@MedicalBooksStore 2017 Cartilage Volume 2 Pathophysiology

Susanne Grässel · Attila Aszódi Editors
Cartilage
Volume 2: Pathophysiology
Cartilage
Susanne Grässel • Attila Aszódi
Editors
Cartilage
Volume 2: Pathophysiology
Editors
Susanne Grässel Attila Aszódi
Orthopädische Klinik für die Universität Department of General, Trauma, and
Regensburg Reconstruction Surgery
Universitätsklinikum Regensburg Ludwig-Maximilians-University
Regensburg Munich
Germany Germany
ISBN 978-3-319-45801-4 ISBN 978-3-319-45803-8 (eBook)

DOI 10.1007/978-3-319-45803-8
Library of Congress Control Number: 2017931314
© Springer International Publishing Switzerland 2017

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Preface
Volume two of this book series comprised of three volumes is dedicated to provide
an overview about the pathophysiology of cartilage, joint tissue, and intervertebral
disks.
The text is designed to be of use to multiple medical and basic science disciplines
as orthopedics, rheumatology, and trauma surgery and all basic investigators work-
ing in the field of cartilage, joint, and intervertebral disk pathophysiology.
This volume focuses on the major cartilage pathophysiologies which include
osteoarthritis and rheumatoid arthritis, degeneration of intervertebral disks, and
genetic skeletal diseases as cartilage collagenopathies and other hereditary chon-
drodysplasias resulting from mutations in structural cartilage proteins.
Chapter 1 provides an overview about osteoarthritis (OA) which is the most
common joint disorder and known as a leading cause of disability in the adult popu-
lation. It is now appreciated that all components of the joint, including the cartilage,
calcified cartilage, synovial joint lining, and periarticular bone, undergo pathologi-
cal changes during the initiation and progression of OA. Some of these alterations
can be attributed to direct injury and mechanical disruption of the tissues, but in
general the mechanisms are dependent on active cell-mediated processes that occur
during the long time course of the disease. A deeper understanding of the specific
and unique roles of complex signaling networks and their downstream targets
involving biochemical crosstalk among the cartilage, synovium, bone, and other
joint tissues will provide mechanistic insights into the pathologic processes that
affect the cartilage and other joint tissues in OA, but also may identify potential
therapeutic targets for treatment of this debilitating disease. Chapter 2 provides
insight into mechanical stress as an obligatory etiological factor in the development
of OA. Understanding how tissues of the joint respond to mechanical injury is likely
to inform our understanding of pathogenesis. Articular cartilage is avascular yet
responds rapidly and strongly to a range of mechanical stresses. It does so by acti-
vating a number of mechanosensitive pathways mediated by release of molecules
trapped within the pericellular matrix as well as by triggering mechanoreceptors at
the cell surface. These pathways appear to be relevant to the in vivo response to
mechanical disruption and affect the course of experimental OA.
The gradual loss of articular cartilage from the surface of articulating joints is a
feature of OA. It is marked by degradation of the cartilage matrix, including the
large aggregating proteoglycan aggrecan, the small leucine-rich proteoglycans
v
vi Preface
known as SLRPs, and the fibrillar type II collagen. Chapter 3 discusses the major
families of cartilage-degrading enzymes, the matrix metalloproteinases (MMPs),
and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)
families. Factors that regulate MMP and ADAMTS activity, with a focus on MMP-
13, ADAMTS-4, and ADAMTS-5 as the major protagonists of cartilage degrada-
tion, are discussed. The important role of degraded matrix fragments in regulating
inflammation in osteoarthritis, via Toll-like receptor signaling, is highlighted.
Chapter 4 puts emphasis on the functions of proteoglycans as one of the main com-
ponents of the articular cartilage ECM. Proteoglycans bind water and provide the
basis for absorbing high compressive loads. Additionally, they bind cytokines, che-
mokines, growth factors, and morphogens, thereby protecting these factors against
proteolysis and/or acting as a depot of regulatory factors when matrix degradation
occurs. They also modulate signaling pathways and create morphogen gradients by
immobilization of ligands in the ECM and regulation of the turnover of ligands.
Given these important roles of proteoglycans in regulating cell functions, it is well
understandable that the loss of ECM and degradation of proteoglycans during OA
induce severe changes in cartilage homeostasis.
The presence and production of soluble factors in the osteoarthritic joint have
always been a focus of research, as they are assumed to play a role in the initiation
and/or progression of the disease. Chapter 5 reviews research data which assign an
important role to chemokines, growth factors, and adipokines in OA; however it
also emphasizes on a traditionally studied subset of inflammatory, anti-inflammatory,
and modulatory cytokines. Differential profiles of these factors compared to healthy
joints were found in the knee and other OA affected joints, whereby joint damage
itself induces a specific change in the secretory pattern of diverse soluble factors.
Genetic skeletal diseases are a diverse and complex group of over 450 rare dis-
eases that affect the development and homeostasis of the skeleton. Although indi-
vidually rare, as a group of related genetic skeletal diseases, they have an overall
prevalence of at least 1 per 4,000 children, which extrapolates to a minimum of
225,000 people in the European Union, and this extensive burden in pain and dis-
ability leads to poor quality of life and high healthcare costs. Dominant-negative
(qualitative) defects in numerous cartilage structural proteins result in a broad range
of genetic skeletal diseases. Chapter 6 will focus on mutations in fibrillar and fibril-
associated collagen genes which cause a wide range of chondrodysplasias, ranging
from premature arthritis to severe early lethal disorders. Mutations of cartilage-
specific collagens can cause cartilage tissue dysfunction by reducing synthesis of
structurally normal protein or through protein misfolding which leads to intracellu-
lar retention and degradation and consequent secretion of reduced amounts of struc-
turally abnormal protein. In addition, collagen misfolding mutations can induce a
cellular unfolded protein response which ultimately promote apoptosis and thus
contribute to the pathology. Chapter 7 will focus on a disease spectrum resulting
from mutations in the glycoproteins, cartilage oligomeric matrix protein (COMP),
type IX collagen, and matrilin-3, which together cause a continuum of pheno-
types that are among the most common of the autosomal dominant genetic skel-
etal diseases. Pseudoachondroplasia (PSACH) and autosomal dominant multiple
Preface vii
epiphyseal dysplasia (MED) define a disease spectrum typified by varying degrees

of short-limbed dwarfism, joint pain with stiffness, and early-onset OA. New insight
into disease-related musculoskeletal complications such as myopathy, ligamentous
laxity, and tendinopathy has been gained through the analysis of mouse models of
the PSACH and MED disease spectrum.
Chapter 8 will summarize and discuss the role of integrins in the physiology and
pathophysiology of the growth plate and articular cartilage. Integrins are membrane
receptors responsible for bidirectional communication between the cells and the
surrounding by transmitting physicochemical signals through adhesion complexes.
In addition, integrins are involved in sensing mechanical stress signals generated by
the extracellular matrix and transduce them into the cell interior converting physical
stimuli to biochemical signaling. Chondrocyte integrins have thus indispensable
roles in cartilage development, skeletal growth, and articular cartilage function.
Chapter 9 will focus on the peripheral nervous system which is critically involved
in the metabolism of joint tissue and intervertebral disks (IVD). Nerve fibers of
sympathetic and sensory origin innervate synovial tissue and subchondral bone of
diarthrodial joints. During endochondral ossification in embryonic limb develop-
ment, sensory and sympathetic neurotransmitters modulate osteo-chondrogenic dif-
ferentiation of mesenchymal progenitor cells, vascularization, and matrix
differentiation indicating a distinct role in skeletal growth and possible limb regen-
eration processes. In adults, sensory and sympathetic neurotransmitters are involved
in the pathology of inflammatory diseases as rheumatoid arthritis which manifests
mainly in joints. In addition, they might play a role in the pathogenesis of a priori
degenerative joint disorders, as OA and intervertebral disk degeneration.
Tissues of intervertebral disks share similarities to those of diarthrodial joints,
such as a thin layer of cartilage that lines the interface between the joint and the
bony elements and a central space rich in extracellular matrix molecules that pro-
motes lubrication and maintains osmotic pressure. Like the pathophysiology of
other cartilaginous joints, intervertebral disks undergo biomechanical and structural
changes as a result of aging and mechanical insults. Due to higher mechanical load-
ing, lumbar disks are more susceptible to degeneration, which can lead to symptom-
atic outcomes such as low back pain, sciatica, and other physical disabilities. These
affect the quality of life as we age and present a significant burden to the healthcare
system globally. Chapter 10 will provide an overview of the intervertebral disk in
health and disease.
Bringing together international experts from diverse fields of musculoskeletal
research was a demanding task requiring patience and persistence not only for vol-
ume one of this book series but also for this volume. For that we are very grateful to
our authors of this volume who managed to complete their chapters and who dedi-
cated their spare free time to writing their reviews.
Regensburg, Germany Susanne Grässel

Munich, Germany Attila Aszódi
Contents
1 Pathogenesis of Osteoarthritis in General �� 1

Mary B. Goldring, Kirsty L. Culley, and Miguel Otero
2 Cartilage Injury and Osteoarthritis �� 27
Heba M. Ismail and Tonia L. Vincent
3 Proteoglycan and Collagen Degradation in Osteoarthritis�� 41
Stephanie J. Gauci, Heather Stanton, Christopher B. Little,
and Amanda J. Fosang
4 Role of Proteoglycans in Osteoarthritis �� 63
Jessica Bertrand and Annelena Held
5 Pro- and Anti-inflammatory Cytokine Profiles
in Osteoarthritis �� 81
Yvonne Bastiaansen-Jenniskens, Daniel Saris,
and Laura B. Creemers
6 Molecular Genetics of the Cartilage Collagenopathies�� 99
Shireen R. Lamandé, Trevor L. Cameron, Ravi Savarirayan,
and John F. Bateman
7 Pseudoachondroplasia and Multiple Epiphyseal Dysplasia:
Molecular Genetics, Disease Mechanisms and
Therapeutic Targets �� 135
Michael D. Briggs, Peter Bell, and Katarzyna A. Piróg
8 Integrin-Mediated Interactions in Cartilage Physiology
and Pathophysiology�� 155
Attila Aszódi
9 The Sensory and Sympathetic Nervous System in Cartilage
Physiology and Pathophysiology�� 191
Susanne Grässel, Rainer H. Straub, and Zsuzsa Jenei-Lanzl
10 Intervertebral Disc Degeneration �� 229
Akansha M. Shah, Sarah Yoon Ji Kwon, Wilson C.W. Chan,
and Danny Chan
ix
Contributors
Attila Aszódi, PhD Laboratory of Experimental Surgery and Regenerative

Medicine, Clinic for General, Trauma and Reconstruction Surgery, Ludwig-
Maximilians-University, Munich, Germany
Yvonne Bastiaansen-Jenniskens, PhD University Medical Center Utrecht,
Department of Orthopaedics, Utrecht, The Netherlands
John F. Bateman, PhD Murdoch Childrens Research Institute, Royal Children’s
Hospital, Parkville, Australia
Biochemistry and Molecular Biology, University of Melbourne, Parkville, Australia
Peter Bell, PhD Institute of Genetic Medicine, Newcastle University, International
Centre for Life, Newcastle upon Tyne, UK
Jessica Bertrand, PhD Otto-von-Guericke University Magdeburg, Department of
Orthopaedic Surgery, Magdeburg, Germany
Michael D. Briggs, PhD Institute of Genetic Medicine, Newcastle University,
International Centre for Life, Newcastle upon Tyne, UK
Trevor L. Cameron, PhD Murdoch Childrens Research Institute, Royal Children’s
Danny Chan, PhD School of Biomedical Sciences, The University of Hong Kong,
Pokfulam, Hong Kong
Wilson C.W. Chan, PhD School of Biomedical Sciences, The University of Hong
Kong, Pokfulam, Hong Kong
Laura B. Creemers, PhD University Medical Center Utrecht, Department of
Orthopaedics, Utrecht, The Netherlands
Kirsty L. Culley, PhD Hospital for Special Surgery Research Institute and Weill
Cornell Medical College, New York, NY, USA
Amanda J. Fosang, PhD University of Melbourne Department of Paediatrics and
Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, VIC,
Australia
xi
xii Contributors
Stephanie J. Gauci, PhD University of Melbourne Department of Paediatrics and

Australia
Mary B. Goldring, PhD Hospital for Special Surgery Research Institute and Weill
Susanne Grässel, PhD Experimental Orthopaedics, Department of Orthopaedic
Surgery, ZMB/BioPark 1, University of Regensburg, Regensburg, Germany
Annelena Held, PhD Otto-von-Guericke University Magdeburg, Department of
Orthopaedic Surgery, Magdeburg, Germany
Heba M. Ismail, PhD Arthritis Research UK Centre for OA Pathogenesis,
Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, UK
Zsuzsa Jenei-Lanzl, PhD Experimental Orthopaedics, Department of Orthopaedic
Surgery and Laboratory of Experimental Rheumatology and Neuroendocrine
Immunology, Department of Internal Medicine I, University of Regensburg,
Regensburg, Germany
Sarah Yoon Ji Kwon, PhD School of Biomedical Sciences, The University of
Hong Kong, Pokfulam, Hong Kong
Shireen R. Lamandé, PhD Murdoch Childrens Research Institute, Royal
Children’s Hospital, Parkville, Australia
Departments of Paediatrics, University of Melbourne, Parkville, Australia
Christopher B. Little, PhD Raymond Purves Bone and Joint Research
Laboratories, Kolling Institute, Institute of Bone and Joint Research, Sydney
Medical School Northern, University of Sydney, St. Leonards, NSW, Australia
Miguel Otero, PhD Hospital for Special Surgery Research Institute and Weill
Katarzyna A. Piróg, PhD Institute of Genetic Medicine, Newcastle University,
International Centre for Life, Newcastle upon Tyne, UK
Daniel Saris, PhD University Medical Center Utrecht, Department of Orthopaedics,
Utrecht, The Netherlands
Ravi Savarirayan, PhD Murdoch Childrens Research Institute, Royal Children’s
Departments of Paediatrics, University of Melbourne, Parkville, Australia
Victorian Clinical Genetics Service, Parkville, Australia
Akansha M. Shah, PhD School of Biomedical Sciences, The University of Hong
Kong, Pokfulam, Hong Kong
Contributors xiii
Heather Stanton, PhD University of Melbourne Department of Paediatrics and

Australia
Rainer H. Straub, MD Laboratory of Experimental Rheumatology and
Neuroendocrine Immunology, Department of Internal Medicine I, University of
Regensburg, Regensburg, Germany
Tonia L. Vincent, FRCP, PhD Arthritis Research UK Centre for OA Pathogenesis,
Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, UK
Pathogenesis of Osteoarthritis
in General 1
Mary B. Goldring, Kirsty L. Culley, and Miguel Otero
Abstract
Osteoarthritis (OA) is the most common joint disorder and is a leading cause
of disability in the adult population. It is now appreciated that all compo-
nents of the joint, including the cartilage, calcified cartilage, synovial joint
lining, and periarticular bone, undergo pathological changes during the ini-
tiation and progression of OA. Some of these alterations can be attributed to
direct injury and mechanical disruption of the tissues, but in general the
mechanisms are dependent on active cell-mediated processes that occur dur-
ing the long time course of the disease. Based on clinical observations and
experimental studies, it is now recognized that it is possible for individual
patients to exhibit common sets of symptoms and structural abnormalities
due to distinct pathophysiological pathways that act independently or in
combination. Recent research focusing on the underlying pathological mech-
anisms has identified complex signaling networks involving biochemical
cross talk among the cartilage, synovium, bone, and other joint tissues. These
complex networks involve interplay among anabolic, catabolic, and inflam-
matory signals within a background of poorly characterized genetic factors.
A deeper understanding of the specific and unique roles of these mediators
and their downstream targets will provide mechanistic insights into the
pathologic processes that affect the cartilage and other joint tissues in OA but
also may identify potential therapeutic targets for treatment of this debilitat-
ing disease.
M.B. Goldring (*) • K.L. Culley • M. Otero

Hospital for Special Surgery Research Institute and Weill Cornell Medical College,
New York, NY 10021, USA
e-mail: goldringm@hss.edu; culleyk@hss.edu; oterom@hss.edu
© Springer International Publishing Switzerland 2017 1

S. Grässel, A. Aszódi (eds.), Cartilage, DOI 10.1007/978-3-319-45803-8_1
2 M.B. Goldring et al.
1.1 Introduction
Osteoarthritis (OA) is the most common joint disorder and the major cause of dis-
ability in the adult population. The pathophysiology of the disease is characterized
by progressive loss of articular cartilage, cartilage calcification, osteophyte forma-
tion, subchondral bone remodeling, and mild to moderate inflammation of the syno-
vial lining. Although cartilage destruction is the hallmark of OA disease, the
involvement of changes in the periarticular tissues, including the subchondral bone,
ligaments, tendons, menisci, and synovial membrane, is now well recognized
(Loeser et al. 2012a; Goldring and Goldring 2007). For example, ligaments and
menisci are important for maintaining biomechanical stability in the joint, and their
injury can lead eventually to cartilage loss. In addition, multiple factors are involved
in the pathogenesis of OA, including genetic susceptibility, biomechanics of the
affected joint, and the presence and extent of inflammation. It has been difficult,
therefore, to identify specific targets for therapy.
Investigations in various in vitro models and preclinical in vivo models during
the past decades have focused primarily on cartilage degradation or repair as a ther-
apeutic target and more recently on how biomechanical and cellular responses in
chondrocytes are modified by interactions with other joint tissues, in particular, the
synovium and bone (Goldring et al. 2011; Goldring and Otero 2011; Goldring and
Berenbaum 2015). This chapter focuses on the role of the chondrocyte in maintain-
ing cartilage homeostasis and responding to adverse events in the whole joint that
modify cartilage integrity and result in the initiation and progress of the osteoarthri-
tis disease program.
1.2 Disease Etiologies and Therapeutic Prospects
Studies in patients that undergo operative procedures indicate that there are different
etiologies and time courses that result in the initiation and development of OA. These
subsets represent a continuum of early, progressive, and end-stage OA and include
(1) anterior cruciate ligament (ACL) injury (<35 years of age), (2) acute meniscal
injury (26–40 years of age), (3) degenerative meniscus (40–65 years of age), and (4)
total joint replacement (>50 years of age). Epidemiologic studies have established
that there is a strong relationship between ACL disruption and the risk for subse-
quent development of OA (Segawa et al. 2001; Buckwalter and Brown 2004; Roos
2005; Lohmander et al. 2007; Meunier et al. 2007). Studies of populations with
meniscal injury have also been useful for identifying OA risk factors (Englund and
Lohmander 2004). Meniscal injuries are commonly seen in association with ACL
injury (Jones et al. 2003; Louboutin et al. 2009).
Current understanding indicates that the aberrant distribution of forces in carti-
lage leads to altered mechanotransduction in the chondrocytes and subsequent acti-
vation of catabolic and inflammatory genes, deregulated matrix synthesis, and
decreased repair capacity (Lotz and Kraus 2010; de Lange-Brokaar et al. 2012) (see
also Chap. 2). The development of posttraumatic cartilage pathology may in turn
1 Pathogenesis of Osteoarthritis in General 3
adversely impact the structural and functional properties of periarticular bone. The
damaged meniscus is an additional source of inflammatory cytokines, chemokines,
and reactive oxygen species that could promote expression and activation of proteo-
lytic enzymes and adversely affect cell survival and synthetic activity of chondro-
cytes and cells of other joint tissues (Englund et al. 2009; Rai et al. 2013).
In addition to trauma or injury, there are other factors that influence the disease
process, including aging, genetic predisposition, abnormal biomechanics, obesity,
and comorbidities such as cardiovascular disease, metabolic syndrome, and dia-
betes. However, OA clinical trial designs have not accounted for these multifacto-
rial aspects in disease subsets, but rather have selected subjects based on joint
location or diagnosis as primary OA or secondary to other types of arthritis (Punzi
et al. 2010; Kloppenburg 2014; Stiebel et al. 2014; Detert et al. 2014). There is a
need, therefore, for optimization of cohort selection based on classifying OA
patients according to disease phenotypes related to distinct pathophysiological
pathways (Bijlsma et al. 2011; Blanco and Ruiz-Romero 2012; Conaghan 2013;
Henrotin 2014).
Given the complexity of OA, it is not surprising that there is no structure-
modifying agent for OA and the available pain therapies have limited efficacy and
associated toxicities (Matthews and Hunter 2011). However, it is unlikely that a
single therapy will be effective against both symptoms and structural changes in the
entire spectrum of OA patients (Mobasheri 2013a, b; Pulsatelli et al. 2013; Thakur
et al. 2014). Most trials have addressed function and pain at later stages of disease
when there is already radiographic evidence of disease progression such as joint
space narrowing and osteophytes. The development of validated diagnostic and
prognostic molecular biomarkers (Lafeber and van Spil 2013; Hsueh et al. 2014;
Lotz et al. 2013; Tonge et al. 2014), as well as novel imaging biomarkers (Hunter
et al. 2013), that could be used for evaluating pre-symptomatic early-stage disease,
may permit therapeutic interventions to halt or slow OA progression prior to irre-
versible joint damage. There is therefore a need for deeper understanding of the
structure and function of articular cartilage and other joint tissues and how they
interact and respond to adverse environmental insults in ways that disrupt normal
joint homeostasis.
1.3 Articular Cartilage Physiology
Adult articular cartilage is composed of a specialized matrix of type II collagen and

the large aggregating proteoglycan, aggrecan, along with several “minor” collagens
and small proteoglycans. Its unique structural organization provides tensile strength
via the collagen network and compressive resistance via the proteoglycans, which
contribute to the capacity of the matrix to accommodate more than 70 % water
(Heinegard and Saxne 2011; Onnerfjord et al. 2012; Hunziker et al. 2014) (for fur-
ther details, see Chaps. 3 and 4). Cartilage is relatively hypocellular compared with
other tissues, with the chondrocytes constituting only 1–2 % of the total cartilage
volume, and it lacks a vascular supply and innervation. In normal adult articular
cartilage, the chondrocyte has limited proliferative capacity and its ability to per-
form low-turnover repair declines with age. Since the half-life of type II collagen
within the cartilage collagen network is more than 100 years, whereas the proteo-
glycan components have half-lives of weeks to years, the chondrocyte is involved
mostly in replacing the glycosaminoglycans on the aggrecan and other small pro-
teoglycan core proteins. The importance of these matrix proteins in determining the
structural and functional properties of the articular cartilage can be observed in
chondrodysplasias and other heritable disorders where mutations or deficiencies in
cartilage matrix genes result in altered skeletal development often associated with
the premature development of OA (Sandell 2012) (see also Chap. 6) (Fig. 1.1).
Chondrocytes in articular cartilage exist in lacunae as single cells encased in a
pericellular matrix (PCM) consisting of collagen VI microfibrils, hyaluronan, per-
lecan, biglycan, aggrecan as monomers or small aggregates, and type IX collagen
but virtually no type II collagen (Wilusz et al. 2014). The PCM helps to maintain the
a b
Superficial zone
Alarmins
DAMPs Alarmins Proteases
Adipokines DAMPs
Cytokines Adipokines
Chemokines Cytokines
Intermediate zone ECM fragments Chemokines ECM
proteolysis
Cristals
ECM fragments
Proteases Adipokines Proteases
Cytokines
Radial zone Chemokines
Tidemark → Catabolic factors

