paradigms have been established
that support a signal transduction scheme for stress to exert cell
effects. How these stresses affect the formation of limbs, AC, and
436? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 2 February 2008
joints is not well understood, and represents an important area
of future research. Understanding how the subtle application of
normal mechanical stress leads to appropriate limb and cartilage
development is important for envisioning therapies that facilitate
cartilage repair, including tissue engineering approaches.
Address correspondence to: Regis J. O’Keefe, Box 665, Depart-
ment of Orthopaedics, University of Rochester Medical Center,
601 Elmwood Avenue, Rochester, New York 14642, USA. Phone:
(585) 273-1261; Fax: (585) 275-1121; E-mail: regis_okeefe@
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