Alarmins
DAMPs
Calcified zone
Subchondral bone
Fig. 1.1 Schematic representation of cartilage organization in the healthy joint and in osteoarthri-
tis. (a) Each of the four different zones of healthy articular cartilage, the superficial, intermediate,
radial, and calcified zones, is characterized by distinct chondrocyte morphology and extracellular
matrix organization and composition. The calcified zone differs from the three other zones by the
mineralization of its extracellular matrix, by the presence of vessels (red), and by nerve fibers
(green) that originate from the subchondral bone. The calcified zone interfaces with the non-
mineralized cartilage, from which it is separated by the tidemark, and the subchondral bone. (b) In
OA, there is progressive loss of cartilage matrix from the superficial zone associated with chondro-
cyte phenotypic modifications, including the formation of clusters, catabolic activation, and hyper-
trophic differentiation. Cytokines, chemokines, alarmins, DAMPs, adipokines, and other mediators
released into the synovial fluid from the cartilage, synovium, and other joint tissues amplify a
vicious circle of cartilage damage. In addition to cartilage damage, remodeling of the subchondral
bone occurs with the development of vessels (red) located in vascular channels, which also contain
osteoblasts, osteoclasts, and sensory nerves (green) (From Houard et al. (2013) with permission)
chondrocyte in a low-turnover, survival state by protecting it from interacting with

molecules in the interterritorial cartilage matrix via cell-surface receptors such as
integrins, cell determinant 44 (CD44), annexins, syndecans, and discoidin domain
receptor 2 (DDR2) (Loeser 2014; Pap and Bertrand 2013; Xu et al. 2011, 2014).
Chondrocytes exist in a low-oxygen tension environment and intracellular survival
factors such as HIF-1α are required for maintenance of homeostasis and adaptation
to the mechanical environment (Maes et al. 2012). Primary cilia located on the
chondrocyte surface are required for chondrocytes to respond to mechanical forces
and to maintain hedgehog signaling (Wann et al. 2012; Ruhlen and Marberry 2014;
Ho et al. 2013; Thompson et al. 2016). These organelles contain mechanosensitive
receptors, including the transient receptor potential vanilloid 4 (TRPV4), piezo
channels, and connexin 43, as well as integrins, which permit the chondrocytes to
sense and adapt their metabolic activity in response to physical forces (Loeser 2014;
O’Conor et al. 2014; Knight et al. 2009; Mayan et al. 2015).
Cartilage also provides a unique articulating surface with a very low coefficient
of friction, facilitated by a boundary layer of lubricants, including lubricin, encoded
by the PRG4 gene, and hyaluronic acid that are produced by chondrocytes and
synovial cells (Waller et al. 2013; Jay and Waller 2014). Joint motion and mechani-
cal loading induce fluid movement between the cartilage and the synovial fluid,
facilitating the diffusion of molecules across cartilage and thus providing nutrition.
Soluble products transferred from the underlying subchondral bone could also be an
important source of nutrients, especially for the deeper layers of the articular
cartilage.
1.4 Articular Cartilage Pathology in OA
Of clinical importance is the ability of the chondrocyte to react to mechanical stim-

uli and to structural changes in the surrounding cartilage matrix through responding
to and producing anabolic and catabolic cytokines, which may act to influence car-
tilage homeostasis in a positive or negative manner (Goldring and Otero 2011;
Goldring et al. 2011) (for further details, see Chaps. 2 and 5). Cartilage destruction
with loss of proteoglycans and the degradation of the collagen network are the piv-
otal events that determine the irreversible progression of OA disease. Cartilage
destruction in OA is believed to be chondrocyte mediated in response to biome-
chanical insults and may occur directly or indirectly through the production of cyto-
kines and cartilage matrix-degrading proteinases in cartilage and other joint tissues
(Anderson et al. 2011). Joint injury alters the biomechanical dynamics of the joint
and joint kinematics, thereby stimulating chondrocyte mechanotransduction, activa-
tion of catabolic and inflammatory genes, deregulated matrix synthesis, and
decreased repair capacity (Lohmander et al. 2007; Lotz and Kraus 2010; Louboutin
et al. 2009) (Fig. 1.1).
Direct analysis of cartilage or chondrocytes from OA patients undergoing joint
replacement has yielded evidence that chondrocytes respond not only to proinflam-
matory cytokines but also inhibitory and anabolic cytokines that modulate
responses. The impact of cytokines on chondrocyte function, particularly with

respect to their various roles in cartilage destruction, has been reviewed exten-
sively (Goldring and Otero 2011; Goldring et al. 2011; Loeser et al. 2012a) (see
also Chap. 5). Studies of cartilage degradation in genetically modified mouse
strains that are resistant to or accelerate the development of OA spontaneously
with aging or when subjected to trauma and biomechanical injury have provided
information about the molecular effectors of the disease and potential targets for
therapy (Little and Hunter 2013; Vincent et al. 2012; Malfait et al. 2013; Fang and
Beier 2014) (for details, see Chap. 2).
The sequence of pathological changes in the cartilage matrix associated with OA
development has been elucidated by histological and biochemical analyses of carti-
lage from human subjects with OA and various animal models of OA (Table 1.1)
(Goldring and Goldring 2007; Houard et al. 2013; Loeser et al. 2012a). The earliest
changes include the loss of negatively charged glycosaminoglycans resulting in
increased water content associated with swelling of the cartilage matrix. Macroscopic
changes in the cartilage matrix composition are accompanied by the appearance of
surface fibrillations, characterized by microscopic cracks in the superficial zone. As
the disease progresses, exfoliation of fragments of cartilage and deep fissures
extending into the deeper cartilage layers lead to exposure of the underlying zones
of calcified cartilage and subchondral bone.
Accompanying or possibly preceding these matrix changes, chondrocytes
undergo marked phenotypic changes, which have been attributed to the adverse
effects of excessive mechanical loading that directly injures the cells or modulates
chondrocyte function via cell-surface mechanosensors. A requisite early event is the
disruption of the PCM associated with the biomechanically induced release of
inductive factors such as transforming growth factor (TGF)-β from the matrix and
action of proteinases such as HTRA1. This results in the exposure of receptors such
as DDR2 and syndecan-4 to extracellular matrix ligands, which are not normally in
contact with the chondrocyte. Activation of these receptors and downstream signal-
ing then induces gene expression of proteinases that can directly degrade the major
interterritorial matrix components, type II collagen, and aggrecan and produce frag-
ments that can further amplify the degradative process through binding to integrins
(Pap and Bertrand 2013; Xu et al. 2014; Loeser 2014) (for further details, see Chap.
8). There is also evidence of increased chondrocyte synthetic activity in regions of
enhanced pericellular staining for proteoglycans, reflecting attempts at repair. With
progressive matrix depletion, the chondrocytes appear to form clonal clusters sug-
gesting earlier proliferative events. Many of the differentially expressed genes iden-
tified in genomic analyses encode proteins that can be detected in these clonal
clusters in human OA cartilage (Sandell 2012). There are also vast regions of
decreased chondrocyte density containing cell membrane “ghosts” or fragmented
chondrocyte nuclei consistent with apoptosis. The accumulation of advanced glyca-
tion end products, a feature of aging cartilage that alters the composition and mate-
rial properties of the matrix, can also be observed. Chondrocytes with features of a
senescence-associated secretory phenotype have also been noted in aging cartilage
predisposed to OA development (Loeser 2013).
Table 1.1 Changes in the cartilage, synovium, calcified cartilage, and bone during the evolution
of osteoarthritis
Alterations in cartilage matrix and Increased water content with swelling of the cartilage
chondrocyte function in OA matrix
Increased chondrocyte anabolic and catabolic activities
(MMPs, ADAMTSs, other proteases)
Decreased proteoglycan content: increased degradation
and decreased synthesis
Development of surface fibrillations
Disruption of the collagen network (increased
breakdown)
Fissuring and fragmentation of cartilage matrix
Chondrocyte death and development of chondrocyte
clusters
Chondrocyte hypertrophy
Contributions of synovium and Low-grade synovitis associated with the infiltration of
synovial fluid mononuclear cells, e.g., activated B and T lymphocytes
Production of reactive oxygen species, nitric oxide,
DAMPs, alarmins, and proinflammatory cytokines
Production of IL-2, IL-7, IL-15, and IL-21 involved in
recruitment, survival, and activity of lymphocytes
Novel chemokine signature: IL-8, CCL5, CCL19, and its
receptor CCR7, as well as MCP-1 and MIP-1β and γ
Induction of Toll-like receptors by DAMPs and alarmins
and activation of NF-kB signaling
Induction of complement cascade
Alterations in the calcified Penetration of the calcified cartilage by vascular channels
cartilage and osteochondral with neural elements
junction in OA Expansion of the zone of calcified cartilage with
advancement into the articular cartilage
Duplication of the tidemark
New bone formation at the osteochondral junction
Bone changes in OA Increased cortical plate and subchondral cancellous bone
remodeling
Increased cortical plate thickness (initial increase in
porosity and reduced stiffness with progression to
increased cortical plate stiffness)
Decreased subchondral cancellous bone mass and
alterations in architecture
Formation of osteophytes
Development of bone marrow lesions
Formation of bone cysts
Bone attrition (altered bone contour)
Proteoglycan degradation is mediated by aggrecanases of the a disintegrin and

metalloproteinase with thrombospondin motifs (ADAMTS) family that cleave the
aggrecan core protein (Fosang and Beier 2011) (see Chap. 3). The collagen network
also becomes susceptible to disruption either by physical forces or by increased
activity of matrix metalloproteinases (MMPs) (Wang et al. 2013). The increased
activities of degradative enzymes produced by chondrocytes not only disrupt the
integrity of the cartilage matrix but also lead to the generation of cartilage matrix
breakdown products and secreted damage-associated molecular patterns (DAMPs)
and alarmins that contribute to further deregulation of chondrocyte function through
Toll-like receptors, integrins, and other cell-surface receptors (Houard et al. 2013;
Loeser et al. 2012a; Liu-Bryan and Terkeltaub 2015) (see also Chap. 8). They also
act on the adjacent synovial tissue to induce inflammation and the release of proin-
flammatory products, including cytokines and reactive oxygen species that feed
back on the chondrocytes to enhance the catabolic state.
Many of the signaling events activated in chondrocytes by excessive mechanical
loading are similar to those induced by inflammatory and oxidative stress (Perera
et al. 2010; Nam et al. 2009). During oxidative stress, HIF-2α, induced by NF-kB
signaling, directly targets hypoxia-responsive DNA elements in the promoter
regions of MMP13, COL10A1, VEGFA, and other genes that are involved in chon-
drocyte dysfunction in OA (Saito et al. 2010; Yang et al. 2010; Hashimoto et al.
2013). In contrast, the upregulation of HIF-3α by lubricin is associated with down-
regulation of HIF-1α and HIF-2α target genes as part of a mechanism that protects
against aging-related or posttraumatic OA development in mouse models (Ruan
et al. 2013). Hypoxia and oxidative stress via induction of the metal regulatory tran-
scription factor-1 (MTF1) and the Zn2+ transporter, ZIP8, also regulate the so-called
Zinc-ZIP8-MTF1 axis to promote cartilage destructive proteinases in vitro and
in vivo (Kim et al. 2014).
Taken together, all of the changes observed in the cartilage matrix and resident
chondrocytes over the course of OA initiation and progression have been interpreted
as reflecting the influences of local biomechanical conditions to produce both dys-
functional catabolic and anabolic cellular responses. However, these changes in the
cartilage cannot be considered in isolation, as the adjacent synovium and subchon-
dral bone play significant roles in OA pathology (Loeser et al. 2012a; Scanzello and
Goldring 2012; Goldring and Goldring 2010).
1.5 I nfluence of Synovitis and Inflammation on OA

Pathology
The cartilage is particularly responsive to factors released by the synovium, and the
alterations in the cartilage composition and properties induced by the synovial prod-
ucts may in turn adversely affect the adjacent subchondral and periarticular bone.
Inflammation, characterized by synovitis, is one of the risk factors for the develop-
ment of OA (Scanzello and Goldring 2012), and several studies have shown that the
presence of synovitis is correlated with OA symptoms and progression of OA joint
pathology (Ayral et al. 2005; Hill et al. 2007; Guermazi et al. 2011). The inflamed
synovium releases cytokines, chemokines, and other soluble mediators that can be
detected in OA synovial fluids and influence chondrocyte functions via cell-surface
receptors to induce catabolic and inflammatory mediators (Fig. 1.1). The synovium
is also a source of degradative enzymes, including MMPs and aggrecanases that can
directly degrade the cartilage matrix (Loeser et al. 2012a), although this occurs
more characteristically in RA where there is direct interaction between tissues at the
cartilage-synovial pannus junction (Otero and Goldring 2007; Goldring and Marcu
2009).
Synovitis is also observed frequently following joint injury and may be a prog-
nostic indicator of the rate of development of posttraumatic OA (PTOA) (Scanzello
et al. 2009, 2011, 2013). Magnetic resonance imaging (MRI) findings correlate with
microscopic and macroscopic evaluations of synovitis and suggest that synovitis is
often present soon after a traumatic event (Loeuille et al. 2005). The low-grade
synovitis following injury is associated with the infiltration of mononuclear cells,
including activated B and T lymphocytes, and with the production of inflammatory
cytokines (de Lange-Brokaar et al. 2012; Benito et al. 2005; Pearle et al. 2007). Of
interest, there is evidence that cartilage damage may not progress rapidly without
inflammation in the synovial compartment (Scanzello et al. 2011, 2013; Scanzello
and Goldring 2012).
Many proinflammatory cytokines, adipokines, and chemokines have been impli-
cated in the initiation and development of OA joint pathology, including the per-
petuation of synovitis and cartilage damage (Wojdasiewicz et al. 2014). In contrast
to the findings in rheumatoid arthritis, the involvement of IL-1 and TNF-α in the
pathogenesis of OA synovitis and pathology remains controversial. There is evi-
dence, however, implicating other cytokines, including the common γ-chain family
of cytokines, IL-2, IL-7, IL-15, and IL-21, which are involved in recruitment, sur-
vival, and activity of lymphocytes (Scanzello et al. 2009).
Multiple chemokines have also been identified in synovial tissues and synovial
fluid from patients with OA, including IL-8, CCL5, CCL19, and its receptor CCR7,
as well as MCP-1 and MIP-1β and γ (Scanzello et al. 2009, 2011; Haringman et al.
2006; Gobezie et al. 2007; Endres et al. 2010; Ritter et al. 2013) (see also Chap. 5).
Of interest is the finding that several of the chemokines isolated from synovial fluid
are chemotactic for human subchondral bone progenitor cells (Endres et al. 2010).
There is increasing evidence implicating a role for innate immunity in the patho-
genesis of the synovial inflammation in OA (de Lange-Brokaar et al. 2012; Scanzello
and Goldring 2012; Berenbaum 2013; Haseeb and Haqqi 2013). In the context of
OA, activation of the innate response is initiated by stimulation of TLRs, which are
pattern-recognition receptors that were identified initially as receptors for microbial
products generated during infection. These receptors are now known to play key
roles during cellular stress and are responsive to products of extracellular matrix
damage, including cartilage degradation products, during sterile tissue injury
(Piccinini and Midwood 2010; Liu-Bryan and Terkeltaub 2015). TLRs have been
detected in both chondrocytes and synovial cells in tissues from OA patients where
they can be activated by the endogenous DAMPS released from injured cartilage
and other joint tissues, including the bone (Schelbergen et al. 2012; Sohn et al.
2012; Nair et al. 2012; Gomez et al. 2014). Ligand-mediated TLR activation in
chondrocytes and synovial cells is an important stimulus for canonical NF-kB sig-
naling, the major pathway mediating the gene expression of a broad spectrum of
proinflammatory mediators and products, including chemokines and cytokines
(e.g., IL-1, IL-6, and TNF-α) (Marcu et al. 2010).
Gene profiling studies of cartilage or whole joints from animal models of OA
have helped to identify pathways that are involved in deregulation of chondrocyte
function in OA pathogenesis (Appleton et al. 2007; Olex et al. 2014). Both age-
related and injury-induced OA pathologies (Loeser et al. 2012b, 2013), as well as
spontaneous OA models such as the Str/ort mouse (Poulet et al. 2012), are associ-
ated with upregulation of genes of the senescence-associated secretory phenotype,
including inflammatory cytokines, chemokines, MMPs, and immune and defense
response genes (Loeser et al. 2012b). The prominence of the NF-kB pathway as a
common thread among the different gene signatures has been emphasized in these
models, suggesting a key regulatory role for stress and inflammatory signaling via
canonical NF-kB signaling in human OA (Goldring et al. 2011; Marcu et al. 2010).
The findings that mechanical stimuli modulate NF-kB signaling (Nam et al. 2009)
provide an explanation for why NF-kB-related gene signatures may be upregulated
in mouse models of PTOA in the absence of overt signs of inflammation such as
synovitis and immune cell infiltration. Profiling studies have revealed that inflam-
matory signatures are present before the appearance of overt OA and, in some mod-
els, are associated with increased numbers of activated T and B lymphocytes in the
spleens of the mice destined to develop OA (Poulet et al. 2012).
The complement cascade is one of the major effector mechanisms of the immune
system, and activation of the complement cascade plays an important role in the
pathogenesis of a broad spectrum of inflammatory and autoimmune disorders
(Holers 2014). There is evidence that both chondrocytes and synovial macrophages
can produce complement components and inhibitors, and previous studies have
demonstrated increased synovial complement component deposition in the setting
of acute flare-ups of OA and in synovial fluids from patients with OA (Konttinen
et al. 1996; Wang et al. 2011). A recent study by Wang et al. (2011) showed that
mice with impaired ability to activate the complement system were partially pro-
tected from the development of OA, providing direct evidence for a role of the
complement system in OA pathogenesis, at least in murine models. A role for the
complement system in human subjects requires further study.
1.6 Osteophytes
Osteophytes do not originate from the cartilage, but are bony outgrowths that grow
by a process of endochondral ossification involving TGF-β and bone morphogenetic
protein-2 (BMP-2), through proliferation of periosteal cells at the joint margin, dif-
ferentiation into cells with morphological features of hypertrophic chondrocytes,
mineralization, and replacement of the endochondral tissue with the bone (van der
Kraan and van den Berg 2007). Osteophytes are localized to the joint margins and
are a radiographic hallmark of OA. However, rather than contributing to joint dys-
function and OA progression, they may serve to stabilize the joint (Guyton and
Brand 2002; Messent et al. 2007; van der Kraan and van den Berg 2007), based on
the finding that removal of osteophytes from the knee joint in animal models induces
joint instability (Pottenger et al. 1990). In addition, there is no relationship between
osteophyte size and the risk for development of structural progression of knee OA
in human subjects (Felson et al. 2005).
1.7 ross Talk Between Articular Cartilage

C
and Subchondral Bone
The articular cartilage, calcified cartilage, and subchondral cortical and trabecular
bone in diarthrodial joints form a biocomposite that is uniquely adapted to allow the
transfer of load. The concept that an impermeable barrier consisting of the calcified
cartilage and tidemark, the histologically defined demarcation between the deep
articular and calcified cartilage, prevents the transfer of solutes and soluble products
between the articular cartilage and subchondral bone has been challenged by studies
showing direct contact between bone and cartilage cells and tissues in localized
regions in the osteochondral junction (Brower et al. 1962; Imhof et al. 2000; Lyons
et al. 2006; Pan et al. 2009). Microcracks produced by mechanical stress and exac-
erbation of naturally occurring pores in the subchondral bone plate provide conduits
for vascular invasion into the calcified cartilage and enable cross talk through diffu-
sion of small molecules (Lane et al. 1977; Burr and Schaffler 1997; Bullough 2004;
Imhof et al. 2000). TGF-β-mediated angiogenesis, potentially among the earliest
events driving OA (Zhen et al. 2013), is followed by vascular invasion into the
osteochondral junction (Pan et al. 2012; Saito et al. 2012). Examination of OA
mouse models by microCT shows early and temporal subchondral plate porosity
and increased perforation with enhanced biochemical and mechanical interactions
among the subchondral trabeculae, bone marrow cells, and articular cartilage
(Botter et al. 2011; Gu et al. 2012) (Fig. 1.1).
The expansion of the calcified cartilage, associated with tidemark duplication,
into the deep zones of the articular cartilage leads to local cartilage thinning, thereby
compromising the mechanical function of the osteochondral unit (Burr and Gallant
2012). This process may be mediated by chondrocytes that appear in the deep zone
OA cartilage contiguous with the calcified cartilage and have phenotypic features
that recapitulate a developmental molecular program, including expression of genes
encoding type X collagen, MMP13, and transcription factors such as Runx2 (van
der Kraan and van den Berg 2012). These hypertrophic-like chondrocytes also are a
source of proangiogenic factors, including VEGF, that play a role in promoting
vascular penetration of the calcified cartilage at the osteochondral junction through
channels originating at sites of microcracks and fissures, accompanied by sensory
and sympathetic nerves that may play a role in joint pain (Ashraf et al. 2011a, b;
Stoppiello et al. 2014) (for details, see Chap. 9). Cuffs of new bone form around
these channels involving increased osteoclast activity, infiltration of inflammatory

cells into the marrow spaces, increased endothelial cell proliferation and vascular
density, and localized bone marrow replacement by fibrovascular tissue expressing
VEGF (Suri and Walsh 2012; Walsh et al. 2010).
Additional in vitro findings provide direct evidence that interactions between
bone cells and chondrocytes can modulate phenotypic expression in each cell type
and also demonstrate the differential effects of osteoblasts from normal versus OA
bone. Coculture of bovine articular cartilage explants with fragments of subchon-
dral bone can increase chondrocyte survival compared to explants cultured alone
(Amin et al. 2009). In cocultures of chondrocytes and osteoblasts separated by
semipermeable membranes to allow transfer of soluble mediators, osteoblasts
derived from the “sclerotic” bone from patients with OA have the capacity to reduce
the expression of genes encoding aggrecan (ACAN), collagen type II (COL2A1),
and the transcription factor Sox9 and to increase expression of MMP3 and MMP13
compared to osteoblasts from normal, “nonsclerotic” bone. Pretreatment of the
osteoblasts with IL-1, IL-6, or oncostatin M can further enhance MMP3 and MMP13
production (Sanchez et al. 2008). Coculture also upregulates MMP13, COL2A1,
osteopontin, and alkaline phosphatase in osteoblasts. Gene expression profiling of
osteoblasts cultured from OA bone specimens compared to osteoblasts obtained
from comparable regions of normal bone shows differential gene expression that
may account for the findings obtained in coculture studies (Hilal et al. 1998; Zhang
et al. 2012; Chou et al. 2013).
Among the important molecular networks that play roles in cartilage-bone cross
talk in OA are the TGF-β/BMP, fibroblast growth factor (FGF), and Wnt/β-catenin
pathways. All three pathways are involved in regulating cartilage and bone develop-
ment, are dysregulated during OA pathogenesis, and have been used as additive fac-
tors in tissue engineering strategies (Baron and Kneissel 2013; Lories et al. 2013).
TGF-β signaling has roles in cartilage and bone homeostasis, as well as in OA
pathogenesis (Bush and Beier 2013). This may be explained partly by the avail-
ability of receptors and downstream intracellular signaling molecules. In normal
articular chondrocytes, the canonical receptor, ALK5, activates Smad2/3 to inhibit
chondrocyte hypertrophy, whereas in OA chondrocytes the decreased ratio of
ALK5 to ALK1, which signals through BMP-related Smad1/5/8, results in induc-
tion of MMP13 and increased cartilage catabolism (Blaney Davidson et al. 2009;
van der Kraan et al. 2012; van den Bosch et al. 2014; van der Kraan 2014), associ-
ated with increased nerve growth factor (NGF) (Blaney Davidson et al. 2014).
These aberrant chondrocyte responses may account for the off-target effects of
TGF-β injections in joints that result in osteophyte formation and synovial fibro-
sis. Studies in animal models of OA also show the differential effects of TGF-ß
inhibitors depending upon the dose and mode of administration. For example, the
implantation of a TGF-ß-specific antibody in the subchondral bone attenuates
both cartilage loss and OA bone changes. Furthermore, systemic administration
of low doses of a TGF-ß inhibitor can prevent the migration of mesenchymal stem
cells in the subchondral bone of operated limbs and attenuate neovascularization
and cartilage loss (Zhen et al. 2013).
FGF signaling is important in skeletal development and cartilage and bone

homeostasis (Vincent 2012), where the differential use of receptors determines a
temporal and spatial program of cellular differentiation. FGF-2, for example, pre-
serves chondrocyte phenotype through FGFR3 but promotes cartilage destruction
via FGFR1 (Yan et al. 2011). Of the four receptors, FGFR1 and 3 are the most
abundant, and OA cartilage has a reduced ratio of FGFR3 to FGFR1. In vitro and
in vivo studies have shown that FGF-18 via FGFR3 protects against cartilage dam-
age (Barr et al. 2014; Mori et al. 2014). The potential of FGF-18 and other anabolic
factors to promote cartilage regeneration tissue engineering approaches is a subject
of intensive study (Ellman et al. 2013). Proof-of-concept clinical trials in humans to
address structural end points by MRI and radiographic joint space narrowing and
pain are needed (Lohmander et al. 2014).
Wnt signaling in the cartilage and bone is also complex and is determined by the
availability of Wnt ligands in different tissues and their use of distinct, canonical
versus noncanonical pathways during skeletal development and growth versus OA
pathogenesis (Baron and Kneissel 2013; Lories et al. 2013). A number of Wnt
antagonists have been identified in the bone and cartilage and explored as OA thera-
peutics or targets. These include dickkopf-related protein-1 (DKK-1), secreted
frizzled-related protein 1 (sFRP1), and sclerostin (SOST). DKK-1 and sFRP1, in
addition to affecting bone homeostasis, also can have profound affects in cartilage
by modulating hypertrophic differentiation (Leijten et al. 2012; van den Bosch et al.
2014) and expression of proteinases (Oh et al. 2012; Bougault et al. 2014). DKK-1
has differential effects in the cartilage and bone depending on dosage and mode of
delivery. The inhibitory effect of DKK-1 on cartilage degradation is related to its
capacity to inhibit VEGF production by osteocytes and osteoblasts in the bone
(Funck-Brentano et al. 2014).
SOST, identified originally as specific to osteocytes, can be detected in cartilage
and in osteochondral tissues, plasma, and synovial fluid in OA patients and animal
models (Chan et al. 2011; Mabey et al. 2014). SOST inhibitors increase bone forma-
tion (Lewiecki 2014), but their effects in cartilage and OA are controversial (Roudier
et al. 2013; Lories et al. 2013). Conflicting findings may be due partially to cross
talk between Wnt signaling and TGF-β/BMP pathways that disrupt homeostasis by
inducing endochondral ossification during osteophyte formation (Gelse et al. 2012)
or promoting the switch toward BMP-related ALK1 and Smad1/5/8 (van den Bosch
et al. 2014). In addition, TGF-β is required for the suppression of SOST by mechan-
ical load (Nguyen et al. 2013).
The differential capacity of the bone and cartilage to adapt to changes in local
biomechanics and injury is an important factor in the development of OA joint
pathology and has implications for designing therapeutic interventions. In animal
models of traumatic joint injury, the onset of bone pathology can be linked to a
specific initiating event (Anderson et al. 2011; Christiansen et al. 2015).
Importantly all of the studies demonstrate a strong association between local
changes in subchondral bone and cartilage pathology, thereby supporting the con-
cept that the cellular and morphological adaptations to local mechanical factors
occur in both tissues.
There is, however, considerable controversy about whether OA pathology

begins in the subchondral bone or the overlying articular cartilage. Although the
articular cartilage is designed to deform under compressive loads without failure,
it has a lower capacity to withstand tension or shear stresses that occur during joint
motion (Burr and Gallant 2012). Under normal conditions, the subchondral bone
can protect the overlying articular cartilage from damage by distributing loads.
During OA development, alterations in the contour of the subchondral bone or
heterogeneity in bone density or stiffness create local shear stresses that increase
deformation of the cartilage and predispose it to splitting and fibrillation, since it is
not able to adapt as rapidly as the bone by modifying its matrix composition and
mechanical properties (Burr and Gallant 2012). The initial fibrillation and splitting
of the articular cartilage reflect the acute effects of the loading and are followed by
alterations in cartilage proteoglycan turnover and progressive activation of a cata-
bolic program in chondrocytes. The increased thickness of the zone of calcified
cartilage also contributes to the thinning of the articular cartilage (Radin et al.
1984; Radin and Rose 1986).
Insights into the sequential changes in both articular cartilage and periarticular
bone have been obtained in noninvasive tibial loading models in mice in which the
magnitude and duration of cyclic compressive loading can be defined (Poulet et al.
2011; Ko et al. 2013). The bone changes involving increased subchondral cortical
bone plate thickness occurs in the predicted region of increased load transfer and
corresponds to the region with cartilage pathology. In contrast to the effects on the
cortical bone, the trabecular epiphyseal bone mass decreases, consistent with stress
shielding of this region by the thickened subchondral plate (Ko et al. 2013). These
findings are consistent with the observations in patients with established OA, in
whom there is loss of subchondral cancellous bone associated with trabecular thin-
ning (Karvonen et al. 1998; Burr and Gallant 2012).
In contrast, a single loading session, which does not acutely alter chondrocyte
viability or articular cartilage or bone integrity, induces proteoglycan loss and carti-
lage thinning consistent with activation of a progressive catabolic chondrocyte
response (Ko et al. 2016); however, bone remodeling increases only transiently and
begins to resolve at later time points. These findings provide evidence that a single
event of traumatic loading produces sustained adverse effects on chondrocyte func-
tion that are not reversible and lead to progressive cartilage loss, whereas the bone
responds rapidly to the initial loading by changing cellular remodeling activities
and eventually reestablishes a physiological state.
Further evidence of an association between the cartilage and bone in the
response to trauma is provided by the association of bone marrow lesions with
regions of OA cartilage pathology and their persistence as predictive of progressive
cartilage loss and joint pain (Kothari et al. 2010; Roemer et al. 2009, 2011, 2015;
Hunter et al. 2006). Originally termed “bone marrow edema” by Wilson and col-
leagues in 1988 (Wilson et al. 1988), they can be detected using fluid-sensitive
MRI sequences in localized regions of increased signal intensity in the subchon-
dral bone in human subjects with OA. However, histological analyses of regions
corresponding to the anatomical sites of the bone marrow lesions have revealed the
presence of fat necrosis, localized marrow fibrosis, and vascular changes associ-
ated with microfractures of the trabecular bone at various stages of healing (Zanetti
et al. 2000; Taljanovic et al. 2008; Leydet-Quilici et al. 2010). These findings sug-
gest that the MRI signal is not generated by actual “edema” but rather by an active
cellular process associated with local regions of bone damage. Bone marrow
lesions are especially common in regions of denuded cartilage (Bowes et al. 2015),
indicating that the overlying cartilage has a role in distributing mechanical forces
and protecting the subchondral bone from the adverse effects of excessive load.
The anatomic association of the cartilage and bone pathology provides further evi-
dence that both tissues are responsive to local environmental conditions and that
interactions between them affect their composition and structural properties in a
reciprocal manner.
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Cartilage Injury and Osteoarthritis
2
Heba M. Ismail and Tonia L. Vincent
Abstract
Mechanical stress is an obligatory aetiological factor in the development of OA
so understanding how tissues of the joint respond to mechanical injury is likely
to inform our understanding of pathogenesis. Much is known about how vascular
tissues respond to damage, a process that involves activation of platelets on the
exposed endothelium and recruitment of leukocytes to the site of injury. The
articular cartilage is avascular yet responds rapidly and strongly to a range of
mechanical stresses including cutting, avulsion, impact loading and shearing. It
does so by activating a number of mechanosensitive pathways mediated by
release of molecules trapped within the pericellular matrix as well as by trigger-
ing mechanoreceptors at the cell surface. In this way injury drives a number of
intracellular signalling pathways, leading to a broad range of cellular responses.
These pathways appear to be relevant to the in vivo response to mechanical dis-
ruption and affect the course of experimental OA.
2.1 Introduction
The role of mechanical overload/injury in osteoarthritis development cannot be

overstated. The epidemiology of this is compelling; paralysed joints do not develop
OA and excessive mechanical strain accelerates OA development. Increased
mechanical strain is not only due to increased load traversing the joint (obesity, high
impacts, direct cartilage trauma) but also importantly includes loss of joint mecha-
noprotective mechanisms (through muscle weakness, loss of gait reflexes with age,
H.M. Ismail • T.L. Vincent (*)

Arthritis Research UK Centre for OA Pathogenesis, Kennedy Institute of Rheumatology,
NDORMS, University of Oxford, Roosevelt Drive, Oxford OX3 7FY, UK
e-mail: heba.ismail@keneedy.ox.ac.uk; tonia.vincent@kennedy.ox.ac.uk

28 H.M. Ismail and T.L. Vincent
Table 2.1 How mechanical factors contribute to OA risk

Increased load through the joint Intra-articular fracture
High-impact injuries
Obesity
Joint malalignment
Developmental shape abnormalities, e.g.
chondrodysplasias
Loss of joint mechanoprotective Joint destabilising injuries, e.g. meniscal and
mechanisms cruciate ligament injury
Loss of gait reflexes upon ageing
Muscle atrophy (age, immobilisation, genetic)
tendon/meniscal injuries leading to joint destabilisation, etc.) (reviewed in Brandt

et al. 2009) (Table 2.1). A somewhat outdated explanation for this association is that
simple ‘wear and tear’ leads to passive attrition of the articular surface with exces-
sive use. This theory has largely been discounted by showing that disease is highly
dependent upon the induction and regulation of specific matrix-degrading enzymes
(see Chap. 3) and that mice in which these enzymes have been deleted genetically
are substantially protected from developing OA despite the fact that their joints have
been surgically destabilised (Glasson et al. 2005; Little et al. 2009). Our subsequent
observation that induction of inflammatory response genes (including metallopro-
teinases) in surgically destabilised joints is highly mechanosensitive allowed us to
speculate that excessive mechanical stress in vivo is sufficient to activate pathways
that drive matrix degradation and initiate disease (Burleigh et al. 2012).
2.2 rticular Cartilage: Structure and Mechano-Sensing

A
Function
The articular cartilage is a paucicellular type II collagen- and proteoglycan-rich tis-

sue. It is a highly lubricated connective tissue with exceptionally low surface fric-
tion that allows smooth articulation of the opposing joint surfaces. Lubrication is
achieved by a synergistic relationship between the proteoglycan, the lubricin and
the glycosaminoglycan hyaluronan, together with phosphatidylcholine lipids (Seror
et al. 2015). Intact lubrication protects the surface from shear stress. Loss of lubricin
leads to increased shear stress and superficial chondrocyte apoptosis (Waller et al.
2013). The type II collagen fibres are highly cross-linked and provide the matrix
with tensile strength. They are also oriented to aid articulation and reduce shear-
associated damage, lying parallel to the articular surface in the most superficial
regions of the matrix and perpendicular to the surface in deeper regions. The col-
lagenous component of the matrix is laid down around the time of skeletal maturity
and is not thought to turn over in later adult life. Although osteoarthritic chondro-
cytes do secrete type II collagen, it is not clear whether this can become successfully
incorporated into the damaged matrix. Proteoglycan turnover, on the other hand, is
2 Cartilage Injury and Osteoarthritis 29
relatively rapid, and it is this component of the matrix that can change rapidly as the
cartilage volume shrinks with loss of weightbearing activity (so-called atrophy)
(Palmoski et al. 1979; Vanwanseele et al. 2002).
The chondrocyte is regarded as the single cell of the cartilage although there are
different morphological and molecular features of chondrocytes through the differ-
ent layers of the tissue. These cells are responsible for developing and maintaining
the matrix. Although they rarely divide in the healthy tissue and appear relatively
‘quiescent’, chondrocytes are exquisitely mechanosensitive. The chondrocyte is a
spiculated cell that extends multiple short processes into the immediate pericellular
matrix (PCM). The PCM, just 3–5 μm wide, is rich in type VI collagen, perlecan
and other minor proteoglycans such as decorin and biglycan (Kvist et al. 2008;
Soder et al. 2002; Tesche and Miosge 2004). As it is devoid of type II collagen, it
exhibits a much reduced Young’s modulus (stiffness) compared with the further
remote matrix, and this is thought to allow selective compression of the PCM upon
cartilage loading (Guilak et al. 2006). Both cell surface ‘mechanoreceptors’ and
mechanisms controlled directly by the PCM drive mechano-responses in chondro-
cytes. These will be considered next.
2.3 Chondrocyte Injury Assays
A number of different methods for applying injurious mechanical stress to a chon-

drocyte in vitro have been described (see Table 2.2). These include injury to the
chondrocyte embedded within its native matrix (cartilage explant), e.g. by cutting
with a scalpel, explantation of the cartilage from the intact joint or compression
(Dell’accio et al. 2008; Gruber et al. 2004; Quinn et al. 1998; Redman et al. 2004).
Cartilage injury models using murine tissues allow to interrogate the cellular response
to injury in a pathway-dependent manner and have been done by ‘avulsing’ the
immature femoral condyle of 4–6-week-old mice (Chong et al. 2013). Alternatively,
mechanical stress can be applied to isolated cells either grown in monolayer or
Table 2.2 In vitro cartilage injury models

In vitro cartilage
injury models Tissue Injury stimulus
Cutting injury Articular cartilage explants Explantation (cutting the tissue from the
intact joint surface)
Cultured articular cartilage Recutting explants that have been
explants cultured for >24 h post explantation
Injurious loading Articular cartilage explants Injurious load (excessive magnitude of
load or high velocity (impact))
Isolated cells grown in artificial Injurious shear or compressive stress
matrix (alginate, agarose)
Avulsion injury Whole femoral heads Avulsion – shearing through the growth
(murine) plate
Stretch? Cells in monolayer Stretch activation (? injurious)
embedded within three-dimensional artificial matrices, e.g. by stretching or mechan-

ical compression (Salter et al. 2002; Wright et al. 1996; Vincent et al. 2007). The
frequency of load and speed of load application can also be altered. High-velocity
loads create impact-associated cartilage injuries in vitro (Aspden et al. 2002).
2.4 ellular Response to Cartilage Injury: Activation

C
of Intracellular Signalling
Most common cell signalling systems have been described in the context of carti-
lage/chondrocyte injury. For the purposes of this chapter, we will restrict our précis
to those studies that have specifically addressed the response to injurious mechani-
cal stress rather than covering mechano-responses of chondrocytes to modest cyclic
and static load that are likely to be regarded as non-injurious (Fig. 2.1).
2.4.1 Focal Adhesions: Src, FAK and Integrin Signalling
Integrins have been defined as ‘mechanoreceptors’ in other cell types, triggered by

perturbations in the extracellular matrix and transducing extracellular signals to the
cell (Huveneers and Danen 2009; Wang et al. 1993). Upon activation, integrins
Fig. 2.1 Intracellular responses to cartilage injury. Multiple pathways are activated by a variety of
injurious stimuli. Some involve known receptor systems on the cell membrane, e.g. FGF receptor.
Others are unknown. Cellular outcomes that have been investigated largely include mechanisms of
cell death upon injury or regulation of genes that determine tissue health
cluster in focal adhesions to create transmembrane protein complexes that form

tyrosine kinase signalling hubs. Articular chondrocytes express multiple integrins
for binding to type II collagen, fibronectin and other ECM molecules (Vinall et al.
2002) (for further details, see Chap. 8). α5β1-integrin is responsible for cell attach-
ment to fibronectin and plays a role in proliferation and adhesion in chondrocytes
(Enomoto-Iwamoto et al. 1997). The interaction between α5β1-integrin, stretch-
activated ion channels and interleukin-4 in response to cyclic mechanical strain has
been reported in chondrocytes in a number of studies (reviewed in Chen et al. 2013).
Integrin-cytoskeleton interactions are implicated in death-signalling pathways in
chondrocytes following impact injury. Cytoskeletal dissolution by treatment with
cytochalasin B or nocodazole considerably decreased impact-induced chondrocyte
death, which suggests that the cytoskeleton is required for impact-induced chondro-
cyte death (Sauter et al. 2012). Additionally, Jang et al. (2014) found that excessive
compression of the articular cartilage led to cytoskeleton-dependent chondrocyte
death. This process involved integrin activation and signalling via focal adhesion
kinase (FAK) and Src family kinases. Treatment with FAK and Src kinase inhibitors
significantly improved chondrocyte viability after impact loads.
Sterile cutting injury to the surface of the intact articular cartilage caused rapid
(within seconds) activation of protein tyrosine kinases of the Src kinase family
including focal adhesion kinase, paxillin and cortactin (Watt et al. 2013). The Src
inhibitor, PP2, blocked injury-induced tyrosine phosphorylation and modulated
inflammatory gene expression patterns induced in response to explantation injury.
Interestingly Src activation was not seen in response to recutting cartilage explants
that had been cultured post explantation (recut) suggesting that injury to the intact
joint surface may be different to injuring cartilage in vitro (Fig. 2.1).
2.4.2 MAPK and NFkB Signalling
The mitogen-activated protein (MAP) kinases, p38, extracellularly regulated kinase

(ERK) and c-Jun N terminal kinase (JNK) are implicated in multiple cellular
processes. In chondrocytes p38 and ERK MAPKs are induced by shear and com-
pressive forces (Fitzgerald et al. 2008, Fanning et al. 2003) as well as by explanta-
tion and recutting injury (Gruber et al. 2004; Vincent et al. 2002). Using an
osteochondral explant blunt injury model, Ding et al. (2010) demonstrated enhanced
phosphorylation of the three MAPKs 20 min post impaction. The activation of MAP
kinases has been observed in various osteoarthritic-cartilage injury models also
(Chowdhury et al. 2008; Ding et al. 2008; Guo et al. 2009). Cartilage explantation
and murine femoral condyle avulsion are also able to activate NFkB signalling
although, interestingly, these pathways do not appear to be activated upon recutting
injury (Chong et al. 2013; Gruber et al. 2004).
Treatment with specific p38 and ERK MAP kinase inhibitors partly reversed loss
of cell viability and proteoglycan content in impacted explants. Similar findings
were reported by Rosenzweig et al. (2012). In this study, mechanical injury to
bovine osteochondral explants led to MAPK activation and decreased chondrocyte
viability especially in the superficial zone. Reduction in cell viability post-injury

correlated with increased caspase-3 activity and could be reversed by ERK and p38
inhibition. Inhibition of p38 in vitro also caused a reduction in metalloprotease acti-
vation, although inactivation of p38 in vivo was not chondroprotective. Indeed,
genetic loss of function of p38 (by overexpressing a dominant negative form of p38)
was associated with higher grades of osteoarthritis in the knee (Namdari et al. 2008).
Sustained ERK activation caused by excising or cutting the cartilage is at least
partly attributed to the release of FGF2 from the pericellular matrix (PCM) of chon-
drocytes (Vincent et al. 2002). FGF2 is sequestered on the heparan sulphate chains
of perlecan and released within seconds of injury or compression (Vincent et al.
2004, 2007). Interestingly, neither NFkB nor JNK activation upon injury is due to a
soluble factor released from the injured cartilage (our unpublished observations)
(Fig. 2.1).
2.4.3 Smad and Wnt Signalling
Other factors such as TGFβ are also released from the matrix upon mechanical
compression and drive phosphorylation of Smad2/Smad3 (Madej et al. 2016). This
pathway becomes less responsive with age perhaps explaining why cartilage health
declines over time. Activation of BMP and Wnt signalling has also been reported in
response to cartilage injury, and this drives gene regulation in injured explants (see
below) (Dell’Accio et al. 2006, 2008) (Fig. 2.1).
2.4.4 Ion Channel Signalling
Mechanically induced Ca2+ signalling has been studied extensively in chondrocytes.

Han et al. (2012) developed a system to measure Ca2+ signalling of chondrocytes in
situ and found that Ca2+ signalling is regulated not only by mechanical force but also
by the cellular environment, such as ECM topography and temperature.
A number of mechanosensitive Ca2+ channels have been identified in chondrocytes
including the intracellular PLC-inositol 1,4,5-trisphosphate pathway, stretch-activated
ion channels, the transient receptor potential vanilloid 4 (TRPV4) cation channel
(reviewed in Chen et al. 2013) and the newly identified family of cation-permeable,
directly mechanically activated (MA) ion channels, named ‘Piezo’ (Lee et al. 2014).
TRPV4 is responsible for mediating the anabolic response of chondrocytes to osmotic
or mechanical stress (Phan et al. 2009; O’Conor et al. 2014) and may therefore
respond to physiological rather than injurious mechanical stress. Blocking TRPV4
in vitro makes chondrocytes less responsive to hyposmotic stress and impairs intra-
cellular calcium flux (Phan et al. 2009). TRPV4 knockout mice show a loss of the
Ca2+ response to hypo-osmotic challenge and have significantly increased OA indi-
cating that this pathway is chondroprotective in vivo (Clark et al. 2010). Chondrocytes
express the two channels Piezo 1 and Piezo 2, which respond synergistically to injuri-
ous mechanical strain. Inhibition of Piezos by GsMTx4, a biologically derived pep-
tide that specifically inhibits mechanically activated cation channels, caused inhibition
of the chondrocyte’s response to injury. GsMTx4 significantly protected chondro-

cytes from cell death following injury (Lee et al. 2014) (Fig. 2.1).
2.5 Cellular Response to Cartilage Injury: Cell Death
Mechanical injury to the articular cartilage can cause chondrocyte cell death
(reviewed in Kuhn et al. 2004). Death occurs through two principal mechanisms,
necrosis or apoptosis; the latter being more likely to occur if the injury is sharp
rather than blunt (Redman et al. 2007; Tew et al. 2000). Since chondrocytes repre-
sent only 1–10 % of the cartilage tissue volume and have low regenerative capacity
(Stockwell 1978), cell death is considered a potentially important pathological
event in OA. The use of inhibitors of caspases, key mediators of apoptosis, has been
used in OA and osteochondral injury models in rabbits to significantly reduce chon-
drocyte cell death (D’Lima et al. 2006; Dang et al. 2006; Costouros and Kim 2007).
Goodwin et al. (2010) demonstrated that the enhanced release of mitochondrial
reactive oxygen species (ROS) following mechanical injury to the articular cartilage
would induce cell death. Suppressing the ROS production either by rotenone, an
electron transport chain inhibitor, or by N-acetylcysteine (Martin et al. 2009), an
oxidant scavenger, dramatically reduced the rates of cell death. This suggests that
oxidative stress is responsible for chondrocyte death following cartilage mechanical
injury. Similarly, Killian et al. (2014) have shown that cartilage trauma caused by
high impaction triggered acute cell death in rabbit articular chondrocytes in addition
to an increase in nitric oxide release by joint tissues.
Bartell et al. (2015) developed a technique to measure cartilage mechanics at
microscales of strain fields and rapid time frames. In response to injurious load, the
effect on chondrocytes can be measured as early as 3 ms following localised impact.
The authors found that the high rates of strain were highly correlated with chondro-
cyte cell death. Interestingly, chondrocyte cell death was only detected after 2 h
following impact and the removal of the superficial zone increased the penetration
of cell death to deeper zones suggesting a protective role for the surface layer.
Another form of cell death has also been reported in OA chondrocytes called
chondroptosis. Chondroptosis, first described by Roach et al. (2004), includes some
elements of the apoptotic and autophagic processes. Chondroptosis involves signifi-
cant morphological changes within the endoplasmic reticulum and Golgi apparatus,
which functions to segment the cytoplasm into several smaller autophagic-like vac-
uolised compartments (Roach et al. 2004). Studies of growth plate chondrocytes
provide evidence that this process may help prevent a chondrocyte from entering a
committed stage of apoptosis (Bohensky et al. 2007a, b) and may explain the long
life of a chondrocyte.
Almonte-Becerril et al. (2010) have used a Wistar rats OA model followed by a
high-impact exercise. They showed that following injury, apoptotic cell death com-
bined with autophagy was induced in chondrocytes. At early-stage OA, apoptosis
was induced in the superficial and midzones of the cartilage, whilst an increase in
both apoptosis and autophagy (as measured by active caspase-3 and LC3II expres-
sion) was observed at later stages of OA.
On the other hand, Carames et al. (2012) reported that mechanical impact injury
to the bovine and human cartilage causes inhibition of autophagy (decreased expres-
sion of the autophagic markers ULK1, Beclin1 and LC3) in the cartilage superficial
zone, and this was associated with increased release of the sulphated glycosamino-
glycan (GAG) indicating cartilage damage. Reactivation of the autophagic pathway
by rapamycin resulted in inhibition of cell death and a reduction in GAG loss in
response to mechanical injury (Fig. 2.1).
2.6 Cellular Response to Cartilage Injury: Gene Regulation
Several groups have studied gene regulation in response to mechanical cartilage

injury in vitro. Dell’Accio et al. performed a microarray study of the recut human
articular cartilage and identified a key role for the Wnt pathway in the injury response.
Injury was associated with the up-regulation of a number of Wnt activators and their
inhibitors as well as leading to activation of the canonical pathway mediated by cyto-
plasmic accumulation of β-catenin (Dell’accio et al. 2008). Several studies have sug-
gested a proteolytic cartilage phenotype following injury. Lee et al. studied the
regulation of selective proteases following explantation injury, either alone or with
the addition of a traumatic compressive load. Both explantation and compression
regulated protease gene expression over the first 24 h of injury (Lee et al. 2005).
Injurious compression also caused a rapid early release of glycosaminoglycan that
was deemed to be due to matrix damage. This was followed by an active release of
GAG that could be suppressed by metalloproteinase inhibition (DiMicco et al. 2004).
Explantation also regulates IL1α and IL1β mRNA levels and induces pro-protein
production by the chondrocyte, albeit not causing its secretion (Gruber et al. 2004).
By injecting joints with specific inhibitors, it has been possible to investigate the
role of individual pathways/molecules in the explantation injury response. Such
studies have revealed that several injury genes such as TIMP1, activin A and TSG-6
are FGF2 dependent (Chong et al. 2013). Importantly, FGF2-dependent genes iden-
tified in vitro were also regulated upon joint destabilisation in vivo (Chong et al.
2013). Regulation of inflammatory response genes upon explantation such as
inflammatory cytokines and metalloproteinases is largely Src kinase dependent,
although some genes are also FGF2 dependent (Chong et al. 2013; Watt et al. 2013).
Some of these molecules likely contribute to promoting tissue catabolism upon car-
tilage injury, whilst others appear to be anti-catabolic or repair-promoting. Both
may contribute to pathology in OA (Fig. 2.1).
2.7 In Vivo Models of Cartilage Injury
Increasingly, we have seen an attempt to validate injury pathways in vivo

(Table 2.2). In vivo models of injury have been around for many decades, and the
response to injury has been described in detail histologically and metabolically
(reviewed in Campbell 1969). For instance, experiments of rabbit articular carti-

lage scarification by Meachim showed that chondrocytes around lesions become
metabolically more active, increasing their proteoglycan production and clonal
proliferation. Longer-term outcome of in vivo injury is inconsistent, with some
studies showing repair of focal lesions and others showing limited repair responses.
Successful outcome is in part thought to be due to whether the osteochondral
junction has been breached (Meachim 1963). Eltawil et al., more recently, suggest
that genetic strain and age are other modifiable factors that dictate successful
repair in the mouse (Eltawil et al. 2009). In these studies they created full-thick-
ness cartilage defects in the intercondylar groove of the femur and measured fill-
ing of the defect after 8 weeks. They also assessed the quality of repair tissue, as
well as neo-epitopes of matrix turnover, and showed that non-repairing strains
demonstrated evidence of OA changes in the matrix around the lesion. Similarly,
Rai et al. demonstrated a strain-dependent ability of focal cartilage lesions to
repair, a response that could be partly recapitulated in the injured auricular carti-
lage (Rai et al. 2012).
Surgically induced models of cartilage injury through joint destabilisation offer
a model to study chronic cartilage injury in vivo. Microarray studies of genes regu-
lated in the articular cartilage within the first few weeks following surgery have
identified many genes and pathways previously identified in in vitro cartilage injury
studies and in OA (Appleton et al. 2007; Gardiner et al. 2015). These models of
post-traumatic OA have been used in multiple studies to investigate candidate path-
ways in the disease (Vincent et al. 2012).
Another experimental system that has been utilised to investigate the immediate
and longer-term response to acute traumatic injury in vivo is that induced by cyclic
forced compression of the hyper-flexed knee joint in mice (Poulet et al. 2011). In
these experiments, mice undergo cyclic injurious joint compression under general
anaesthetic. This leads to damage to the posterior condylar cartilage and later,
osteoarthritic changes more widely within the joint. Other non-surgical models of
cartilage injury have also been developed and are reviewed in (Christiansen et al.
2015) (Table 2.3).
Table 2.3 In vivo cartilage injury models

Invasive Primary cartilage outcome
In vivo cartilage injury models (surgical)? assessed
Focal cartilage defect Yes Repair
Articular cartilage scarification Yes Repair and degeneration
Destabilisation of the joint (several different Yes Degeneration
models)
Cyclic tibial compression load No Degeneration
Intra-articular tibial fracture No Degeneration
ACL rupture by tibial compression overload No Degeneration
ACL anterior cruciate ligament
Conclusion
Cartilage injury is arguably the most important aetiological factor in the develop-
ment of OA, and studying the cellular response of cartilage to injury offers us
insights into how mechanical injury might drive pathological processes that
characterise disease. It is worth bearing in mind that the injury responses
described in these studies are likely to be pertinent to a more generic response of
connective tissues to injury, and therefore conclusions drawn from them are
likely also to be relevant to other tissues in the damaged joint.
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Proteoglycan and Collagen Degradation
in Osteoarthritis 3
Stephanie J. Gauci, Heather Stanton, Christopher B. Little,
and Amanda J. Fosang
Abstract
The gradual loss of articular cartilage from the surface of articulating joints is a
feature of osteoarthritis. It is marked by degradation of the cartilage matrix,
including the large aggregating proteoglycan aggrecan, the small leucine-rich
proteoglycans known as SLRPs and the fibrillar type II collagen. Aggrecan pro-
vides the water-holding capacity of cartilage, while the collagen II scaffold pro-
vides elastic restraint, aided by a protective coat of small leucine-rich proteoglycans.
Damaged aggrecan is readily replaced by synthesis of new aggrecan; however,
type II collagen can resist only a limited amount of proteolysis before cartilage
function is compromised. In this review the major enzyme families of cartilage-
degrading enzymes, the matrix metalloproteinases (MMPs) and a disintegrin and
metalloproteinase with thrombospondin motifs (ADAMTS) families, are dis-
cussed. We examine factors that regulate MMP and ADAMTS activity, with a
focus on MMP-13, ADAMTS-4 and ADAMTS-5 as the major protagonists of
cartilage degradation. We also compare the effects of blocking aggrecanolysis and
collagenolysis separately, or together, on cartilage erosion in a mouse model of
osteoarthritis. The role of degraded matrix fragments in regulating inflammation
in osteoarthritis, via Toll-like receptor signalling, is also discussed.
S.J. Gauci, PhD • H. Stanton, PhD • A.J. Fosang, PhD (*)

University of Melbourne Department of Paediatrics and Murdoch Childrens
Research Institute, Royal Children’s Hospital, Parkville 3052, VIC, Australia
e-mail: stephanie.gauci@mcri.edu.au; heather.stanton@mcri.edu.au;
amanda.fosang@mcri.edu.au
C.B. Little, PhD
Raymond Purves Bone and Joint Research Laboratories, Kolling Institute, Institute
of Bone and Joint Research, Sydney Medical School Northern, University of Sydney,
Level 10 Kolling Building – B6, Royal North Shore Hospital, St. Leonards
2065, NSW, Australia
e-mail: christopher.little@sydney.edu.au

42 S.J. Gauci et al.
3.1 Cartilage Matrix: Composition and Function
Articular cartilage is predominantly an extracellular matrix with no nerves or blood

vessels and only a limited population of sparsely distributed chondrocytes. This
small proportion of cells combined with the lack of vasculature means that once it is
damaged, cartilage has a poor capacity for self-repair. The cartilage matrix has two
key functions in the diarthrodial joint. One function is to provide a smooth, friction-
less surface to facilitate movement; this function is conferred mostly by the lubricat-
ing glycoprotein, lubricin, secreted at the cartilage surface. The second function of
cartilage is to distribute mechanical load across joint surfaces; this function is criti-
cally dependent upon water, which is attracted by the high concentration of nega-
tively charged aggrecan trapped within a network of fibrous type II collagen. The
type II collagen fibres are also decorated with minor collagens and small leucine-rich
proteoglycans (SLRPs). Proteinase-mediated degradation of aggrecan, collagens and
SLRPs severely compromises the mechanical properties of articular cartilage in
osteoarthritis (OA) and other cartilage pathologies. These proteinases are produced
by chondrocytes and also by synovial fibroblasts and infiltrating macrophages.
3.1.1 Aggrecan Structure and Function
Aggrecan is a large proteoglycan containing chondroitin sulphate (CS) and keratan

sulphate (KS) glycosaminoglycan side chains. The aggrecan core protein comprises
three globular domains, including G1 and G2 at the N-terminus and a C-terminal G3
globular domain separated from G2 by an extended CS-attachment region (Fig. 3.1).
Multimolecular aggregates of aggrecan containing as many as 100 aggrecan mono-
mers per aggregate (MWaggregate~200 × 106 Da) are immobilised in cartilage by bind-
ing to hyaluronan via their G1 domains (Fig. 3.1). The hyaluronan-aggrecan
complex is further stabilised by binding of link protein to form a trimeric complex.
The vast size of the aggrecan aggregates, together with their water-binding capacity
and their inability to dissociate under physiological conditions, traps aggrecan in
cartilage and provides the osmotic swelling pressure that is required for weight-
bearing and cartilage function. Aggrecan is degraded by proteolysis at multiple sites
along the core protein, but the most detrimental cleavage occurs within the rod-like
interglobular domain which separates the tissue-anchored G1 domain from the
water-binding CS and KS chains. Proteolysis within the CS-rich region is consid-
ered less detrimental to cartilage function and likely reflects normal maturation and
ageing, since most aggrecan in adult cartilage lacks the G3 domain and varying
portions of the CS-rich region (Ilic et al. 1998).
3.1.2 Type II Collagen Structure and Function
Type II collagen is the major collagen in cartilage; its expression is restricted to

cartilaginous tissues and the vitreous of the eye (further details, see Chap. 2 in
Volume 1 (Grässel 2016)). Type II collagen is a homotrimer of three identical α
3 Proteoglycan and Collagen Degradation in Osteoarthritis 43
Fig. 3.1 Schematic cartoon showing arrangement of aggrecan and collagen in cartilage. The
aggrecan core protein contains three globular domains including a C-terminal G3 domain, a G2
domain (green) and an N-terminal G1 domain (black). Aggrecan is anchored in the cartilage matrix
by binding to link protein (blue diamond) and hyaluronan to form large multi-molecular aggre-
gates (Adapted, with publisher’s permission, from Fosang and Beier (2011))
chains, each with an uninterrupted helical domain characterised by repeating

Gly-X-Y motifs where X and Y are bulky amino acids, often proline and hydroxy-
proline. The helical domain is flanked by N- and C-terminal propeptide domains
that are removed during secretion and N- and C-terminal telopeptides that form
inter- and intra-helical cross links, essential for helix stability (Metsaranta et al.
1991; Bateman et al. 1996). Collagen monomers assemble outside the cell into het-
erofibrils with collagens IX and XI, in a process that is aided by the SLRPs. Type II
collagen fibres in the cartilage superficial zone are aligned parallel with the cartilage
surface, whereas type II collagen fibres in the midzone have a cross-over pattern;
fibres in the deep zone, adjacent to the calcified cartilage, are aligned perpendicular
with the cartilage surface. This arrangement of type II collagen provides tensile
strength and a structural scaffold for the cartilage. Proteolytic clipping of the colla-
gen network loosens the elastic restraint within the cartilage matrix, causing it to
soften, and, at the same time, allows aggrecan to swell and occupy increasingly
greater hydrodynamic domains (Maroudas 1976). Only a limited amount of type II
collagenolysis is tolerated before the onset of irreversible cartilage damage.
3.1.3 SLRP Structure and Function
The SLRP core proteins are characterised by a central region of contiguous leucine-
rich repeat motifs, flanked by cysteine-rich domains (further details, see Chap. 1 in
Volume 1 (Aspberg 2016)). The leucine-rich repeats adopt a horseshoe shape that
binds to extracellular matrix molecules, in particular, the collagens. The variable

N-termini of the SLRPs defines the unique function of family members and may
contain CS, KS or dermatan sulphate chains. The SLRPs biglycan, decorin, lumi-
can, fibromodulin and keratocan are common to cartilage, tendon and meniscus
(Melrose et al. 2008; Rees et al. 2009a). Epiphycan, mimecan, osteoadherin, chon-
droadherin, chondroadherin-like, asporin, osteoglycin, opticin and prolargin are
also found in cartilage (for structure and classification of SLRPs found in joint tis-
sues, see Chap. 1, Volume 1 (Aspberg 2016)). SLRPs are best known for their role
as structural constituents in joint matrices. SLRPs such as biglycan, decorin, fibro-
modulin and lumican bind to collagen fibrils, likely aiding fibril formation and
modulating tissue organisation. SLRPs coat the surface of collagen fibrils, provid-
ing a steric barrier that potentially limits the access of collagen-degrading enzymes.
Aside from their structural role, SLRPs directly regulate cell function by binding a
range of cytokines, growth factors and cell surface receptors. The importance of
SLRPs in OA is evident from the phenotypes of the SLRP-deficient mice. Major
phenotypes of SLRP single and double knockout mice are tendon and ligament
defects leading to joint laxity, ectopic ossification and early-onset OA (reviewed in
Ni et al. (2014)).
3.2 Cartilage-Degrading Proteinases in OA
In contrast to the aggressive inflammation that typifies rheumatoid arthritis, the hall-
mark of OA is radiographic joint space narrowing caused by proteinase-driven car-
tilage erosion in a relatively mild inflammatory environment. Cartilage erosion in
OA can progress for many years in the absence of symptoms. Given that type II
collagen in adult human cartilage has a half-life of ~117 years (Verzijl et al. 2000),
any significant damage to the collagen network is generally considered irreparable
(Stoop et al. 1999). Data from mouse models of arthritis have confirmed that carti-
lage can resist only a limited attack on type II collagen before the onset of irrevers-
ible damage (Stoop et al. 1999). In contrast, in vivo studies have shown that large
glycosaminoglycan-bearing aggrecan fragments lost from cartilage following stim-
ulated aggrecanolysis are readily replaced by newly synthesised aggrecan (Thomas
1956). The major protagonists of cartilage degradation in OA are the metal-
dependent proteinases of the matrix metalloproteinase (MMP) and a disintegrin and
metalloproteinase with thrombospondin motifs (ADAMTS) families. Cathepsins,
and cysteine proteinases such as m-calpain, might also have roles in aggrecan catab-
olism (Struglics and Hansson 2010; Troeberg and Nagase 2012) but are not dis-
cussed further.
3.2.1 MMPs
The MMPs are members of the metzincin superfamily. They have a zinc ion at the
active site that is essential for catalytic activity and a conserved motif of histidine
residues (HExxHxxGxxH) that coordinate the zinc ion. Following this motif is a
conserved methionine that forms a characteristic turn important for structure and
function. The MMPs are expressed as inactive zymogens, with an N-terminal pro-
peptide that maintains latency, a catalytic domain and, in the majority of MMPs, a
flexible hinge region linked to a hemopexin domain. The membrane-bound MMPs
have an additional transmembrane domain and a cytoplasmic tail or are GPI
anchored. Dysregulated MMP activity against type II collagen and aggrecan is
thought to contribute to cartilage pathology in OA (Troeberg and Nagase 2012;
Goldring et al. 2011). Specific cleavage sites and the products of MMP-mediated
aggrecanolysis and collagenolysis have been well characterised, and fragments of
type II collagen have been investigated for their utility as OA biomarkers
(Nemirovskiy et al. 2007). SLRPs are also substrates for MMPs in cartilage.
Much attention has focused on MMP-13 (collagenase-3) due to its potent activity
against type II collagen. Selective inhibitor studies have shown that MMP-13 is the
major cartilage collagenase in vivo (Billinghurst et al. 1997; Dahlberg et al. 2000).
MMP-13 expression is increased in cartilage and synovium during late-stage OA
(Mitchell et al. 1996; Bau et al. 2002; Kevorkian et al. 2004; Davidson et al. 2006),
and animal studies have shown that overexpression of MMP-13 in mouse cartilage
induces OA-like pathology in the knee joint (Neuhold et al. 2001). Mmp13−/− mice
are protected against cartilage erosion in a surgical model of OA (Little et al. 2009).
Accordingly, MMP-13 is a prime target for OA therapies. Early attempts to develop
MMP inhibitors focused on chelation of the active site zinc ion, using hydroxamic
acid derivatives. These inhibitors failed in clinical trials due to poor selectivity and
tendinitis-like side effects (reviewed in Jacobsen et al. (2010)). Since then, inhibitor
designs have targeted secondary binding sites, known as exosites, with the aim of
improving selectivity. Several pharmaceutical companies have reported the devel-
opment of potent and highly selective inhibitors to MMP-13 (Gao et al. 2010; Nara
et al. 2014; Engel et al. 2005; Gege et al. 2012; Jungel et al. 2010; Ruminski et al.
2016; Fischer and Riedl 2016), but to date there are no reports of clinical trials of
these new classes of MMP-13 inhibitors (reviewed in Fields (2015)).
3.2.2 ADAMTS Enzymes
Like MMPs, the ADAMTS enzymes are a subset of the metzincin superfamily.
There are 19 members of the mammalian ADAMTS family, all of which are secreted
and active extracellularly. All ADAMTS enzymes comprise an N-terminal pro-
domain with a furin-recognition sequence, a catalytic domain with the consensus
sequence HEBxHxBGBxH, a disintegrin-like domain, a cysteine-rich domain, a
spacer domain and one or more thrombospondin repeats. ADAMTS-4 and
ADAMTS-5 degrade aggrecan and they are considered the most important
ADAMTS enzymes in OA. These aggrecanases are also active against SLRPs,
including biglycan, fibromodulin and keratocan. ADAMTS-4 and ADAMTS-5 have
a similar domain structure, with ADAMTS-4 having one and ADAMTS-5 having
two thrombospondin motifs. The non-catalytic ancillary domains of ADAMTS-4
and ADAMTS-5 have roles in substrate recognition and matrix localisation

(Gendron et al. 2007; Fushimi et al. 2008). Mice with ADAMTS-5 deletion or
expressing catalytically inactive ADAMTS-5 have significantly reduced cartilage
erosion in models of OA and inflammatory arthritis, respectively (Glasson et al.
2005; Stanton et al. 2005). Monoclonal antibodies targeting the catalytic and disin-
tegrin domains of ADAMTS-5 have shown efficacy in inhibiting cartilage erosion in
a mouse model of OA (Larkin et al. 2015).
3.2.3 Regulation of MMP and ADAMTS Enzymes
In healthy cartilage the expression of MMPs and ADAMTS enzymes is tightly regu-
lated to allow low turnover remodelling of the matrix. Evidence from in vitro and
in vivo studies has shown that in OA, cells of the joint produce, and respond to, a
range of pro-inflammatory mediators, particularly interleukin (IL)-1α, IL-1β and
tumour necrosis factor α (TNFα) (reviewed in Goldring and Otero (2011)). IL-1α
and β are well known for their ability to upregulate MMP expression in OA cartilage
(Mitchell et al. 1996; Kobayashi et al. 2005; Bau et al. 2002). For the aggrecanases,
studies with inflammatory mediators have produced inconsistent results, with many
studies supporting the constitutive expression of ADAMTS-5 mRNA in joint tissues
and cells, but others reporting regulation of ADAMTS-5 mRNA by IL-1α and β,
oncostatin M and FGF-2 (reviewed in Fosang et al. (2008); Fosang and Rogerson
(2010)). ADAMTS-4 mRNA, in contrast, is reproducibly upregulated by cytokines
in many experimental systems. Interestingly, the regulation of ADAMTS-4 and
ADAMTS-5 expression differs according to species, cell type, tissue origin (hip or
knee) and perhaps disease stage.
The post-transcriptional regulation of MMP and ADAMTS enzymes in OA car-
tilage is mediated by epigenetic modification to DNA (Tsezou 2014), histone modi-
fication (Troeberg and Nagase 2012) and alternative splicing in the case of
ADAMTS-4 (Wainwright et al. 2013). One area of post-transcriptional regulation
that is rapidly evolving is the contribution that non-coding microRNAs (miRNAs)
make to the regulation of gene expression in OA. miRNAs bind to the 3′-untrans-
lated region of target RNAs and silence gene expression. Over 40 miRNAs have
now been reported as differentially regulated in OA (Nugent 2016). Compared with
wild-type mice, miR-140 −/− mice develop OA-like pathology with increased levels
of ADAMTS-5 mRNA and increased aggrecan loss in vitro (Miyaki et al. 2009).
Conversely, overexpression of miR-140 in chondrocytes reduced aggrecan loss in
IL-1β-treated cells in vitro and also reduced in vivo aggrecan loss in the antigen-
induced model of inflammatory arthritis (Miyaki et al. 2010). miR-146a (Li et al.
2011) and miR-148a (Vonk et al. 2014) also negatively regulate ADAMTS-5
mRNA, whereas miR-125b regulates ADAMTS-4 (Matsukawa et al. 2013). MiR-
33a (Kostopoulou et al. 2015) and miR-27b (Akhtar et al. 2010) function as positive
and negative regulators, respectively, of MMP-13 in human chondrocytes, and
miRNA-127-5p suppresses IL-1β-induced MMP-13 production in human chondro-
cytes (Park et al. 2013). Most recently, miR-30a has been identified as an essential
regulator of IL-1β-induced ADAMTS-5 expression and cartilage degradation

(Ji et al. 2016).
In normal tissues, the post-translational regulation of the MMP and ADAMTS
enzymes is tightly controlled to keep levels of enzyme activity in check. The first
checkpoint is activation, which requires proteolytic processing at the N-terminus to
remove the propeptide. For most MMPs, this activation occurs via a ‘cysteine-
switch’ mechanism, involving proteolytic disruption of a pairing between a con-
served cysteine residue in the propeptide and the zinc ion at the active site. A range
of serine proteinases are candidates for MMP propeptide removal in arthritic joints,
including plasmin, matriptase-1, activated protein-C, mast cell tryptases and chy-
mases (reviewed in Fosang and Beier (2011)). Active MMPs are also capable of
initiating MMP activation cascades; in particular, membrane-type MMP-1 (MT1-
MMP) can activate proMMP-2 and proMMP-13 (Murphy et al. 1999). Some MMPs,
including MMP-11, MMP-28 and several MT-MMPs, as well as ADAMTS-4 and
ADAMTS-5 have a furin-recognition sequence (RXK/RR) in their propeptide
domains, which is cleaved by proprotein convertases. There are seven proprotein
convertases in mammals, including the archetypal furin, which compartmentalises
to various subcellular, cell surface and extracellular locations, providing spatial
control of enzyme activation. PACE4 is the most abundant proprotein convertase in
extracts of human osteoarthritic cartilage and is likely to be the primary activator of
ADAMTS-4 and ADAMTS-5 in cartilage (Malfait et al. 2008).
For ADAMTS-4 and ADAMTS-5, C-terminal truncation by proteolytic (Gao
et al. 2004) or autocatalytic mechanisms (Zeng et al. 2006; Flannery et al. 2002)
provides a further level of control. Autocatalytic cleavages occur in the spacer
domain of ADAMTS-4 and ADAMTS-5 (Flannery et al. 2002; Zeng et al. 2006).
The spacer and cysteine-rich domains are important for locating the enzymes to the
cell surface or to the pericellular or extracellular matrices. The endogenous tissue
inhibitors of metalloproteinases (TIMPs) inhibit MMPs and ADAMTS enzymes.
All four members of the TIMP family inhibit MMPs, showing relatively little selec-
tivity, with the exception of some membrane-type MMPs that are poorly inhibited
by TIMP-1. ADAMTS-4 and ADAMTS-5, in contrast, are only inhibited by TIMP-
3, but it is effective at sub-nanomolar concentrations and is the only TIMP that
localises to the cartilage matrix, via binding to heparan sulphate-bearing proteogly-
cans, including perlecan (Troeberg et al. 2014).
The levels of MMPs and ADAMTS enzymes in cartilage are further controlled
by endocytotic clearance via low-density lipoprotein receptor-related protein-1
(LRP-1). ADAMTS-4 and ADAMTS-5 share the same binding site on LRP-1, but
ADAMTS-5 has the greater affinity for LRP-1 (Yamamoto et al. 2016). TIMP-3 is
also internalised by LRP-1, and, interestingly, TIMP-3 binding to sulphated glycos-
aminoglycans reduces TIMP-3 clearance rates and increases TIMP-3 affinity for
ADAMTS-5 (and likely ADAMTS-4 and several MMPs), making synthetic glycan
mimetics potential targets for blocking cartilage destruction in OA (Troeberg et al.
2014). More recently, Yamamoto and colleagues showed that MMP-13 is also inter-
nalised by LRP-1 and that it can be co-endocytosed with ADAMTS-4, ADAMTS-5
or TIMP-3 (Yamamoto et al. 2016). A rapid, LRP-1-mediated clearance of
MMP-13, ADAMTS-5 and TIMP-3 might explain why detection of these proteins
in chondrocyte-conditioned medium is difficult, despite measurable levels of mRNA
expression. Rapid clearance might also provide tight control over proteinase activity
in healthy cartilage. But in OA, shedding of LRP-1 protein, along with elevated
expression of MMP-13, ADAMTS-4 and ADAMTS-5, is expected to tip the balance
in favour of cartilage degradation.
3.2.4 Aggrecan Degradation
Following the detection of aggrecanase fragments in human synovial fluids from

patients with OA, joint injury and inflammatory joint disease (Lohmander et al.
1993; Sandy et al. 1992), two aggrecanase enzymes were cloned and identified as
ADAMTS-4 (Tortorella et al. 1999) and ADAMTS-5 (Abbaszade et al. 1999). Both
enzymes cleaved at the E373↓374A bond in the aggrecan interglobular domain.
ADAMTS-5 is the primary aggrecanase in the mouse, but it is not yet clear whether
ADAMTS-4 or ADAMTS-5 is the predominant human aggrecanase. Monoclonal
antibodies against a conformational epitope spanning both the catalytic and
disintegrin-like domains of either ADAMTS-4 or ADAMTS-5 have been raised to
further explore the roles of ADAMTS-4 and ADAMTS-5 in vivo (Larkin et al. 2015).
The anti-ADAMTS-5 antibodies blocked aggrecan loss from human cartilage
explants cultured with or without cytokine stimulation, whereas anti-ADAMTS-4
antibodies only blocked aggrecan loss from unstimulated cartilage explants. These
results do not provide a clear-cut verdict on whether ADAMTS-4 might also be a
functional aggrecanase in humans; however, they reveal the interesting possibility
that in both ADAMTS-4 and ADAMTS-5, the active site spans the catalytic and the
disintegrin-like domains. Other studies have also suggested that both catalytic and
disintegrin-like domains of ADAMTS-5 are required for maximum aggrecanase
activity (Kosasih et al. 2016; Santamaria et al. 2015). It is unclear which ADAMTS
is responsible for aggrecan degradation in tendon. Tendon explant cultures secrete
aggrecanase activity in the absence of catabolic stimulation suggesting a role for
aggrecanases in constitutive turnover of aggrecan (Rees et al. 2009a).
Cleavage at E373↓374A by ADAMTS enzymes is considered the most detrimental
to aggrecan function because it releases all the CS chains from the aggrecan aggre-
gate. Although this large and highly glycosylated 374ARGS fragment is considered a
defining product of cartilage erosion, it is not the preferred cleavage site for the
ADAMTS enzymes. Instead ADAMTS-4 and ADAMTS-5 cleave bovine aggrecan
in the CS-rich region, at E1666↓1667G and E1480↓1481G, prior to cleavage at E373↓374A
(Tortorella et al. 2000). Subsequent cleavage events at E1819↓1820A and E1919↓1920L
also occur in the CS-rich region. These cleavage sites are conserved in human, pig,
mouse and rat aggrecan. Several fragments are further catabolised, for example, the
G1-TEGE373 fragment is cleaved by MMPs to generate a 32-amino-acid fragment
with a 342FFGV N-terminus and a TEGE373 C-terminus, which we have named the
‘32mer’ (Lees et al. 2015). The 32mer has pro-inflammatory and pro-catabolic
activity, as discussed below.
3.2.5 Aggrecan Neoepitopes as Markers of Cartilage Catabolism
Neoepitope antibodies are valuable and well-documented tools for identifying

proteinase families involved in protein degradation. Aggrecan neoepitope anti-
bodies recognise amino acid sequences at the newly created N- or C-terminus of
cleaved aggrecan, but critically, they do not recognise the same sequence of amino
acids in undegraded aggrecan or aggrecan fragments derived from different
enzyme families. Aggrecan 374ARGS fragments are the most prominent fragments
in OA synovial fluids and are widely used as a marker of ADAMTS activity in
human OA studies and other experimental systems. In contrast, aggrecan 374ARGS
fragments are poorly represented in children with juvenile arthritis, suggesting
that enzymes other than aggrecanases might have a greater role in childhood
arthritides (Struglics et al. 2012). MMP-derived aggrecan 342FFGV fragments are
also present in human and animal synovial fluids. Similarly, the corresponding G1
domains with either IPEN341 or TEGE373 C-termini are retained in cartilage by
binding to hyaluronan and are readily detected by immunohistochemistry in sec-
tions from human and animal cartilage.
3.2.6 Type II Collagen Degradation
The collagenases are a specialised subfamily of MMPs that cleave type II collagen
at a single site in the triple helix, approximately three-quarters of the way from the
N-terminus at G775↓776L (human sequence). Cleavage at this highly conserved site
produces a three-fourths length TCA N-terminal fragment and a one-fourth length
TCB C-terminal fragment (TCB) and is the initiating event in collagenolysis (Miller
et al. 1976), in all species. Thereafter the thermally unstable TCA and TCB fragments
denature to become substrates for further proteolysis, for example, TCB fragments
are cleaved by MMPs at G778↓779Q (Mitchell et al. 1996; Billinghurst et al. 1997)
and G781↓782 (Billinghurst et al. 1997). TCA cleavage products have been detected in
the urine of OA patients by mass spectrometry (Nemirovskiy et al. 2007). Although
MMP-1, MMP-2, MMP-8, MMP-13 and MMP-14 cleave native type II collagen
in vitro, MMP-13 is considered the principal cartilage collagenase (Billinghurst
et al. 1997; Mitchell et al. 1996; Knäuper et al. 1996).
Considering that most enzyme substrates are single polypeptide sequences, the
helix of native collagen presents a considerable challenge for hydrolysis, given that
three polypeptide sequences are tightly wound. The mechanism of hydrolysis is
intriguing because the catalytic cleft of the collagenases is not wide enough to
accommodate a triple helical substrate (Chung et al. 2004), and isolated catalytic
domains of the collagenases fail to cleave collagen (Knäuper et al. 1997; Patterson
et al. 2001), suggesting that a sophisticated protease-substrate interaction is
involved. Manka et al. (2012) used data from biochemical and crystallographic
analyses to construct a model of collagenolysis that is unique in the field of protease-
substrate interactions. Collagenolysis is made possible by the plasticity of the col-
lagenases and by the relative thermoflexibility of collagen at the collagenase
cleavage site, which is sufficient at body temperature to allow localised loosening of

the helix. Mapping has shown that catalytic and hemopexin domains of MMP-1
bind cooperatively to two conserved leucines near the cleavage site, to anchor two
collagen α chains. Modelling suggests that flexing of MMP-1 at the linker region
between the catalytic and hemopexin domains bends the third α chain, facilitating
localised collagen unwinding and positioning the collagenase-cleavable bond in the
catalytic cleft. Following the initial cleavage, it is possible that the other two strands
unravel, making them susceptible to hydrolysis (Stura et al. 2013).
Collagen type II fibrils can be further compromised by cathepsin or MMP cleav-
age within the N- and C-terminal telopeptide domains. For example, peptides with
the sequence EKGPDP, derived from the collagen type II telopeptide region have
been detected in the urine of OA patients (Mazzuca et al. 2006). Similarly, peptide
fragments derived from cathepsin K cleavage at G61↓62K (human sequence) near the
N-terminus of the triple helix have been detected in human and animal cartilage
in vitro (Kafienah et al. 1998) and in vivo (Dejica et al. 2008).
3.2.7 he Relative Contributions of Aggrecanolysis

T
and Collagenolysis to Cartilage Erosion
Cartilage erosion in OA can progress for many years in the absence of clinical
symptoms, and there is evidence to suggest that aggrecanolysis precedes collagen-
olysis, for example:
(i) The consistent finding of weak metachromatic staining in sections of degener-

ate human and animal cartilage, prior to evidence of clinical symptoms.
(ii) The in vivo studies of Lewis Thomas (1956) which showed that papain injected
into an upright rabbit ear produced a loose, floppy ear lacking tissue turgor;
aggrecan is readily degraded by papain whereas fibrillar collagen is resistant to
all proteinases other than collagenases under physiological conditions.
(iii) The data showing that treatment of cartilage explants with IL-α, IL-1ß, TNF-α,
oncostatin M or retinoic acid stimulates marked aggrecan loss within 1–2 days,
followed by collagen breakdown a week or more later (Little et al. 2002;
Kozaci et al. 1997; Beekman et al. 1998; Pratta et al. 2003).
Possibly, the long period of subclinical OA is due to protection conferred on the

collagen scaffold by constantly replenishing levels of aggrecan. Furthermore, it is
possible that cartilage failure and radiographic cartilage damage emerge when
aggrecan synthesis declines, and the collagen scaffold becomes progressively
exposed.
Other than the studies above on the timing of aggrecanolysis versus collagenoly-
sis, there are currently no data to indicate which of these processes is most impor-
tant for driving OA. To explore this aspect, we produced knock-in mice with
mutations at either the aggrecanase cleavage site in the aggrecan IGD (Jaffa mice)
(Little et al. 2007) or the collagenase cleavage site in type II collagen. To block
cleavage of type II collagen, we introduced a mutation at the primary collagenase

cleavage site in mouse collagen II that changes amino acids PQG775↓776LAG to
PPG775↓776MPG in the α1(II) chain. This mutation blocks cleavage at the primary
collagenase cleavage site and thus blocks production of the TCA and TCB fragments
that are the substrates for further collagen processing by gelatinases. This change,
which is identical to the changes made previously in the α1(I) chain of type I col-
lagen (Wu et al. 1990; Liu et al. 1995; Hasty et al. 1993; Zhao et al. 1999), com-
pletely blocked cleavage at the three-fourths to one-fourth collagenase cleavage site
but, importantly, had no impact on collagen fibrillogenesis, which remained normal.
Whereas type II collagen is expressed in cartilage and the vitreous of the eye, type
I collagen is expressed abundantly in many tissues. The type I collagen knock-in
mouse has a mild bone phenotype that resolves with age.
In the experiments described here, Jaffa mice were homozygous for the
aggrecanase-resistant mutation, whereas the collagenase-resistant mutation was
heterozygous in both the single- and double-mutant mice. We first compared pas-
sive and stimulated aggrecan loss in response to short-term IL-1α treatment and
found that while the levels of passive aggrecan loss were low, and similar for each
genotype (data not shown), the levels of stimulated aggrecan loss were different for
each genotype (Fig. 3.2a). The greatest level of aggrecan loss was from wild-type
Fig. 3.2 Aggrecan loss and cartilage erosion in wild-type mice or mice resistant to aggrecanase
activity, collagenase activity or both activities. (a) Femoral head cartilage was harvested from
3-week-old wild-type mice or mice resistant to aggrecanase activity (Agg’ase) (Little et al. 2007),
collagenase activity (Coll’ase) or resistant to both aggrecanase and collagenase activity (Agg’ase
and Coll’ase). The explants were stimulated with IL-1α for 3 days, and the amount of aggrecan
released into the medium was quantitated by the dimethylmethylene blue dye-binding assay. For
each genotype the number of biological replicates is shown in brackets. Statistical significance was
calculated by the unpaired t-test with Welsh’s correction. The relevant P values are shown. (b) Ten-
week-old wild-type mice, or mice resistant to aggrecanase activity (Little et al. 2007), collagenase
activity (Fosang, 2009 unpublished data) or resistant to both aggrecanase and collagenase activity,
received surgery to induce experimental OA by destabilising the medial meniscus (DMM surgery).
After 8 weeks, the operated knees were harvested and processed for histology to score for cartilage
erosion. For each genotype the number of biological replicates is shown in brackets. The whiskers
represent the 5–95 percentiles and outliers are represented by solid circles. Statistical significance
was calculated by unpaired t-test with Welsh’s correction. The relevant P values are shown
cartilage, with 59 % of aggrecan lost to the conditioned medium after 3 days in cul-
ture. In contrast, the amount of aggrecan released from Jaffa cartilage was reduced
to 37 % and reduced to 27 % loss from the collagenase-resistant mice. Aggrecan
released from the double aggrecanase- and collagenase-resistant mice was only
18 % (Fig. 3.2a). While these data confirm the key role of ADAMTS activity in
stimulated aggrecanolysis, it also suggests that there is a relationship between
aggrecanolysis and collagenolysis, with specific inhibition of the latter conferring
protection against the loss of aggrecan from cartilage.
To examine the effect of genotype on in vivo cartilage erosion, male 10-week-
old mice from each genotype had OA surgically induced by destabilisation of the
medial meniscus (DMM), and cartilage erosion was scored for 8 weeks after sur-
gery, as previously described (Little et al. 2007). The results showed that com-
pared with wild-type mice, cartilage erosion was significantly reduced in both the
homozygous aggrecanase-resistant (P < 0.0134) and heterozygous collagenase-
resistant (P < 0.0432) mouse lines. Furthermore, it was clear that blocking both
catalytic activities together substantially reduced the severity of cartilage erosion
(P < 0.00001) (Fig. 3.2b). Since the collagenase-resistant mice were heterozygous
for the knock-in mutation, the results suggest that blocking collagenase activity is
likely to be a more efficacious OA target than blocking aggrecanase activity, and
clearly blocking both activities simultaneously had the greatest effect on reducing
cartilage erosion.
3.2.8 SLRP Degradation
Unlike the fields of aggrecanolysis and collagenolysis which are relatively mature,
the field of SLRP proteolysis is still emerging. Biochemical-style studies in which
recombinant enzymes have been used to digest recombinant or purified SLRPs in
solution have identified specific cleavage sites in SLRP core proteins (Melching
et al. 2006; Monfort et al. 2006; Imai et al. 1997; Gendron et al. 2007; Rees et al.
2009b; Kashiwagi et al. 2004), but such studies have included relatively few SLRPs
found in joint tissues and only a small pool of enzymes. Other studies on SLRP
degradation processes in tissues ex vivo or cultured in vitro have identified SLRPs
that are more susceptible to proteolysis in normal and OA cartilage and allowed the
timing of SLRP degradation to be placed in context with timing of degradation of
other matrix molecules such as aggrecan and collagen (Monfort et al. 2006;
Heathfield et al. 2004; Zhen et al. 2008; Sztrolovics et al. 1999). Analysis of the size
and abundance of SLRP fragments in human joint tissues has revealed increased
fragmentation of SLRPs in OA joints, compared with age-matched non-arthritic
controls (Melrose et al. 2008; Cs-Szabo et al. 1997). However, SLRP cleavage sites
defined in vitro using soluble SLRPs do not always correlate in size with SLRP
fragments detected in tissues ex vivo (Melrose et al. 2008). This might reflect the
degree of glycosylation or the accessibility of cleavage sites in matrix-bound
SLRPs, compared with soluble SLRPs, or that enzymes other than those tested
in vitro are responsible for cleaving SLRPs in tissues.
One consequence of SLRP degradation in OA tissues is breaching of the steric

barrier that SLRPs provide on the surface of collagen fibrils. This activity is pre-
dicted to expose collagen fibrils to proteolytic attack and to accelerate cartilage
degeneration (reviewed in Ni et al. (2014)). Fibromodulin fragmentation precedes
collagen degradation in IL-1α-stimulated cartilage explants, and this fragmentation
occurs long after the peak of aggrecan loss (Sztrolovics et al. 1999; Heathfield et al.
2004). Fibromodulin is cleaved by MMP-13, provided it is matrix bound, producing
a product that is identical to the fibromodulin fragment that is generated in cartilage
explant cultures (Heathfield et al. 2004). Biglycan is also highly susceptible to
MMP-13 digestion (Monfort et al. 2006), suggesting that poorly controlled MMP-
13 activity could degrade the SLRP surface coat leading to degradation of the col-
lagen fibril and progressive degeneration of the fibrillar network. Other MMPs,
including MMP-2, MMP-3, MMP-8, MMP-9 and MMP-12, as well as the aggreca-
nases, can also release SLRP fragments from human cartilage (Zhen et al. 2008).
In a study mapping fragments of decorin, biglycan, fibromodulin, lumican and
keratocan in meniscus, knee and hip cartilage from OA patients (Melrose et al.
2008), the same molecular mass fragments were found in all tissues, suggesting
common proteolytic mechanisms at play, despite differences in cell type, matrix
composition and vascularity between meniscus and cartilage. Furthermore, although
SLRP fragments were less abundant in normal cartilage, the fragment sizes were
similar (with the exception of fibromodulin), suggesting that the enzymes respon-
sible for SLRP proteolysis in OA are the same as those regulating homeostasis in
normal cartilage.
3.3 artilage Degradation Products Promote Inflammatory

C
Mediators
In addition to compromising the structural and mechanical properties of cartilage,

aggrecan degradation generates fragments with bioactivity that can compromise tis-
sue function. A synthetic 32-amino acid peptide (342FFG-EGE374), representing the
peptide product of MMP cleavage at the amino terminus and aggrecanase cleavage
at the carboxy terminus, is pro-inflammatory and pro-catabolic when added to cul-
tures of primary mouse chondrocytes, synovial fibroblasts or peritoneal macro-
phages (Lees et al. 2015). Furthermore, native, KS-containing 32mer purified from
pig articular cartilage, also upregulates the expression of MMP-3 and MMP-13
mRNA and protein in human chondrocytes. These results suggest that the 32mer
aggrecan degradation product is a DAMP (damage (or danger)-associated molecu-
lar pattern).
DAMPS are host-derived molecules found often (but not always) ‘out of context’
for the cells. For example, DAMPs are molecules that may be hydrolysed, reduced,
oxidised, denatured, fragmented, overexpressed or mislocalised, such as DNA out-
side the nucleus or mitochondria. DAMPs mediate their actions via interactions
with members of the Toll-like receptor (TLR) family, leading to activation of major
signalling pathways including the nuclear factor kB and mitogen-activated protein
kinase pathways (reviewed in O’Neill (2006) and details in Chap. 1). The TLRs
expressed in human cartilage include TLR1, TLR2, TLR3, TLR4 and TLR6 (Zhang
et al. 2008). Activation of these TLRs can upregulate collagenase expression and
activation, leading to resorption of cartilage collagen. The aggrecan 32mer signals
via TLR2 in mouse chondrocytes (Lees et al. 2015). Because the 32mer is derived
from the G1-EGE373 fragment, which is retained in the cartilage matrix with a half-
life of ~20 years (Verzijl et al. 2001), repeated cycles of glycosaminoglycan loss
and new aggrecan synthesis produce a readily renewable supply of G1-EGE373 sub-
strate, making the aggrecan 32mer an important addition to the list of OA-relevant
DAMPs.
Other DAMPs present in degraded, inflamed or OA cartilage include fibronectin
fragments (Su et al. 2005), tenascin C (Chevalier et al. 1994; Midwood et al. 2009)
and fragments of low MW hyaluronan (Liu-Bryan and Terkeltaub 2010). From the
proteoglycan family, soluble biglycan, decorin and versican show capabilities or
characteristics of DAMPs (reviewed in Schaefer (2014)), and recently the proteo-
glycan lubricin was added to the list (Alqurqini et al. 2015). In chondrocytes, big-
lycan signalling (mostly via TLR-4) upregulates MMP and inflammatory cytokine
mRNA and protein expression and downregulates aggrecan and type II collagen
mRNA expression (Barreto et al. 2015). Biglycan must be both soluble and intact
to act as a DAMP, since matrix-bound biglycan does not signal, nor does deglyco-
sylated biglycan core protein or biglycan-derived glycosaminoglycan chains
(Schaefer et al. 2005) (reviewed in Nastase et al. (2012)). Intact, soluble biglycan
is present in the circulation (Moreth et al. 2010) and in synovial fluids from patients
with OA and is most abundant in joint fluids from patients with advanced OA
(Barreto et al. 2015).
Conclusions
Together, aggrecan and collagen II provide the compressive resilience and
mechanical strength required for weight bearing in articular cartilage. SLRPs
decorating the surface of collagen fibrils further augment collagen resilience by
modulating fibril formation and providing a steric barrier against proteinase
attack. The proteolytic activities of MMP-13, together with ADAMTS-5 and
possibly ADAMTS-4 activities, compromise cartilage function and are therefore
valid targets for the development of OA therapies. The modulation of these pro-
teinase activities by cytokines and non-coding RNAs provides a layer of regula-
tion that may also be amenable to pharmacological intervention. Early results
from genetic approaches targeting aggrecan and collagen-degrading activity
in vivo suggest that simultaneous blockade of both activities provides greater
efficacy in limiting cartilage damage, than either activity alone.
Several products of cartilage erosion are recognised as DAMPs. DAMPs are
‘danger signals’ derived not from bacteria or pathogens but from pathogen-free cell
or tissue injury, and they drive inflammation. While low levels of inflammation
provide benefits to tissues and organisms by eliminating cellular and tissue debris
following a physical or biological insult, persistently high levels of harmful stimuli
can produce DAMPS that act as renewable danger signals, triggering ongoing and
escalating cycles of inflammation, that can become chronic. Specific fragments of

degraded cartilage molecules including aggrecan, hyaluronan and fibronectin are
now recognised as DAMPs, and the degradative events that generate these DAMPS
are also well known. Other DAMPs are likely to emerge as we learn more about the
dynamic signalling between healthy and unhealthy cartilage in OA.
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Role of Proteoglycans in Osteoarthritis
4
Jessica Bertrand and Annelena Held
Abstract
The articular cartilage covers the end of the long bones and ensures frictionless
motion of the joints. Chondrocytes produce and maintain in a homeostatic equi-
librium a large amount of extracellular matrix (ECM), thereby ensuring the
cartilage function in locomotion and the absorption of biomechanical stress
throughout life. To achieve this, chondrocytes are extremely sensitive to mechan-
ical stress and injury signals and can rapidly adapt their metabolic activity
ultimately ensuring that remodeling and repair of the tissue follow injury.
Proteoglycans as one of the main components of the ECM have diverse functions
in the cartilage. They bind water and provide the basis for absorbing high compres-
sive loads. Additionally, they bind cytokines, chemokines, growth factors, and mor-
phogens, thereby protecting these factors against proteolysis and/or acting as a
depot of regulatory factors when matrix degradation occurs. They also modulate
signaling pathways and create morphogen gradients by immobilization of ligands in
the ECM and regulation of the turnover of ligands. The extracellular domains of
proteoglycans can be shed from the cell surface, generating soluble decoy receptors,
and they can act as co-receptors for various tyrosine-kinase-type growth factor
receptors. Furthermore, they can act as endocytic receptors and contribute to the
clearance of bound ligands and cooperate with integrins and other cell adhesion
receptors to facilitate cell attachment, cell–cell interactions, and cell motility.
Given these important roles of proteoglycans in regulating cell functions, it is
well understandable that loss of ECM and degradation of proteoglycans during
OA induce severe changes in cartilage homeostasis. The following review will
discuss the role of different proteoglycans in the aforementioned processes with
special emphasis on the role in osteoarthritis.
J. Bertrand, PhD (*) • A. Held, M Sc

Department of Orthopaedic Surgery, Otto-von-Guericke University Magdeburg,
Leipziger Straße 44, 39120 Magdeburg, Germany
e-mail: jessica.bertrand@med.ovgu.de; Annelena.held@med.ovgu.de

64 J. Bertrand and A. Held
4.1 Introduction
The articular cartilage is an avascular, aneural tissue that covers the end of the long
bones and ensures frictionless motion of the joints. Its only cell type, the chondro-
cyte, produces and maintains in a homeostatic equilibrium a large amount of extra-
cellular matrix (ECM). The ECM is rich in highly sulfated glycosaminoglycans
(GAGs), which provide capacity for shock absorption. Because of the cartilage
function in locomotion, the absorption of biomechanical stress and the maintenance
of homeostatic integrity of this tissue are important throughout life. To achieve this,
chondrocytes are extremely sensitive to mechanical stress and injury signals and
can rapidly adapt their metabolic activity ultimately ensuring that remodeling and
repair of the tissue follow injury. If the stress factors and damage to the cartilage,
however, are too extreme for repair by chondrocytes, the degeneration of cartilage,
called osteoarthritis (OA), is the ultimate result. OA is widely considered a degen-
erative disease that is characterized by progressive structural changes in joint tis-
sues, principally in articular cartilage, but also in the subchondral bone, the synovial
membrane, and the synovial fluid. Articular cartilage fibrillation and erosions
accompanied by chondrocyte dedifferentiation and loss of ECM components such
as proteoglycans are key features associated with OA.
The ECM is principally composed of water, different collagens, various proteo-
glycans, and other non-collagenous proteins and glycoproteins present in lesser
amounts (Buckwalter and Rosenberg 1988; Buckwalter and Lane 1997). This com-
position helps to retain water in the cartilage ECM and thereby provides the basis
for the biomechanical properties of the cartilage. In healthy articular cartilage, tis-
sue fluid represents between 65 and 80 % of the total weight (Mow et al. 1992),
proteoglycans account for 25–35 %, and collagens account for about 60 % of the
cartilage dry weight (Buckwalter et al. 2005).
Proteoglycans are the second-largest group of macromolecules in the ECM of
the articular cartilage (Aspberg 2016). They consist of a core protein with one or
more covalently attached linear glycosaminoglycan (GAG) chains. These side
chains can be very long as they might be composed of more than 100 monosaccha-
rides. Proteoglycans can be separated into different subgroups according to the
nature of GAG side chains attached to the core protein or the size of the core pro-
tein. The side chains can be composed of heparan, chondroitin, dermatan, and kera-
tan sulfate. According to their size, aggrecan, versican, agrin, and perlecan belong
to the large-sized proteoglycans, and syndecan, glypican, decorin, biglycan, aspo-
rin, fibromodulin, and lumican belong to the group of small-sized proteoglycans,
whereby the last five proteoglycans form the subgroup of small leucine-rich proteo-
glycans (SLRPs).
The most important function of interstitial proteoglycans is their capacity to bind
water and provide the basis for absorbing high compressive loads by water desorp-
tion and resorption. Furthermore, proteoglycans can bind cytokines, chemokines,
growth factors, and morphogens such as bone morphogenetic proteins (BMPs)
(Ruppert et al. 1996), fibroblast growth factors (FGFs) (Rapraeger et al. 1991), and
Wnt ligands (Wnts) (Reichsman et al. 1996). These bound factors are protected
4 Role of Proteoglycans in Osteoarthritis 65
against proteolysis and act as a depot of regulatory factors that can be liberated
either by selective degradation of the matrix or in disease when matrix degradation
occurs. It has been shown that heparan sulfates modulate signaling pathways, e.g.,
Wnts, through several mechanisms (Whitelock and Iozzo 2005). First, Wnts avidly
bind to heparan sulfate proteoglycans (HSPGs), thereby creating morphogen gradi-
ents (Han et al. 2005) (for details, see (Hartmann 2016)). Second, HSPGs immobi-
lize and regulate the turnover of ligands that act at the cell surface (Filion and Popel
2004). Third, the extracellular domains of these proteoglycans can be shed from the
cell surface, generating soluble heparan sulfate proteoglycans that can inhibit inter-
actions at the cell surface (Bernfield et al. 1999). Biochemical studies have impli-
cated HSPGs not only in the binding of growth factors but also in Wnt signal
transduction (Reichsman et al. 1996; Uren et al. 2000). Cell surface or transmem-
brane proteoglycans can act as co-receptors for various tyrosine-kinase-type growth
factor receptors, lowering the threshold for activation of signaling cascades or
changing the duration of signaling reactions. Furthermore, they can be internalized
together with the respective receptor and ligand(s) and thereby act as endocytic
receptors and contribute to the clearance of bound ligands. They can also cooperate
with integrins and other cell adhesion receptors to facilitate cell attachment, cell–
cell interactions, and cell motility (Esko and Linhardt 2009). Finally, HSPG binding
to Wnt ligands is strictly required to maintain these hydrophobic molecules in solu-
tion, thereby allowing their biological activity (Fuerer et al. 2010) (Fig. 4.1).
Given these important roles of proteoglycans in regulating cell functions, it is
well understandable that loss of ECM and degradation of proteoglycans during OA
induce severe changes in cartilage homeostasis. The following paragraphs will dis-
cuss the role of different proteoglycans in the aforementioned processes with spe-
cial emphasis on the role in osteoarthritis.
4.2 Large Proteoglycans
Prominent large proteoglycans in the cartilage are aggrecan, versican, agrin, and
perlecan (Aspberg 2016).
4.2.1 Aggrecan
Aggrecan belongs to the family of chondroitin sulfate proteoglycans and is the

largest in size and the most abundant proteoglycan in the cartilage. It possesses
more than 100 GAG chains and is composed of three globular domains (G1, G2,
and G3). The attachment sites for the GAG chains are located between G2 and G3.
The G1 domain interacts with hyaluronan (HA) and the link protein, mediating the
formation of large proteoglycan aggregates (Watanabe et al. 1998; Schwartz et al.
1999) (for details, see Chap. 3). Aggrecan occupies the interfibrillar space of the
cartilage ECM and provides the cartilage with osmotic properties, which are criti-
cal to its ability to resist compressive loads. In former times it has been suggested
Fig. 4.1 Proteoglycans have diverse functions in modulating signal transduction and biomechani-
cal properties in the cartilage. Induction of some signal pathways can be influenced by proteogly-
cans in various ways. Proteoglycans can act as co-receptor (a) or create morphogen/ligand
gradients (b). Under inflammatory/pathological conditions, fragments of some proteoglycans can
induce signaling cascades (c). Proteoglycans are also important for the biomechanical properties
of the cartilage. Due to their predominantly negative charge, given by their side chains, proteogly-
cans comprise an inevitable osmotic function in the cartilage. Furthermore, proteoglycans are able
to sense mechanical loading (d) and biomechanical stress (e), thereby initiating downstream sig-
naling pathways
that aggrecan is not immediately incorporated in aggregates and that this incorpo-
ration is age dependent (Oegema 1980). Newer studies, however, suggest that
newly synthesized aggrecan is processed in pools with different capacities for
aggregation, which might be defined by factors like extracellular pH, age, and
disease state (Bayliss et al. 2000). The C-terminal G3 domain of aggrecan includes
different modules such as an epithelial growth factor (EGF) module, a C-type lec-
tin module, and a C-reactive protein (CRP) module. Interestingly, it has been
shown that with aging, there is an increase in the population of aggrecan monomers
lacking the G3 domain, which is most likely due to extracellular proteolytic degra-
dation. This cleavage results in the production of one fragment that remains bound
to HA and one free fragment. The HA bound fragment is retained within the carti-
lage tissue, whereas the latter fragment is lost by diffusion into the synovial fluid.
This cleavage of aggrecan results in a net decrease in the anionic charge of the
aggrecan molecule and therefore a decreased ability of the cartilage to resist com-
pression due to the reduction in osmotic force (Roughley and Mort 2014). The
major contributors to aggrecan degradation in vivo are members of the matrix
metalloproteinase (MMP) family, in particular stromelysin (MMP3) and MMP13,
which are produced under inflammatory conditions and during OA (Troeberg and
Nagase 2012). Furthermore, the two members of the ADAMTS (a disintegrin and
metalloprotease with thrombospondin motifs) family of metalloproteases,
ADAMTS-4 and ADAMTS-5, are also potent aggrecan-degrading enzymes
(Nagase and Kashiwagi 2003), which are also highly expressed during OA (for
further details, see Chap. 3).
A very recent study investigated the extent of aggrecan cleavage between indi-
viduals and found a high variability in the aggrecan cleavage, which was greatest in
areas adjacent to sites of cartilage erosion compared to sites more remote within the
same joint. The proportion of aggrecan cleavage attributable to MMPs or aggreca-
nases was also variable, but aggrecanase action was usually predominant in late-
stage OA (Mort et al. 2016). Therefore, the proteolytic cleavage of aggrecan due to
the increased expression of MMPs and aggrecanases under disease conditions is
directly linked to a reduction and deterioration of biomechanical properties of the
cartilage.
Lees and coworkers have investigated another aspect of aggrecan cleavage
in the perpetuation of OA. They analyzed the bioactivity of an aggrecan frag-
ment that is generated by ADAMTS-4/ADAMTS-5 cleavage and subsequent
cleavage of the fragment by MMPs, resulting in a 32-amino-acid fragment
(Lees et al. 2015). The originated 32-mer peptide caused a pro-catabolic, anti-
anabolic, and pro-inflammatory response in murine and human chondrocytes
in vitro. The incubation of chondrocytes with this 32-mer aggrecan fragment
increased mRNA expression for several proteases, including MMP-13 and
ADAMTS-5, and decreased mRNA expression for matrix molecules, including
type II collagen alpha 1 (COL2A1) and aggrecan (ACAN). The authors could
show that this effect was mediated through binding of the aggrecan fragment to
the Toll-like receptor (TLR)-2. It has long been known that some matrix mol-
ecules and fragments of these proteins, including fibronectin, tenascin C, and
hyaluronan fragments, can act as damage-associated molecular patterns
(DAMPs) and activate Toll-like receptors (TLRs) that are locally expressed in
the joint, initiating a cascade of inflammatory cytokine production (Liu-Bryan
and Terkeltaub 2015).
4.2.2 Versican
Versican, like aggrecan, consists of globular structures at the N-terminal (G1

domain) and C-terminal (G3 domain). Five splice variants of versican are known,
which are expressed under specific conditions or in different tissues. The V3 variant,
however, is generally expressed at low levels in adult tissues (Cattaruzza et al.
2002). The G1 domain of versican has the capability to bind hyaluronan, and the G3
domain consists of various modules. Between G1 and G3, the chondroitin sulfate
GAG side chains attach to the core protein (Zimmermann and Ruoslahti 1989). The
G3 domain promotes cell adhesion (Wu et al. 2002), and the G1 promotes cell pro-
liferation, migration and adhesion, and inflammation (Yang et al. 1999; Wight et al.
2014). The first description of versican in association with OA was by Nishida et al.
(Nishida et al. 1994). The authors showed that the cytoplasm of inflammatory cells
invading the OA cartilage matrix was stained for a protein with an extremely high
homology to versican (Nishida et al. 1994). Some other studies also found an upreg-
ulation of versican expression in OA cartilage, mainly linking this phenomenon to
an attempt of chondrocytes to repair the cartilage damage (Heard et al. 2013; Martin
et al. 2001; Cs-Szabo et al. 1997). Versican can be degraded by the aggrecanases
ADAMTS-4/ADAMTS-5 (Westling et al. 2004; McCulloch et al. 2009), but the
degradation or the relevance of degradation in cartilage has not been investigated.
That cleavage of versican also leads to a release of a free fragment like it has been
shown for aggrecan degradation, has also not been proven for cartilage, but is very
likely. However, it has been shown that versican also acts as an endogenous ligand
of TLRs, generating a rapid inflammatory response (Frey et al. 2013), although this
has not been shown for the cartilage yet.
4.2.3 Agrin
Agrin is a large HS proteoglycan that is abundantly distributed at most basement

membranes. It is predominantly known to exhibit its function in neurotransmission
(Iozzo and San Antonio 2001) and in mechanotransduction, connecting the cyto-
skeleton to other members of the basement membrane (Bezakova and Ruegg 2003;
Denzer et al. 1997, 1998). Microarray screening of cartilage samples after injury
identified agrin gene expression to be differentially regulated (Dell’accio et al.
2008). Besides, a transgenic mouse model showed that agrin deficiency leads to
changes in the growth plate cartilage, specifically affecting the viability of hypertro-
phic chondrocytes (Hausser et al. 2007). In the context of OA, Eldridge et al.
observed a progressive loss of agrin in human OA cartilage and in a surgical mouse
model of this disease (destabilization of the medial meniscus (DMM)) compared to
preserved cartilage or sham-operated joints (Eldridge et al. 2015). According to this,
in vitro loss-of-function experiments indicated decreased glycosaminoglycan con-
tent and a downregulation of the cartilage-specific transcription factor SOX9 and of
COL2A1 and ACAN as marker genes for the chondrocyte homeostasis (Eldridge
et al. 2015). The regulation of SOX9 expression by agrin seems to depend on the
availability of α-dystroglycan (DAG1) and the low-density lipoprotein receptor-

related protein 4 (LRP4) (Eldridge et al. 2015). LRP4 is a receptor for the Wnt and
BMP inhibitors Sost (Choi et al. 2009) and Sostdc1 (Wise) (Ohazama et al. 2008),
as well as for the Wnt inhibitor Dickkopf1 (Dkk1) (Choi et al. 2009) in osteoblasts.
Therefore, the LRP4 receptor constitutes an interesting link to BMP and Wnt sig-
naling pathways (Eldridge et al. 2015), which are proposed to affect chondrocyte
differentiation and may interfere in the progression of OA (rev. in (van der Kraan
et al. 2010; Luyten et al. 2009; Corr 2008)). This study emphasizes the role of a
pericellular HS proteoglycan in indirect modulation of morphogen gradients, which
are essential for cartilage homeostasis.
4.2.4 Perlecan
Perlecan is a large multi-domain proteoglycan to which three glycosaminoglycan

chains – often heparan sulfate but also chondroitin sulfate – are attached. It is pres-
ent in all basement membranes, cartilage, and bone marrow stromal cells (Hassell
et al. 1980; SundarRaj et al. 1995). Perlecan is associated with bone formation dur-
ing endochondral ossification. Consequently, functional null mutations of the per-
lecan gene in humans and mice are associated with dyssegmental dysplasia of the
Silverman-Handmaker type, Schwartz-Jampel syndrome (SJS) (Giedion et al.
1997), and perlecan-null mice develop severe chondrodysplasia (Arikawa-Hirasawa
et al. 1999, 2001; Costell et al. 1999). Perlecan protein and mRNA levels are upreg-
ulated in OA cartilage adjacent to the main cartilage defect. This might be seen as
an attempt on the part of the cartilage tissue to stabilize the extracellular matrix
(Tesche and Miosge 2004). Another study shows that perlecan is required for the
chondrogenic and adipogenic differentiation from synovial mesenchymal cells
(SMCs) via its regulation of SOX9 and PPARγ gene expression but not for osteo-
genic differentiation via RUNX2 (Sadatsuki et al. 2016). Another study also
describes a role for synovial perlecan, which is required for osteophyte formation in
knee osteoarthritis in mice (Kaneko et al. 2013). Although these findings seem to be
at least in part contradictionary, the role of perlecan in chondrocyte differentiation
and bone formation is consistent.
Perlecan is enriched mainly in the pericellular environment of chondrocytes
(Melrose et al. 2002) and has been identified as an HS proteoglycan that sequesters
FGF-2, which is a regulator of chondrocyte proliferation (Sahni et al. 1999;
Takahashi et al. 2005). In experiments using chondrocytes embedded in alginate,
pericellular perlecan and FGF-2 were accumulated and delivered an FGF-dependent
activation of the extracellular signal-regulated kinase (ERK) when loaded (Vincent
et al. 2007), giving rise to the assumption that perlecan might have a function in
biomechanical-induced OA. This finding was corroborated by another study show-
ing that the HS side chains of perlecan play a significant role in directing the devel-
opment of posttraumatic OA. The authors demonstrated that via the alteration of
FGF/HS/FGFR signaling, perlecan exhibits a chondroprotective function, at least in
part, via the preservation of FGFR-3 and increased FGF signaling (Shu et al. 2016).
4.3 Small Proteoglycans
Small proteoglycans in the cartilage comprise glypican, syndecan, decorin, bigly-

can, asporin, fibromodulin, and lumican.
4.3.1 Glypicans
Glypicans are glycosylphosphatidylinositol (GPI) anchored and can bind extracel-

lular ligands via heparan sulfate chains (David et al. 1990; Bernfield et al. 1999).
Using glypicans as an example, the role of proteoglycans and their side chains as
modulators of signal transduction and facilitators of tissue homeostasis by affecting
the local formation of morphogen gradients can be highlighted. Glypican-3 (GPC3)
knockout mice showed an inhibited noncanonical Wnt/c-Jun N-terminal kinase
(JNK) and an activated canonical Wnt/β-catenin signaling (Song et al. 2005). The
formation of glypican–Wnt complexes has been reported (Ohkawara et al. 2003),
but there are conflicting reports on the involvement of HS chains in this process
(Song et al. 2005; Ohkawara et al. 2003) (De Cat et al. 2003). De Cat et al. clearly
indicated that both the protein and the HS chains seem to be required for the proteo-
glycan function in signal transduction (De Cat et al. 2003).
Grover and Roughley showed low expression of glypican in human articular car-
tilage (Grover and Roughley 1995). Although there is a skeletal phenotype as muta-
tions in the glypican-3 (GPC3) gene have been linked to the Simpson–Golabi–Behmel
syndrome (SGBS), characterizing pre- and postnatal overgrowth (gigantism) with
visceral and skeletal anomalies (Pilia et al. 1996), no reports for the function of
glypican in OA are published until now.
4.3.2 Syndecans
Syndecans represent a family of type I transmembrane HS proteoglycans, which

comprises four members in vertebrates (Couchman 2003). They interact via their
GAG chains with a variety of extracellular matrix molecules and can act as co-
receptors for integrins and for a growing number of growth factor receptors like FGF
receptors (Tkachenko et al. 2005). Due to these interactions, syndecans are involved
in growth control, cell spreading, cellular recognition, cellular adhesion, and signal-
ing (Choi et al. 2011; Couchman 2010). Each syndecan has a tissue-specific and
developmentally regulated expression pattern. While syndecan-1 is mainly expressed
in epithelial cells, syndecan-2 is detectable in mesenchymal cells. Syndecan-3 is
found during bone development and in the nervous system, whereas syndecan-4 is
ubiquitously expressed (Kim et al. 1994). Of the four mammalian syndecan family
members, syndecan-1 and syndecan-3 and syndecan-2 and syndecan-4 are closely
related and can be considered to form subfamilies (Couchman 2010).
Syndecan-4 is expressed ubiquitously and is highly distributed during develop-
ment (David et al. 1992); furthermore, it is upregulated under inflammatory
conditions (Wang et al. 2011). Syndecan-2, however, is mainly expressed during

embryogenesis in the bone, where it can compensate for the loss of syndecan-4
(Bertrand et al. 2013). Under inflammatory conditions syndecan-2 is downregu-
lated, so it cannot compensate the loss of function of syndecan-4 in knockout mice
(Bertrand et al. 2013). Syndecans can interact with several ligands such as growth
factors, cytokines, proteinases, adhesion receptors, ECM components, and morpho-
gens most likely through their HS chains (Pap and Bertrand 2013). Thus, they are
able to modulate tissue homeostatic processes and tissue remodeling upon injury.
Syndecan-4 expression is specifically induced in hypertrophic chondrocytes both in
human OA and in murine models of this disease (Echtermeyer et al. 2009).
Syndecan-4 controls the activation of ADAMTS-5 through direct interaction via its
HS chains, as it immobilizes ADAMTS-5 at the cell surface. Furthermore, it regu-
lates the activation of ADAMTS-5 by regulating the IL-1β-induced expression of
MMP-3, via reduced activation of mitogen-activated protein kinase (MAPK) upon
IL-1β stimulation (Echtermeyer et al. 2009). The role of syndecan-4 in matrix
remodeling during OA was confirmed using wild-type mice, which were treated
with a blocking syndecan-4 antibody, as well as syndecan-4-deficient mice, which
are protected from OA-induced cartilage degradation, most likely because of
decreased MAPK-initiated expression of MMP-3, resulting in reduced ADAMTS-5
activation (Echtermeyer et al. 2009). In line with this, ADAMTS-5-deficient mice
are also protected from cartilage degradation in a murine model of OA (Stanton
et al. 2005; Glasson et al. 2005) (for further details, see Chap. 3).
Another mechanism of syndecan-4 in cartilage protection during OA might be its
function in regulating the dynamic recycling of receptors, e.g., α5β1 and αVβ3 integ-
rins, by disrupting the dynamin- and caveolin-dependent endocytosis (Bass et al.
2011). It was shown that cells, expressing a mutagenized syndecan-4 in one of the
signaling domains, exhibited a severe reduction in syndecan-4/integrin receptor inter-
nalization. These data suggest that syndecan-4-dependent endocytic pathways may be
intimately associated with receptor internalization and therefore regulating signal
transduction (Bass et al. 2011; Humphries et al. 2005). Both integrins have been asso-
ciated with OA and cartilage homeostasis (Garciadiego-Cazares et al. 2015).
4.4 Small Leucine-Rich Repeat Proteoglycans (SLRPs)
The small leucine-rich repeat proteoglycan (SLRP) family is represented by deco-

rin, biglycan, asporin (Lorenzo et al. 2001), fibromodulin (Hedlund et al. 1994), and
lumican (Sztrolovics et al. 1999) in the articular cartilage (Martel-Pelletier et al.
2008). Decorin and biglycan contain one or two chondroitin sulfate GAG chains
(Iozzo 1999), while fibromodulin exhibits keratin sulfate GAG chains (Jepsen et al.
2002). Although asporin is highly identical with decorin and biglycan, by strict defi-
nition, it is no proteoglycan, as the consensus sequence for glycosaminoglycan
attachment between the propeptide and the amino-terminal cysteine motif is miss-
ing (Lorenzo et al. 2001; Henry et al. 2001; Nakajima et al. 2007). The SLRP mem-
bers asporin and decorin consist of collagen binding sites (Kalamajski et al. 2007,
2009; Svensson et al. 1995) and can interact with soluble factors such as members
of the transforming growth factor β superfamily (TGFβ) (Martel-Pelletier et al.
2008; Embree et al. 2010).
Several studies have proposed that SLRPs play a role in cartilage remodeling
during OA (Young et al. 2005; Kizawa et al. 2005; Bock et al. 2001), as increased
mRNA expression of these SLRPs has been described in OA cartilage (Poole et al.
1996; Cs-Szabo et al. 1997). Young et al. reported also an enhanced expression of
biglycan and lumican, although he found a decreased synthesis of decorin in the
articular cartilage in a murine model of OA (Young et al. 2005). In total mRNA
expression and protein synthesis of most SLRPs seem to be increased in late stages
of OA (Bock et al. 2001).
4.4.1 Asporin
Asporin has been shown to bind TGFβ1 and inhibit TGFβ1-induced expression of
COL2A1 and ACAN in ATDC5 cells in vitro. Presumably, asporin thereby is able
to interfere with chondrocyte differentiation and thus cartilage homeostasis (Kizawa
et al. 2005). Kizawa et al. showed an increased expression of asporin in human
OA. Furthermore, the study proposed that a functional polymorphism in the aspartic
acid repeat of the asporin gene might result in a higher susceptibility to OA (Kizawa
et al. 2005). Interestingly, they could show that the ability of asporin to inhibit TGFβ
signaling was significantly different in the polymorphic variants of asporin (Kizawa
et al. 2005), thereby providing a possible mechanism for the role of asporin in OA.
4.4.2 Decorin
Decorin also has high binding affinities not only to TGFβ1 but also to TGFβ2 (Iozzo
and Sanderson 2011; Goldoni and Iozzo 2008). It has been reported that bone-derived
decorin can enhance the ligand-receptor binding of TGFβ and thereby augment cell
responses (Takeuchi et al. 1994), which might alter the balance in bone formation
and degeneration toward bone resorption. Interestingly, Gronau et al. demonstrated
that decorin-deficient mice are less susceptible to develop OA in a forced exercise
mouse model. The study shows higher TGFβ activity and enhanced compressive
stiffness of the articular cartilage accompanied by increased sulfation of GAG chains
(Gronau 2016). They propose that decorin regulates the TGFβ-dependent expression
of the sulfotransferase CHST11, which has been linked to OA by a genome-wide
association study (GWAS) of OA cartilage samples (Reynard and Loughlin 2013).
4.4.3 Biglycan
Biglycan can interact (and sequester) with type I, II, III, and VI collagen, tumor
necrosis factor α (TNFα) (Tufvesson and Westergren-Thorsson 2002), BMP-2,
BMP-4, and BMP-6 (Chen et al. 2004; Mochida et al. 2006), and TGFβ (Hildebrand
et al. 1994) via its tissue-specific chondroitin or dermatan sulfate GAG chains.
Consequently, biglycan-deficient mice show an osteoporosis-like phenotype and
abnormalities of collagen fibrils (Xu et al. 1998).
The study of Schaefer et al. showed for the first time that an SLRP, in this case
biglycan, can act as ligand of TLR-2 and TLR-4. They found that upon binding of
biglycan, the mitogen-activated protein kinase p38, ERK, and nuclear factor kappa-
light-chain enhancer of activated B cells (NF-kB) got activated, leading to the secre-
tion of TNFα, thereby triggering an inflammatory response (Schaefer et al. 2005).
Furthermore, biglycan is capable of clustering several types of receptors and thereby
orchestrating the signal transduction of these receptors. Babelova et al. showed that
soluble, unsequestered biglycan can stimulate receptor interaction of TLR-2/TLR-4
and purinergic P2X (4)/P2X (7) receptors, resulting in the activation of the NLRP3
inflammasome. Although these data were generated in macrophages, the signaling
mechanism most likely will be the same in the cartilage, giving an explanation of
how tissue injury is sensed by innate immune receptors by binding of ECM compo-
nents and translating this signal into an inflammatory response (Babelova et al.
2009). The expression of TLRs (TLR-2 and TLR-4, but also others) has been proven
for chondrocytes (Sillat et al. 2013; Radwan et al. 2013). Interestingly, TLR-2 gene
expression gets upregulated upon stimulation with IL-1β and fibronectin proteolytic
fragments (Su et al. 2005).
4.5 Perspectives
Given the described functions of proteoglycans in the cartilage, the importance of

these molecules in OA is not surprising. Due to their electrostatic loading, they
retain water and largely determine the biomechanical properties of the cartilage.
Upon degradation of proteoglycans, biomechanical properties of the cartilage
change due to the loss of negative charges from the GAG chains. Furthermore,
fragments of various proteoglycans can act as ligands for TLR receptors, inducing
inflammatory cascades. Proteoglycans also bind various ligands, acting as co-
receptor and control the sensitivity of cells for extracellular stimuli. One interest-
ing function of proteoglycans has not been mentioned before, as the responsible
proteoglycan has not been identified yet. Sherwood et al. have shown that the
chemokine CXCL6 is expressed in healthy cartilage and retained through binding
to heparan sulfate proteoglycans. The study shows that CXCL6 signaling via
CXCR1/CXCR2 is important for maintaining cartilage homeostasis and that the
loss of ELR+CXC chemokines during cartilage breakdown in osteoarthritis con-
tributes to the characteristic loss of chondrocyte phenotypic stability and hyper-
trophic differentiation (Sherwood et al. 2015). This study highlights the role of
proteoglycans in keeping a reservoir of different factors bound in the ECM. Whether
all these ligands are needed to keep the cartilage homeostasis, or whether they
might act as morphogens or cytokines upon release during cartilage degradation,
is unknown. The understanding of proteoglycan biochemistry, however, under OA
conditions is of great importance in order to understand the pathomechanisms of
this disease (Fig. 4.1).
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Pro- and Anti-inflammatory Cytokine
Profiles in Osteoarthritis 5
Yvonne Bastiaansen-Jenniskens, Daniel Saris,
and Laura B. Creemers
Abstract
The presence and production of soluble factors in the osteoarthritic (OA) joint
have always been a focus of research, as they are assumed to play a role in initia-
tion and/or progression of disease. Many tissue and cell types in the joint are
capable of their production, with the synovial fluid serving as a reservoir into
which they can be secreted. Although an increasing interest is directed towards
chemokines, growth factors and adipokines, traditionally, a subset of inflamma-
tory, anti-inflammatory and modulatory cytokines has been studied. Differential
profiles compared to healthy joints were found in the knee and other OA joints,
whereby also joint damage induces a specific change in secretory pattern.
However, for the cytokines commonly assumed to play a role in OA, such as IL-1
and TNFα, their consistently low levels, frequent lack of association with disease
and the presence of natural inhibitors suggest that other soluble factors may be
more promising as possible targets.
Y. Bastiaansen-Jenniskens, PhD
Erasmus MC, University Medical Center Rotterdam, Department of Orthopaedics,
Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands
e-mail: y.bastiaansen@erasmusmc.nl
D. Saris, PhD
University Medical Center Utrecht, Department of Orthopaedics,
HP G05.228, PO Box 85090, 3508 GA Utrecht, The Netherlands
MIRA Institute, University Twente,
ME125, PO Box 217, 7500 AE Enschede, The Netherlands
e-mail: D.saris@umcutrecht.nl
L.B. Creemers, PhD (*)
University Medical Center Utrecht, Department of Orthopaedics,
HP G05.228, PO Box 85090, 3508 GA Utrecht, The Netherlands
e-mail: L.B.Creemers@umcutrecht.nl

82 Y. Bastiaansen-Jenniskens et al.
5.1 Cytokines in Osteoarthritis
Cytokines are commonly soluble, but sometimes cell surface-associated proteins

capable of autocrine, paracrine and endocrine signalling (Liles and Van Voorhis
1995; Turner et al. 2014). In osteoarthritis (OA), pro-inflammatory cytokines alleg-
edly play a crucial role in initiation and/or progression of disease (Goldring and
Otero 2011; Goldring and Goldring 2004; Rahmati et al. 2016). Whether inflamma-
tion is the trigger leading to OA, or biomechanical overloading is the initiating
event, cytokine production at some point is the converging element in these two
theories (Berenbaum 2013; Estes et al. 2004; Guilak 2011; Houard et al. 2013). Pro-
inflammatory cytokines can mediate cartilage destruction by enhancing the produc-
tion and activity of several matrices degrading matrix metalloproteinases (MMPs),
in particular the type II collagen degrading MMP-1 and MMP-13 and the aggrecan
degrading ADAMTS-4 and ADAMTS-5 (Durigova et al. 2008; Flannery et al. 2000;
Koshy et al. 2002a, b; Long et al. 2008; Stove et al. 2000). Although initially
assumed to mainly be produced by immune cells, it has become clear that many cell
types in the body are capable of their production (Turner et al. 2014). In the joint
space, many of these factors will end up in the synovial fluid, which connects all
tissues and thereby can mediate solute-drive communication between the joint tis-
sues. Here, combinations of pro- and anti-inflammatory cytokines as part of so-
called cytokine networks are likely to act in concert and may synergistically enhance
or attenuate cartilage matrix degradation (Schett et al. 2013). Together with their
possible role as target in OA, the use of cytokine analysis as biomarker or even
prognostic tool incited many studies evaluating their presence inside and outside the
joint (Table 5.1).
5.2 rigin (Synovial Membrane, Cartilage, Fat Pad, Bone/

O
Cell Types)
An articular joint typically contains the cartilage, bone and synovium and is also
surrounded by different adipose tissue bodies, such as Hoffa’s fat pad (also called
the infrapatellar fat pad) in the knee joint. Next to fibroblast-like synoviocytes
(FLSs), the synovium contains many other cells (de Lange-Brokaar et al. 2015)
such as macrophages (Beekhuizen et al. 2011; Deligne et al. 2015; Fahy et al.
2014), mast cells (de Lange-Brokaar et al. 2015), T-cells (Beekhuizen et al. 2011;
Deligne et al. 2015), B-cells and neutrophils (Deligne et al. 2015) (Fig. 5.1). The
latter three lymphocytes are however mainly present in the sub-intimal layer and
not detectable in healthy synovium (Deligne et al. 2015). The presence of mast
cells is also correlated with the degree of synovitis (de Lange-Brokaar et al.
2015). With all of these different types of immune cells, the synovium is a usual
suspect in contributing to the cytokine levels in synovial fluid. Indeed, synovium
explants in culture secrete many different cytokines, adipokines, chemokines and
growth factors, including IL-1β, IL-1RA, IL-6, IL-8, IL-10, IL-17, IL-18, IL-22,
IL-23, TGFβ1, RANTES/CCL5, CCL18, osteoprotegerin (OPG) and visfatin
5
Table 5.1 Comparisons of cytokine levels in synovial fluid between healthy and diseased joints
Concentration
range in OA
Cytokine OA vs healthy (Acute) damage vs healthy OA vs damage Late vs early (pg/ml)
↑ ↓ = ↑ ↓ = ↑ ↓ = ↑ ↓ =
TNFα Sauerschnig Bigoni et al. Cuellar Irie et al. Ribbens UD Heard et al.
et al. (2014), (2013) and et al. (2003) et al. (2013) -105
Teunis et al. Cameron (2010), (2000) Hussein et al.
(2013), and et al. (1997) Cuéllar and (2008)
Tsuchida et al. Scanzello
et al. (2014) (2015), et al.
Higuchi (2009)
et al.
(2006),
Marks and
Donaldson
(2005), and
Teunis
et al.
(2013)
IL-1α Heard et al. Cameron Tsuchida Heard et al. UD Beekhuizen
(2013) and et al. et al. (2013) et al. (2013),
Tsuchida (1997) (2014) Kahle et al.
et al. (2014) (1992)-15
Pro- and Anti-inflammatory Cytokine Profiles in Osteoarthritis
Tsuchida et al.
(2014)
(continued)
83
Table 5.1 (continued)
84
Concentration
range in OA
↑ ↓ = ↑ ↓ = ↑ ↓ = ↑ ↓ =
IL-1β Sauerschnig Beekhuizen Bigoni et al. Cameron Irie et al. Heard et al. UD Teunis et al.
et al. (2014) et al. (2013), (2013) and et al. (2003) (2013) (2013)-21 Kahle
Heard et al. Marks and (1997), et al. (1992)
(2013), and Donaldson Cuellar
Tsuchida (2005) et al.
et al. (2014) (2010), and
Higuchi
et al.
(2006)
IL-6 Beekhuizen Bigoni et al. Teunis Irie et al. Ribbens Heard et al. 120 Beekhuizen
et al. (2013), (2013), et al. (2003) et al. (2013) et al. (2013)-4747
Heard et al. Cameron (2013) (2000), Heard et al.
(2013), et al. (1997), Scanzello (2013)
Teunis et al. Cuellar et al. et al.
(2013), and (2010), (2009),
Tsuchida Higuchi et al. and
et al. (2014) (2006), Tsuchida
Tsuchida et al.
et al. (2014), (2014)
andCuéllar
et al. (2015)
IL-8 Beekhuizen Teunis et al. Bigoni et al. Cuellar Irie et al. Teunis 52 Tsuchida et al.
et al. (2013), (2013), and (2013) and et al. (2003) et al. (2014)-714 Heard
Heard et al. Tsuchida Cameron (2010), (2013) et al. (2013)
(2013) et al. (2014) et al. (1997) Teunis and
et al. Tsuchida
(2013), and et al.
Tsuchida (2014)
et al.
(2014)
Y. Bastiaansen-Jenniskens et al.
5
IL-10 Beekhuizen Tsuchida Cuellar Teunis et al. Irie et al. Tsuchida Heard et al. 2 Beekhuizen
et al. (2013) et al. (2014) et al. (2013) (2003) et al. (2013) et al. (2013)-58
(2010), (2014) Heard et al.
Cuéllar (2013)
et al.
(2015), and
Tsuchida
et al.
(2014)
IL-13 Tsuchida Tsuchida Cuellar Tsuchida Heard et al. 6 Heard et al.
et al. (2014) et al. (2014) et al. et al. (2013) (2013)-18
(2010) (2014) Tsuchida et al.
(2014)
IL-15 Scanzello 4 Scanzello et al.
et al. (2009)
(2009)
IL-17 Cuellar UD (Heard et al.
et al. (2013)-0.5
(2010) Hussein et al.
(2008)
IL-1RA Teunis et al. Cameron Teunis Irie et al. Teunis Heard et al. UD Beekhuizen
(2013) et al. (1997), et al. (2003) et al. (2013) et al. (2013)-811
and Marks (2013) (2013) Irie et al. (2003)
and
Pro- and Anti-inflammatory Cytokine Profiles in Osteoarthritis
Donaldson
(2005)
IFNγ Teunis et al. Beekhuizen Cuellar et al. Teunis Teunis et al. Tsuchida UD Heard et al.
(2013) and et al. (2013) (2010) and et al. (2013) et al. (2013)-51
Tsuchida Tsuchida (2013) (2014) Tsuchida et al.
et al. (2014) et al. (2014) (2014)
(continued)
85
86
Concentration
range in OA
↑ ↓ = ↑ ↓ = ↑ ↓ = ↑ ↓ =
OSM Tsuchida Teunis et al. Tsuchida Teunis Tsuchida Teunis UD Beekhuizen
et al. (2014) (2013) et al. (2014) et al. et al. (2014) et al. et al. (2013)-38
(2013) (2013) Tsuchida et al.
(2014)
Leptin Beekhuizen 1637 Beekhuizen
et al. (2013) et al. (2013)-2959
Mabey et al.
(2014)
VEGF Heard et al. Cuéllar Rübenhagen Heard et al. 307 Heard et al.
(2013) et al. et al. (2012) (2013) and (2013)-1,000
(2015) Rübenhagen Rübenhagen et al.
et al. (2012) (2012)
An arrow down means decreased concentrations in OA compared to healthy (first column), joints with damage versus healthy joints, etc. An arrow up vice versa
means increased in OA compared to healthy joints, etc. UD means undetectable, so the cytokine was analysed but the values fell below detection limits. Numbers in
superscript denote the accompanying references. Cytokines which were not detectable in a particular study were not included in the overview. For the cytokine
concentrations in OA joints, only studies using ELISA for detection were included, quoting the average levels per patient group
Y. Bastiaansen-Jenniskens et al.
5 Pro- and Anti-inflammatory Cytokine Profiles in Osteoarthritis 87
macrophage fibroblast like synovial cell (FLS)

bone cell
T-cell
chondrocyte
mast cell
adipocyte
Fig. 5.1 Overview of the cells in the various joint structures capable of cytokine production
(Beekhuizen et al. 2011; Deligne et al. 2015; Fahy et al. 2014; Laiguillon et al.
2014). TNFα and nerve growth factor (NGF) immunopositive cells are also seen
in the osteoarthritic synovium (Kc et al. 2016). As might be expected, IL-1β,
IL-6, IL-8, and IL-18 but also TGFβ1 release are increased with increased inflam-
mation of the synovium, characterised by hypervascularised areas containing
hypertrophic and hyperemic villi (Deligne et al. 2015). However, IL-23 and
RANTES secretion was not dependent on the inflammatory status of synovium
explants (Deligne et al. 2015). When the OA synovial tissue was digested and
single cells were cultured, the cells spontaneously secreted the factors mentioned
above, including TNFα, IL-1β and large amounts of IL-6, IL-8 and MCP1.
However, when macrophages were depleted from the digest, IL-1β, TNFα, IL-6
and IL-8 production by the remaining FLSs was strongly decreased. As IL-6 and
IL-8 are known to be produced by FLSs, this indicates that the synovial macro-
phages are required for this secretory activity. No effect was seen after T-cell
depletion (Bondeson et al. 2006). As shown by immunohistochemistry, the
synovium of OA patients also contains IL-35. However, these levels are rela-
tively low when compared to the synovium of rheumatoid arthritis (RA) patients
(Filková et al. 2015).
Next to the synovium, Hoffa’s fat pad contains many immune cells such as mac-
rophages (Bastiaansen-Jenniskens et al. 2012; Wei et al. 2015), T-cells, mast cells
and a few B-cells (Klein-Wieringa et al. 2011). Hoffa’s fat pad produces IL-1β,
IL-6, IL-8, interferon (IFN)γ, MCP1, IL-4, IL-10, IL-17, TNFα, the adipokines
adipsin, adiponectin, visfatin, resistin and leptin and the growth factors fibroblast
growth factor (FGF)2 and vascular endothelial growth factor (VEGF) (Clockaerts
et al. 2012; Distel et al. 2009; Klein-Wieringa et al. 2011; Ushiyama et al. 2003).
Inflammation in the fat pad explants induced in vitro with IL-1β results in an
increase of TNFα release, indicating that protein release might also be altered in the
inflamed knee joint (Clockaerts et al. 2012). The CD4+ and CD8+ T-cells and mac-
rophages in Hoffa’s fat pad produce TNFα, IL-6 and IL-10, and the T-cells also
produce IFNγ (Klein-Wieringa et al. 2011).
Many cytokines, growth factors and adipokines are expressed by the osteoar-
thritic cartilage, as shown in tissue culture or ex vivo tissue extracts, namely, IL-1α,
IL-1β, IL-1RA, IL-4, IL-6, IL-7, IL-8 (actually a chemokine), IL-10, IL-13, TNFα,
oncostatin M (OSM), leukaemia inhibitory factor (LIF), adiponectin, leptin, visfatin
monocyte chemotactic protein 1 (MCP1), RANTES, FGF2, hepatocyte growth fac-
tor (HGF) and VEGF (Beekhuizen et al. 2011; Kc et al. 2016; Laiguillon et al. 2014;
Tsuchida et al. 2014). The severity of OA also seems to influence the production of
IL-1β, TNFα and most likely other cytokines, since, with more severe OA, more
chondrocytes positive for IL-1β and TNFα were observed (Kc et al. 2016; Tetlow
et al. 2001). Surprisingly, most of the aforementioned factors, except for TNFα,
adiponectin, FGF2 and HGF, were produced at a higher level by chondrocytes that
were isolated from the cartilage compared to their production levels inside the tissue
(Tsuchida et al. 2014). This indicates that the surrounding matrix influences the
production of these factors and cell cultures may not be optimal tools to study
OA-related processes.
Finally, the (subchondral) bone is also a source of soluble factors detected in the
synovial fluid; however, little is known about this source. In cell culture, the sub-
chondral bone releases IL-1β, IL-6, IL-8, FGF2 and visfatin (Laiguillon et al. 2014;
Leyh et al. 2014). Although it is not clear from which bone cells these factors arise,
at least IL-6 and IL-1 can be produced by osteoblasts and osteocytes (Bakker et al.
2014; Gordeladze et al. 2002; Maeda et al. 2015).
5.3 ytokine Profiling in Synovial Fluid of Osteoarthritic

C
Joints
Many patient-based comparative studies have aimed to shed light on synovial fluid
cytokine composition in OA (Teunis et al. 2013). Amongst all soluble factors men-
tioned above, mostly the pro-inflammatory, anti-inflammatory and modulatory
cytokines have been studied.
In general, pro-inflammatory cytokine release into the synovial fluid in OA
comes nowhere near the high secretory activity seen in RA joints (Hussein et al.
2008; Kahle et al. 1992; Neidel et al. 1997; Ribbens et al. 2000; Richette et al.
2008). However, if compared to healthy subjects, patients with OA do appear to also
show cytokine upregulation and differential profiles. Amongst those, the cytokines
postulated by many to be instrumental in initiating and/or advancing OA
degenerative processes, IL-1β and TNFα, have been subject to close scrutiny.
However, where TNFα is clearly upregulated in RA (Vervoordeldonk and Tak
2002), its upregulation in OA is far from convincing. Except for two studies on
temporomandibular joint (TMJ) OA (Kaneyama et al. 2002, 2005), its synovial fluid
levels do not seem higher in OA than in healthy joints (Sauerschnig et al. 2014;
Teunis et al. 2013; Tsuchida et al. 2014; Kaneyama et al. 2003), if not entirely unde-
tectable (Heard et al. 2013; Beekhuizen et al. 2013). IL-1β is either upregulated in
the knee and TMJ OA (Sauerschnig et al. 2014; Kubota et al. 1998) or present at
similar levels (Beekhuizen et al. 2013; Heard et al. 2013; Tsuchida et al. 2014)
while undetectable in wrist OA (Teunis et al. 2013). IL-1α did not show any differ-
ential expression at all in the relatively limited numbers of studies describing this
cytokine (Beekhuizen et al. 2013; Heard et al. 2013; Tsuchida et al. 2014). Increased
but also similar levels compared to healthy joints were described for IL-8, which is
actually a chemokine (Beekhuizen et al. 2013; Heard et al. 2013; Kaneyama et al.
2002; Teunis et al. 2013; Tsuchida et al. 2014). Other pro-inflammatory cytokines
found to be upregulated in OA in several studies are oncostatin M (OSM)
(Beekhuizen et al. 2013; Tsuchida et al. 2014) and INFγ (Teunis et al. 2013;
Tsuchida et al. 2014), of which the latter was even suggested to be inversely corre-
lated with acute inflammation (Kahle et al. 1992). However, as for each of these two
factors in which also unchanged levels were demonstrated (Beekhuizen et al. 2013;
Teunis et al. 2013), more research is required to give proper insight on their associa-
tion with OA. This also goes for IL-15 (Scanzello et al. 2009) and leptin (Beekhuizen
et al. 2013), for which even less data are currently available. For the anti-inflamma-
tory cytokines, information is relatively limited too. Despite the wealth of preclini-
cal studies using exogenously applied IL-1RA to interfere with the degenerative
process in OA, very little is known of its concentrations in the osteoarthritic joint
compared to healthy joints, and it may be upregulated (Heard et al. 2013) or rela-
tively unchanged (Teunis et al. 2013). More but also contradictory data are available
for the anti-inflammatory IL-10, with higher (Heard et al. 2013; Teunis et al. 2013;
Fang et al. 1999), similar (Tsuchida et al. 2014) or even lower (Beekhuizen et al.
2013) levels in the OA joint.
Surprisingly, the only cytokine that does show an almost unequivocal elevation
in OA synovial fluid compared to healthy controls is the modulatory cytokine IL-6,
shown for the knee (Beekhuizen et al. 2013; Tsuchida et al. 2012, 2014; Heard et al.
2013), wrist (Teunis et al. 2013) and temporomandibular osteoarthritis (Kaneyama
et al. 2002, 2005; Kubota et al. 1998).
5.4 Profiling in Trauma and Early OA
Apart from characterisation of established OA, synovial fluid cytokine content also
changes during progression from joint trauma via early radiographic to end-stage
OA. As the acute inflammatory response occurring after injury may trigger degen-
eration and hence timely interference may prevent this, many efforts were devoted
to characterising this response. After anterior cruciate ligament (ACL) injury, either
or not combined with meniscal damage, indeed, several cytokines including TNFα,
IL-6, IL-8, IL-10 and IL-1RA are strongly upregulated. This upregulation occurs
already within the first 2 days, thereafter subsiding immediately again over a period
of a few weeks to a few months, when injury is defined as chronic (Bigoni et al.
2013; Cameron et al. 1997; Cuellar et al. 2010; Irie et al. 2003). Changes in IL-1β
levels here again were less unequivocal, while for TNFα levels, these were very
moderate. Joints with isolated cartilage defects were shown to contain upregulated
levels of IL-6, IFNγ, OSM and IL-13 (Tsuchida et al. 2012, 2014), although also
unchanged IL-6 concentrations were reported (Cuéllar et al. 2015).
Interestingly, it appears that after entering the chronic phase and several years
post-ACL and meniscus injury, in particular IL-6 (Cuellar et al. 2010; Cuéllar et al.
2015; Higuchi et al. 2006) and possibly IL-1RA (Marks and Donaldson 2005)
become elevated in the joint again. To what extent this correlates with progression
towards OA is not clear. IL-6 and TNFα were shown to be associated with osteo-
phyte formation (Larsson et al. 2015), and IL-6, IL-10, IL-13 and IL-1RA levels
were higher in late compared to early radiographic OA (Heard et al. 2013). In addi-
tion, also the angiogenic factor VEGF has been shown to be associated with pro-
gression of OA (Heard et al. 2013; Larsson et al. 2015; Mabey et al. 2014;
Rübenhagen et al. 2012).
5.5 Synovial Fluid Cytokines as Therapeutic Targets in OA?
Although many of the pro-inflammatory cytokines mentioned above have been pos-
tulated to play a crucial role in initiation or progression of OA, their true involve-
ment remains unclear. The role of some of the factors traditionally assumed to play
a role in OA, in particular IL-1 and TNFα, has been disputed by a paucity of effects
seen in clinical trials with OA patients using biological inhibitors of these cytokines
(Chevalier et al. 2009, 2014; Cohen et al. 2011; Grunke and Schulze-Koops 2006).
The discrepancy between these clinical results and the plethora of in vitro and
in vivo studies suggesting a causative role of these and other inflammatory cyto-
kines may find its origin in several phenomena. First of all, in vitro studies showing
their effect on cartilage degradation usually depart from concentrations that are
much higher than those found in the osteoarthritic joints. TNFα, IL-1β and OSM are
typically used at concentrations in the 1–50 ng per ml range, whereas their levels in
the joint synovial fluid commonly range from undetectable to up to tens of pico-
grams per ml (Beekhuizen et al. 2013b, b; Heard et al. 2013; Neidel et al. 1997;
Ribbens et al. 2000; Richette et al. 2008; Tsuchida et al. 2014). Though arguably it
is possible that due to the synergy shown between several of these factors (Durigova
et al. 2008; Henderson and Pettipher 1989; Henrotin et al. 2000; Koshy et al. 2002a,
b; Rowan et al. 2001), much lower concentrations are required to achieve cartilage
degrading effects, also this synergy was shown using supraphysiologically high
concentrations only, thus not providing much support for these mechanisms in vivo.
Apart from the fact that most pro-inflammatory cytokines are usually present at
much lower levels in OA than in RA (Fraser et al. 2003; Hussein et al. 2008; Neidel
et al. 1997; Ribbens et al. 2000; Richette et al. 2008), the presence of natural antago-
nists in the osteoarthritic joint may have provided neutralisation of their action. The
IL-1RA/IL-1 ratio was found to be higher in OA than in RA (Richette et al. 2008),
with IL-1RA often present at over 100 times higher levels than IL-1 (Heard et al.
2013; Irie et al. 2003; Rutgers et al. 2010; Richette et al. 2008), which is the ratio
required to inhibit the actions of IL-1 (Arend et al. 1990; Granowitz et al. 1991).
Similarly, the presence of endogenously produced soluble TNF receptors at concen-
trations around 1,000-fold of those of TNFα in the OA joint (Ribbens et al. 2000;
Simao et al. 2014) may preclude biological activity of anti-TNFα treatment in OA
patients. Finally, the levels of cytokines in the synovial fluid may deviate from the
concentrations in the articular cartilage itself, possibly related to fast clearance from
the joint space (Rutgers et al. 2010) and diffusion properties of the cartilage.
One approach to more accurately define the role of cytokines in this complex
environment is by interfering with specific cytokines in the synovial fluid. Using
this approach in vitro, OSM in OA synovial fluid was shown to play a role by inhib-
iting matrix production in the cartilage, but without affecting degradation. Exposure
to OSM alone at the levels found in vivo did not have any effect, while the com-
monly employed high concentrations did reproduce the enhanced degradation
shown before (Beekhuizen et al. 2013). Interference with IL-6 in the synovial fluid
did not affect proteoglycan metabolism in the osteoarthritic cartilage, while adding
this cytokine enhanced regeneration by isolated chondrocytes even slightly
(Tsuchida et al. 2012).
Ideally, the role of cytokines is investigated in vivo. However, although geneti-
cally modified mouse models can provide some information, the similarity of OA
induced in animals to human disease remains a matter of debate (Teeple et al. 2013).
Moreover, accumulating evidence suggests that cartilage degradation in OA is par-
tially related to the phenotype of the underlying bone, via altered biomechanical
properties and secretion of several soluble factors (Favero et al. 2015; Hardcastle
et al. 2015), so any generalised genetic modification of inflammatory cytokine pro-
duction may indirectly rather than directly affect cartilage integrity by targeting the
underlying bone. Using tissue-specific and/or conditional knockout models may
therefore be more informative. In the end, more studies using medium to high
throughput approaches based on large patient cohorts and extensive proteomic pro-
filing will allow to identify factors that consistently correlate with disease up and
may point towards targets as reliable and proven markers that are sufficiently ele-
vated to distinguish with certainty between patient populations.
5.6 Cytokines as Biomarkers
Biomarkers are useful if they can non-invasively predict or diagnose disease pro-
gression. Factors present in the synovial fluid may therefore be less useful as bio-
markers; synovial fluid aspiration is relatively invasive, induces a risk for infection
and is not always possible from relatively healthy joints which typically contain less
synovial fluid (Pascual and Doherty 2009). However, until now, only limited
information is available on cytokines as serum markers for disease. Only one study
suggested several pro- and anti-inflammatory factors to be altered in moderate to
severe OA serum compared to normal, with a decrease for IL-1β, IL-2 and IL-4 and
an increase for IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, TNFα and VEGF (Heard et al.
2013). Although not supported here, serum VEGF and IL-8 have been suggested to
correlate with radiographic severity (Mabey et al. 2014). Also here extensive profil-
ing studies should be carried out with more certainty pinpoint markers that can reli-
ably distinguish subsets of patients with joint disease.
Acknowledgements The authors wish to thank Prof. G. van Osch, Department of Orthopaedics,
Erasmus Medical Centre, for his critical review of the manuscript.
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Molecular Genetics of the Cartilage
Collagenopathies 6
Shireen R. Lamandé, Trevor L. Cameron, Ravi Savarirayan,
and John F. Bateman
Abstract
The cartilage extracellular matrix is rich in fibrillar- and fibril-associated colla-
gens, and mutations in these collagen genes cause a wide range of chondrodys-
plasias, ranging from premature arthritis through to severe early lethal disorders.
Collagen mutations can interfere with matrix organisation and cause cartilage
dysfunction by reducing synthesis of structurally normal protein, or through pro-
tein misfolding which leads to intracellular retention and degradation, and con-
sequent secretion of reduced amounts of structurally abnormal protein. In
addition, collagen misfolding mutations can induce a cellular unfolded protein
response which can alter chondrocyte differentiation and ultimately promote
S.R. Lamandé, PhD (*)

Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville 3052, Australia
Department of Paediatrics, University of Melbourne, Parkville 3052, Australia
e-mail: shireen.lamande@mcri.edu.au
T.L. Cameron, PhD
e-mail: trevor.cameron@mcri.edu.au
R. Savarirayan, MD
Department of Paediatrics, University of Melbourne, Parkville 3052, Australia
Victorian Clinical Genetics Service, Melbourne, Australia
e-mail: ravi.savarirayan@mcri.edu.au
J.F. Bateman, PhD
Department of Biochemistry and Molecular Biology, University of Melbourne,
Melbourne, Parkville 3052, Australia
e-mail: john.bateman@mcri.edu.au

100 S.R. Lamandé et al.
apoptosis and thus contribute to the pathology. The relative impact of intracel-
lular and extracellular consequences is likely mutation and collagen type spe-
cific. Manipulating protein degradation pathways and unfolded protein response
signalling offers hope for treating genetic chondrodysplasias.
6.1 Introduction
Cartilage is a heterogeneous tissue comprised of chondrocytes embedded within a

structurally complex extracellular matrix rich in heterotypic fibrillar collagens and
proteoglycans. In the adult, three types of cartilage occur – elastic cartilage such as
that of the pinna, fibrocartilage as occurs in intervertebral discs and hyaline carti-
lage such as the articular cartilage lining diarthrodial joints. Though permanent and
specialised for its role in facilitating joint movement and resisting compressive load,
articular hyaline cartilage in the adult is developmentally continuous with the tem-
porary hyaline cartilage that first appears in embryogenesis during endochondral
ossification, but which is ultimately degraded and replaced by mineralized bone by
the end of adolescence. For brevity, we will limit our discussion here to hyaline
cartilage since the symptoms of cartilage collagenopathies are most profoundly
manifested in these tissues. This article will review our current knowledge of the
genetic basis of human chondrodysplasias, disorders that affect cartilage develop-
ment, differentiation and function, caused by collagen mutations.
6.2 Cartilage Development and Endochondral Ossification
Since chondrodysplasia mutations affect most aspects of the complex pathway of

cartilage and endochondral bone development, as well as the structural integrity of
mature cartilage, we will provide a brief overview of cartilage development to pro-
vide context for understanding pathological pathways. More detailed descriptions
of cartilage development are found in chapters of volume 1. Long bone formation in
the developing limb constitutes a classic model of endochondral ossification. The
first evidence of endochondral ossification in the developing limb is the appearance
of mesenchymal condensations. These result from mesenchymal cells migrating
into the embryonic limb bud from the lateral plate mesoderm and then aggregating
at sites of future bone growth (Eames et al. 2003; Hall and Miyake 1995). These
processes are precisely regulated by spatiotemporally dependent gene expression in
response to a combination of inputs that include cell matrix interactions, growth
factors and non-coding RNAs. Cells within the condensations express cell-cell
adhesion molecules such as N-cadherin and NCAM (Oberlender and Tuan 1994;
Widelitz et al. 1993), extracellular matrix molecules such as versican (Kamiya et al.
2006), transcription factors including the master regulator of chondrogenesis, SOX9
(Akiyama et al. 2002; Bi et al. 1999) and growth factor receptors including FGFR2
(Ornitz and Marie 2015) and BMPR1B (Lehmann et al. 2003; Baur et al. 2000).
6 Molecular Genetics of the Cartilage Collagenopathies 101
Accordingly, mesenchymal condensations are responsive to the complex interplay

between several signalling pathways, including the FGF (Ornitz and Marie 2015),
GDF5 (Buxton et al. 2001; Storm et al. 1994) and BMP-SMAD (Wang et al. 2016;
Lim et al. 2015) pathways, the latter of which may act in a SOX9-dependent or
SOX9-independent manner, as well as non-coding RNAs such as LncRNA-HIT,
which appears to promote the expression of BMPR1B (Carlson et al. 2015).
The onset of chondrogenic differentiation within mesenchymal condensations is
marked by changes in the morphology and arrangement of cells within the develop-
ing anlage. Whereas condensed mesenchymal cells are relatively small, rounded
and tightly packed, chondrocytes are comparatively larger and characterised by a
classic cobblestone appearance with separation between adjacent cells. This separa-
tion results from a second major hallmark of chondrogenic differentiation, namely,
the dramatic upregulation of genes encoding critical components of the cartilage
extracellular matrix, including aggrecan, type II collagen, COMP, link protein and
matrilin within the early anlage (Cameron et al. 2009). Thus chondrocytes are
responsible for secreting the extracellular matrix within which they become embed-
ded and with which they interact during subsequent differentiation steps.
Surrounding the cartilaginous anlage is a dense perimeter of connective tissue
known as the perichondrium, which is comprised of flattened cells that derive from
the same mesenchymal population as the chondrocytes of the anlage itself, but
which subsequently differentiate into the osteoblasts of the periosteum under the
control of canonical WNT signalling (Day et al. 2005). With further maturation,
chondrocytes at the centre of the developing anlage undergo hypertrophy, express-
ing markers of hypertrophic cartilage such as type X collagen (Kielty et al. 1985);
RUNX2, which drives expression of COL10A1 (Zheng et al. 2003); matrix metal-
loproteinases (MMPs) – including MMP9 and MMP13 that facilitate remodelling
of the matrix (Stickens et al. 2004); and VEGF, which promotes vascular invasion
of the anlage at the site of hypertrophy (Gerber et al. 1999). Ultimately, hypertro-
phic chondrocytes undergo either programmed cell death or transdifferentiation to
form osteoblasts (Tsang et al. 2015), as more osteoblasts enter the area from the
perichondrium (Colnot et al. 2004) to continue the replacement of the hypertrophic
cartilage matrix by mineralized bone, thus forming the primary centre of
ossification.
This process of chondrocyte differentiation, hypertrophy, cartilage matrix remod-
elling, degradation and replacement by bone continues longitudinally in a bidirec-
tional manner at the growth plates, which separate the cartilaginous epiphyses of
growing long bones from the bony diaphysis. Accordingly, growth plate chondro-
cytes appear to be stratified into discrete resting, proliferative and hypertrophic
zones corresponding to different stages of their maturation and characterised by
unique cell morphology and gene expression signatures (Lui et al. 2010; Belluoccio
et al. 2008). The resting zone houses small, round, irregularly arranged chondro-
cytes that will give rise to proliferative chondrocytes and is involved in directing
overall growth plate orientation (Abad et al. 2002). In the proliferative zone, chon-
drocytes orientate under the control of β1 integrin in distinct columns of flattened
cells as they promote longitudinal bone growth through rapid mitotic proliferation
(Aszodi et al. 2003), the extent of which is regulated by a feedback loop involving
PTHrP expressed by cells of the resting zone and perichondrium and IHH produced
by postmitotic, hypertrophic chondrocytes (Kronenberg 2006). FGF signalling is
also critical in regulating the rate of growth plate chondrocyte proliferation and is
dependent on the ligands FGF9 and FGF18 supplied by the perichondrium and
periosteum and FGFR3 expressed in pre-hypertrophic growth plate cartilage (Ornitz
and Marie 2015). As with hypertrophic chondrocytes generated during the forma-
tion of the presumptive cartilage anlage, growth plate hypertrophic chondrocytes
orchestrate the ongoing remodelling and mineralisation of the growth plate through
the secretion of matrix metalloproteinases (Stickens et al. 2004) and its vascular
invasion through the secretion of proangiogenic factors (Gerber et al. 1999).
Subsequent to the formation of the primary ossification centres, a secondary centre
of ossification also forms within each cartilaginous epiphysis of growing long bones
through essentially the same process of chondrocyte hypertrophy, cartilage remod-
elling and mineralisation that takes place at the primary centres of ossification.
6.3 Classificaton of Skeletal Dysplasias
The skeletal dysplasias (inherited disorders of bone and cartilage) are a heteroge-
neous collection of disorders caused by abnormalities in the development, growth
and maintenance of the human skeleton. The current nosologic classification of
these disorders (Bonafe et al. 2015) reflects their diverse manifestations, with over
450 conditions listed in 42 separate groups, some based on common molecular aeti-
ology (i.e. type II collagen group), others on their predominant radiographic fea-
tures (i.e. slender bone group) and others on their most striking clinical features (i.e.
dysplasias with multiple joint dislocations) (see Table 6.1 for an abbreviated nosol-
ogy). Skeletal dysplasias can present in myriad forms, ranging from lethal condi-
tions in utero to late early adult-onset manifestations involving the spine and joints.
Recently, with advances in genomic technologies, many of the genetic causes
underlying the skeletal dysplasias have been elucidated, revealing the multiple
pathogenetic mechanisms that can lead to skeletal defects.
6.3.1 Defects in Local Regulation of Cartilage Growth
Defects in local regulation of cartilage growth include disorders caused by abnor-

malities in growth factors and their receptors. Fibroblast growth factor receptor 3
plays a key role in the negative regulation of bone growth by inhibiting cell growth
in cartilaginous growth plates. Almost all cases of achondroplasia (the most common
form of human dwarfism) are due to one of two specific gain-of-function mutations
in the FGFR3 gene, which result in upregulation of the FGFR3 pathway. The lethal
dysplasia, thanatophoric dysplasia, is due to different mutations in the FGFR3 gene.
Table 6.1 Abbreviated skeletal dysplasia nosology (for details see Bonafe et al. (2015))
Group/disorder Gene/s
FGFR3 chondrodysplasia group FGFR3
Type 2 collagen group COL2A1
Type 11 collagen group COL11A1, COL11A2
Sulphation disorders group DTDST, PAPSS2, IMPAD1, CHST3, CHST14
Perlecan group HSPG2
Aggrecan group AGC1
Filamin group and related disorders FLNA, FLNB, SH3PXD2B
TRPV4 group TRPV4
Ciliopathies with major skeletal EVC1, EVC2, DYNC2H1, WDR34, DYN2H1,
involvement IFT80, TTC21B, WDR19, IFT172, IFT140,
NEK1, WDR35, TCTN3, IFT122, IFT43
Multiple epiphyseal dysplasia and COMP, COL9A1, COL9A2, COL9A3, MATN3
pseudoachondroplasia group
Metaphyseal dysplasias COL10A1, RMRP, PDP1, PTHR1, SBDS,
MMP13, MMP9, RUNX2
Spondylometaphyseal dysplasias ACP5, PCY1A
Spondylo-epi-(meta)physeal dysplasias DYM, RAB33B, SMARCAL1, EIF2AK3, MATN3,
DDR2, SEDL, SLC39A13, LONP1
Severe spondylodysplastic dysplasias TRIP11, SLC35D1, GPX4, SBDS, INPPL1,
MAGMAS
Acromelic dysplasias TRPS1, EXT1, IHH, ADAMTSL2, FBN1,
ADAMTS10, ADAMTS17, LTBP2, SMAD4,
PDE4D, PRKAR1A, GNAS
Acromesomelic dysplasias NPR2, GDF5, BMPR1B, SHOX, GPC6, FZD2,
ROR2, WNT5A, DVL1, SULF1, SLC05A1
Campomelic dysplasia and related SOX9, LIFR
disorders
Slender bone dysplasia group CUL7, OBSL1, CCDC8, TBCE, FAM111A,
RNU4ATAC, PCNT2, CDKN1C
Dysplasias with multiple joint dislocations CANT1, XYLT1, KIF22, B3GALT6
Chondrodysplasia punctata group EBP, ARSE, NSDHL, MGP, LBR, PEX7, DHPAT,
AGPS
Neonatal osteosclerotic dysplasias PTHR1, DHCR24, COL1A1, FAM20C
Osteopetrosis and related disorders TCIRG1, CLCN7, SNX10, DSTM1, RANKL,
PLEKHM1, CA2, LRP5, IKBKG, FERMT3,
RASGRP2, CTSK, LEMD3, WTX, SLC29A3
Other sclerosing bone disorders ANKH, TGFB1, TBXAS1, HPGD, GJA1, OPG,
SOST, LRP4, DLX3, PTDSS1
Osteogenesis imperfecta and decreased COL1A1, COL1A2, CRTAP, LEPRE1, PPIB,
bone density group SERPINH1, BMP1, FKBP10, PLOD2, SP7,
WNT1, TMEM38B, SEC24D, IFTIM5, PLS3,
LRP5, P4HB, XYLT2, B4GALT7, GORAB,
PYCR1, ATP6VOA2
(continued)

Group/disorder Gene/s
Abnormal mineralisation group ALPL, PHEX, FGF23, DMP1, ENPP!, CICN5,
SLC34A3, CASR, ANKH
Lysosomal storage diseases with skeletal IDA, IDS, HSS, NAGLU, HSGNAT, GNS, GALNS,
involvement GLBI, ARSB, GUSB, FUCA, MANA, MANB,
AGA, NEU1, SLC17A5, PPGB, SUMF1,
GNPTAB, GNPTG
Osteolysis group RANK, LMNA, ZMPSTE24, MMP2, NOTCH2,
MAFB
Disorganised development of skeletal EXT1, EXT2, SH3BP2, GNAS, TMEM16E,
components group PTPN11, FGFR1, ACVR1, NF1, TREM2,
TYROBP, IDH1, IDH2
Overgrowth syndromes with skeletal EZH2, NSD1, SETD2, NFIX, AKT1, PIK3CA,
involvement FBN1, FBN2, TGFBR1, TGFBR2, SMAD3,
TGFB2, NPPC, NPR2
Genetic inflammatory/rheumatoid-like WISP3, CIAS1, IL1RN, LPIN2, GALNT3,
osteoarthropathies ANTXR2
Cleidocranial dysplasia and related RUNX2, Fig. 6.4, ALX4, MSX2
disorders
Craniosynostosis syndromes FGFR1, FGFR2, FGFR3, POR, MSX2, TWIST1,
SKI, RECOL4, RAB23, MEGF8, TCF12, ERF
Dysostoses with predominant craniofacial TCOF1, POLR1D, POLR1C, CXORF5, EVC1,
involvement EVC2, ICK, EFNB1, ALX3, ALX4, ALX1,
DHODH, SF3B4, EFTUD2
Dysostoses with predominant vertebral HLXB9, DLL3, MESP2, LFNG, HES7, TBX6,
with and without costal involvement GDF6, GDF3, MEOX1, SNRBP, COG1, BMPER,
NKX3-2
Patellar dysostoses TBX4, LMX1B, KAT6B, ORC1, ORC4, ORC6,
CDT1, CDC6
Brachydactylies (without extraskeletal IHH, BMPR1B, BMP2, GDF5, ROR2, NOG,
manifestations) HOXD13, PTHLH, SOX9
Brachydactylies (with extraskeletal HDAC4, PIGV, PED3A, MYCN, HOXA13,
manifestations) CREBBP, EP300, CHSY1, ARHGAP31, DOCK6,
RBPJ, EOGT, TGDS
Limb hypoplasia-reduction defects group TBX3, NIPBL, SMCA1, SMC3, RAD21, HDAC8,
RMB8A, THPO, TBX5, SALL4, TBX15, ESCO2,
BHLHA9, SHH-ZRS, LMBR1, WNT3, WNT7A,
RECOL4
Ectrodactyly with and without other P63, CDH3, DLX5, DLX6, WNT10B, FGFR1
manifestations
Polydactyly-syndactyly-triphalangism SHH-ZRS, GLI3, FBLN1, HOXD13, SALL1,
group FGFR2, FGFR3, FGF10, KIF7, WNT6, LRP4,
GREM1, FMN1, BHLHA9, FAM58A, IHH, GJA1,
FGF16, CKAP2L, MKS1, TMEM216, TMEM67,
CEP290, RPGRIP1L, CC2D2A
Defects in joint formation and synostoses FGF9, NOG, GDF5, HOXA11, PITX1
Fig. 6.1 Spectrum of type II collagen disorders. The diagram conceptualises phenotypic “islands”
that vary in severity. At the peak of each island is the classic phenotype, with areas between the
islands representing “bridging” phenotypes. The X-rays show examples of early-onset arthritis
(left panels) and achondrogenesis (right panel) (Modified from (Kannu et al. 2010))
6.3.2 Defects in Structural Proteins of Cartilage
Defects in structural cartilage proteins such as collagen types I, II, IX, X and XI and
extracellular matrix proteins such as COMP (cartilage oligomeric matrix protein)
result in various forms of skeletal dysplasias that can be present at varying ages and
with varying severity. Great examples of this are mutations in the gene encoding
collagen type II that cause the group of disorders known collectively as the “type II
collagenopathies”, which comprises achondrogenesis type II, hypochondrogenesis,
spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, Stickler syn-
drome and early-onset arthritis (Kannu et al. 2010) (Fig. 6.1).
6.3.3 Defects in Cartilage Metabolic Pathways
Defects of enzymes, ion channels and transporters that are essential for cartilage
metabolism and homeostasis have been identified as the cause of other skeletal dys-
plasias. An example of this is the TRPV4 gene (transient receptor potential c ation
channel subfamily V, member 4), which encodes a calcium-permeable ion channel.
Dominant mutations in this gene result in activation of the channel leading to

increased concentration of calcium within chondrocytes (Hurd et al. 2015). TRPV4
mutations are the cause of a number of skeletal dysplasias including lethal and non-
lethal metatropic dysplasia, which presents prenatally or in the neonatal period, and
spondylometaphyseal dysplasia Kozlowski type, which presents in childhood
(Andreucci et al. 2011; Kannu et al. 2007). The variable severity of clinical features
seen in the TRPV4 groups of skeletal dysplasias can be correlated with the degree
of activation of the channel (Loukin et al. 2011). Interestingly other mutations
(likely diminishing function) in the TRPV4 gene can present with an arthritic phe-
notype predominately involving the hands and feet (Lamande et al. 2011).
6.3.4 Diagnosis
Making a diagnosis of a specific skeletal dysplasia is often difficult given the vast
number of recognised types. The involvement of physicians with specific skills in
skeletal dysplasias, such as clinical geneticists, orthopaedic specialists and paediat-
ric radiologists, is essential when trying to make an accurate diagnosis in the neona-
tal period and for subsequent management and monitoring.
Based on clinical history and examination, family history and expert evaluation of
radiology (the single most powerful diagnostic tool), it is usually possible to make a
diagnosis in the majority of those who present in childhood with a skeletal dysplasia.
Molecular testing can provide confirmation of a clinical diagnosis. In some cases a
skeletal dysplasia will be unclassifiable in the neonatal period but with time the devel-
opment of additional clinical features and patterns may allow diagnosis. The advent of
new diagnostic tools, such as exome sequencing and extensive skeletal gene panels, has
greatly increased the power to detect the genetic basis of skeletal dysplasias.
6.3.5 Management
The management of skeletal dysplasias requires good understanding of the natural

history and variability of the condition, a multidisciplinary approach and consider-
ation of the psychosocial impacts of conditions that can alter appearance and height.
Many skeletal dysplasias have nonskeletal manifestations (i.e. ocular features in
type II collagen disorders), which require relevant subspecialty input. Ideally,
patients and families should be managed in a centre with such experience and
resources to maximise benefits.
6.4 Cartilage Components and ECMopathies
Cartilage extracellular matrix (ECM) production, architecture and homeostasis is

orchestrated by chondrocytes, and it plays a fundamental role in cartilage morpho-
genesis, development and endochondral bone formation by regulating cell
differentiation, proliferation, adhesion and migration. In the adult, the ECM of the
articular cartilage endows to the articulating surfaces the key biomechanical proper-
ties required for diarthrodial joint function. While the complete landscape of critical
ECM functional structures is not fully defined, it is clear that the correct temporal
and spatial expression of ECM proteins, and the formation of architecturally precise
interacting networks with unique functional and biological characteristics, is funda-
mental. The predominant cartilage ECM components are several members of the
collagen family, proteoglycans and many noncollagenous proteins (Heinegard
2009). More detailed descriptions of cartilage ECM are provided in chapters 1, 2,
and 3 in volume 1 (Aspberg 2016; Grässel 2016; Zaucke 2016).
The main collagenous component of cartilage is collagen II which is extensively
covalently cross-linked in the characteristic fibrillar networks. These structural net-
works are critical for cartilage form and biomechanical function. Collagen II copo-
lymerises with two minor cartilage collagens, types XI and IX, to form the mature
heteropolymeric cartilage collagen fibrils (Eyre 2004; Eyre et al. 2002). Collagen
XI molecules are localised in the interior of the collagen II fibril and act as a tem-
plate for fibrillogenesis. Collagen XI molecules are cross-linked head to tail to other
collagen XI molecules and laterally to the surrounding collagen II. Collagen IX is
found on the surface of the fibril and is also cross-linked to collagen II. Unlike fibril-
lar collagens, II and XI, the collagen IX triple helix contains noncollagenous inter-
ruptions that allow its NC4 domain to project away from the lineal fibril and thus
interact with the adjacent ECM. Collagen IX also has a post-translationally added
glycosaminoglycan side chain, providing additional interaction sites for other ECM
components. Collagen VI is also present in cartilage and its microfibrillar network
is particularly rich in pericellular regions. Chondrodysplasias caused by mutations
in the different cartilage collagen molecules are discussed in detail below.
In addition to the heteropolymeric collagen fibrils, a crucial component of the
cartilage matrix is the large proteoglycan aggrecan. The aggrecan protein core is
richly decorated with sulfated glycosaminoglycan side chains and large numbers
of aggrecan molecules are linked via link protein interactions to hyaluronan to
produce huge multimolecular aggregates. These aggrecan-hyaluron aggregates are
trapped within the fibrillar collagen network, and because they are highly nega-
tively charged, they have the potential to bind large amounts of water, causing the
ECM to swell and thus sustain the high compressive loads that are imposed on
cartilage during joint function (Fosang and Beier 2011). The integrity of the col-
lagen fibril-aggrecan network is the key to cartilage function, and many noncol-
lagenous proteins interact with or serve as adaptors in ECM protein interactions
that are important in establishing network architecture or in maintaining its struc-
ture. Not unexpectedly, because of its key role in cartilage function, mutations in
aggrecan cause several chondrodysplasias, spondyloepiphyseal dysplasia
Kimberley type (Gleghorn et al. 2005), spondyloepimetaphyseal dysplasia aggre-
can type (Tompson et al. 2009), osteochondritis dissecans (Stattin et al. 2010) and
idiopathic short stature (Nilsson et al. 2014) (Table 6.2). Mutations in another large
proteoglycan, perlecan, which is found in many basement membranes throughout
the body but is also richly localised in the chondrocyte pericellular matrix, cause
Table 6.2 Chondrodysplasias caused by ECM gene mutations

Gene Protein Group: representative disorders
Collagens
COL2A1 Collagen II Achondrogenesis type II, hypochondrogenesis,
spondyloepiphyseal dysplasia congenital, Kniest
syndrome, Stickler syndrome type 1
COL9A1 Collagen IX Multiple epiphyseal dysplasia type 6
COL10A1 Collagen X Metaphyseal dysplasia Schmid type
COL11A1 Collagen XI Stickler syndrome type 2, Marshall syndrome,
fibrochondrogenesis type 1
COL11A2 Collagen XI Stickler syndrome type 3, oto-spondylo-mega-
epiphyseal dysplasia, fibrochondrogenesis type 2
ECM structural proteins
HGPS2 Perlecan Dyssegmental dysplasias, Schwartz-Jampel
syndrome
AGC1 Aggrecan Spondyloepiphyseal dysplasia Kimberley type,
spondyloepimetaphyseal dysplasia aggrecan type
COMP Cartilage oligomeric Pseudoachondroplasia, multiple epiphyseal
matrix protein dysplasia type 2
MATN3 Matrilin 3 Multiple epiphyseal dysplasia type 5
MGP Matrix gamma- Chondrodysplasia punctata group, Keutel
carboxyglutamic acid syndrome
ECM post-translational modification
DTDST SLC6A2 sulphate Achondrogenesis type 1B, atelogenesis type 2,
transporter diastrophic dysplasia
PAPSS2 PAPS synthetase 2 Spondyloepimetaphyseal dysplasia PAPSS2 type,
Brachyolmia recessive type
CHST3 Carbohydrate Chondrodysplasia with congenital joint dislocations
sulphotransferase 3 CHST3 type
CHST14 Carbohydrate Ehlers-Danlos syndrome CHST14 type
sulphotransferase 14
ARSE Arylsulfatase E Chondrodysplasia punctata group:
chondrodysplasia punctata, X-linked recessive,
brachytelephalangic type
dyssegmental dysplasias (Arikawa-Hirasawa et al. 2001) and the Schwartz-Jampel

syndrome (Nicole et al. 2000). While these mutations in the genes encoding pro-
teoglycan protein backbones highlight the important structural role of proteogly-
cans, mutations in several of the enzymes involved in the post-translational
modification pathways that generate the highly sulfated glycosaminoglycan side
chains also cause chondrodysplasias (Table 6.2). Mutations in SLC26A2 sulphate
transporter (DTDST) result in achondrogenesis type 1B, atelogenesis type 2 and
diastrophic dysplasia (Rossi and Superti-Furga 2001); PAPS synthetase 2 (PAPSS2)
mutations cause spondyloepimetaphyseal dysplasia PAPSS2 type (Faiyaz ul Haque
et al. 1998) and brachyolmia recessive type (Miyake et al. 2012). Carbohydrate
sulfotransferase 3 (CHST3) mutations cause chondrodysplasia with congenital
joint dislocations CHST3 type (Thiele et al. 2004), and arylsulfatase E (ARSE)
mutations underlie the chondrodysplasia punctata group: chondrodysplasia punc-
tata, X-linked recessive and brachytelephalangic type (Brunetti-Pierri et al. 2003).
Members of the small leucine-rich protein (SLRP) family, including biglycan,
chondroadherin, decorin, epiphycan, fibromodulin, lumican, asporin and PRELP,
are found in cartilage. These proteins have a “horseshoe” shape (McEwan et al.
2006) that is important for protein interactions, allowing them to bind to growth
factors and cytokines (Iozzo and Schaefer 2010; Iozzo and Karamanos 2010), cell
surface receptors (Iozzo and Karamanos 2010) and ECM proteins (Wiberg et al.
2003) and affect signalling as well as ECM structure. Importantly SLRPs interact
with fibril-forming collagens and play a critical role in collagen fibrillogenesis
(Hedbom and Heinegard 1993; Douglas et al. 2006; Danielson et al. 1997). While
human mutations in SLRPs causing chondrodysplasias have not been found to
date, several SLRP knockout mice present cartilage phenotypes (Nuka et al. 2010;
Hessle et al. 2014; Nikitovic et al. 2012) although because of their widespread
expression other phenotypes predominate. Asporin has been linked to osteoarthri-
tis (Xu et al. 2015).
In addition to SLRPs, matrilin-1 and matrilin-3 and COMP (cartilage oligo-
meric matrix protein) are important structural components of cartilage.
Matrilin-1 can interact with the SLRPs biglycan and decorin and act as an adap-
tor protein connecting the collagen II/IX/XI fibrillar and collagen VI microfi-
brillar networks in cartilage (Wiberg et al. 2003). Matrilin-3 (MATN3) mutations
have been described in multiple epiphyseal dysplasia type 5 and spondylo-
epimetaphysial dysplasia MATN3 type implicating matrilin-3 oligomers (and
matrilin-1/matrilin-3 hetero-oligomers) as key interacting structural and signal-
ling components of the cartilage ECM (Jayasuriya et al. 2014; Cotterill et al.
2005). COMP mutations cause pseudoachondroplasia and multiple epiphyseal
dysplasia type 2. The molecular pathology of COMP and matrilin-3 mutations
is discussed in detail in Chap. 7.
6.5 Collagenopathies
The collagen fibrillar network forms the backbone of the cartilage ECM, directly
providing the structure critical for resistance to biomechanical loading and also
providing a scaffold for the myriad of interacting ECM proteins that add further
functionality. It is therefore unsurprising that mutations impacting collagen quan-
tity and quality are a significant cause of chondrodysplasias. In addition to underly-
ing chondrodysplasias, more subtle collagen mutations can predispose cartilage to
osteoarthritic damage. Mutations in the predominant cartilage fibrillar collagens II
(COL2A1), XI (COL11A1, COL11A2), fibril-associated collagen IX (COL9A1,
COL9A2, COL9A3) and hypertrophic cartilage network-forming collagen X
(COL10A1) have been reported in a range of chondrodysplasias (Table 6.2). We
will discuss the two mutation groups, null mutations and structural mutations, sep-
arately since recent data indicates that the pathophysiology is quite different.
Another collagen found in the cartilage pericellular matrix, collagen VI, has not
been associated with human chondrodysplasias, although there is strong evidence
that collagen VI is important in regulating the properties of the chondrocyte matrix
and mechanotransduction (Zelenski et al. 2015). Collagen VI in the pericellular
matrix is lost early in osteoarthritis (Felka et al. 2016) and collagen VI null mice
exhibit skeletal abnormalities including delayed development and ossification, as
well as accelerated osteoarthritic changes in the hip (Alexopoulos et al. 2009). Thus,
while collagen VI is a good chondrodysplasia candidate, its widespread expression
means that cartilage is not the key pathological target, and mutations in each of the
three canonical collagen VI genes COL6A1, COL6A2 and COL6A3 result in the
muscle disorders Ullrich congenital muscular dystrophy and Bethlem myopathy
(Bushby et al. 2014). In these disorders the reduced biomechanical challenges to
cartilage due to patient immobility may preclude the onset of a cartilage clinical
phenotype.
6.6 Mutations Leading to Reduced Protein Synthesis
Mutations that introduce premature stop codons either via point mutations or frame-
shifting indels are a common cause of gene loss-of-function. In COL2A1, heterozy-
gous premature termination mutations are often found in Stickler syndrome, type I,
which is characterised by ocular, auditory, orofacial and skeletal abnormalities
including mild spondyloepiphyseal dysplasia and early-onset osteoarthritis
(Table 6.2). Premature termination mutations most commonly cause nonsense-
mediated mRNA decay (NMD) resulting in functional haploinsufficiency of colla-
gen II in the affected cartilage. This mechanism was confirmed in Stickler syndrome
type I patients using the protein truncation test of in vitro transcribed and translated
mutant and control alleles. Mutant allele protein was not produced unless NMD was
inhibited with cycloheximide (Freddi et al. 2000). Heterozygous premature stop
codon mutations in COL11A2 cause Stickler syndrome, type III, which is a non-
ocular form of Stickler syndrome. COL11A2 is not expressed in the eye, accounting
for this phenotype (Mayne et al. 1993). Homozygous COL11A2 premature termina-
tion mutations have been described in otospondylomegaepiphyseal dysplasia, fibro-
chondrogenesis type 2 (Melkoniemi et al. 2000) and Weissenbacher-Zweymuller
syndrome (Harel et al. 2005). While not tested directly, the assumption is that, in
these patients with homozygous mutations, there would be little or no expression of
the collagen α2(XI) chain. In COL11A1, heterozygous nonsense mutations in com-
bination with a structural mutation on the other allele cause the severe recessive
fibrochondrogenesis type 2 (Tompson et al. 2010). Early-onset hearing loss appears
to be the major phenotype in heterozygous carriers of COL11A1 loss-of-function
alleles (Tompson et al. 2010). In the cho/cho mouse, homozygous for a Col11a1
null allele, a lethal chondrodysplasia results (Li et al. 1995). In the absence of the
α1(XI) chain, collagen II fibrillogenesis is severely disrupted and the resulting
fibrils have larger diameters (Fernandes et al. 2007), indicating that collagen XI
containing the α1(XI) chain, buried in the interior of collagen II fibrils, is required
to maintain the thin diameter fibrils seen in vivo. The heterozygous cho/+ mouse,
with α1(XI) haploinsufficiency, develops early-onset osteoarthritis (Xu et al. 2003).
Heterozygous null mutations in COL10A1 cause metaphyseal chondrodysplasia-
type Schmid which is characterised by irregularities of the metaphyseal ends of
bones resulting in bow legs, coxa vara and short stature (Lachman et al. 1988).
Collagen X forms a homotrimeric network restricted to the epiphyseal growth plate
cartilages of long bones. Both frameshift mutations leading to premature translation
termination and nonsense mutations have been characterised (Bateman et al. 2005).
Interestingly, these are all localised within a relatively small area of exon 3, in the
region coding for the C-terminal trimerisation domain. Studies on patient cartilage
have shown that these mutations lead to complete NMD of mRNA from the mutant
allele, so that COL10A1 is truly haploinsufficient in the patient growth plate carti-
lage (Chan et al. 1998; Bateman et al. 2003). This is surprising for several reasons.
Firstly, exon 3 is the most 3′ exon of the COL10A1 gene, and the current NMD
dogma is that mRNAs containing premature termination codons in the region
encoded by the most 3′ exon are immune to NMD because these mRNAs do not
have downstream exon-exon boundaries defined by protein deposition during splic-
ing that define the stop codon as premature. In COL10A1 NMD competency is
determined by the position of the premature termination mutation relative to the 3′
UTR (Tan et al. 2008). It was also expected that more 5′ mutations in exon 3 and
exon 2 would lead to NMD. However, studies using targeted mutagenesis demon-
strated that this was not the case, and only mutations in the small region of exon 3
where all patient mutations have been localised were susceptible to NMD (Tan et al.
2008). These data on collagen X NMD suggest that the mutant mRNA surveillance
mechanism might be fundamentally different to the current mechanistic models.
This may be due, in part, to the structure of collagen X mRNA, where almost all of
the protein is encoded by a large 3′ terminal exon. Understanding the molecular
mechanisms of collagen X NMD will offer insights into how cells deal with prema-
ture termination mutations in other genes with a similar large 3’ terminal exons
(Fang et al. 2013).
6.7 tructural Mutations Affect Collagen Assembly

S
at Multiple Levels
While the reduced collagen expression resulting from null mutations has the obvi-
ous effect of compromising the cartilage matrix arrangement, structural mutations
in the collagen chains have more complex qualitative and quantitative impacts. To
begin to understand these impacts, we need to consider the assembly framework of
collagens. Collagens assemble via recognition sequences at the C-terminus
(Khoshnoodi et al. 2006). In the case of collagen II, recognition directed by the
noncollagenous C-propeptide sequence determines the selection of three proα1(II)
chains to form collagen II homotrimers and in collagen XI selection of proα1(XI),
proα2(XI) and proα1(II) [called proα3(XI) before it was shown to be the protein
product of the COL2A1 gene] to form collagen XI heterotrimers. In collagen IX
assembly, proα1(IX), proα2(IX) and proα3(IX) chain selection information is con-
tained within the C-terminal region of the COL1 domain and the C-terminal NC1
domains (Mechling et al. 1996). Correct registration of the proα-chains is critical to
initiate collagen triple helix formation which occurs from the C- to N-terminus
(Ishikawa and Bachinger 2013). While collagen sequences contain the information
to fold into a triple helix in vitro, albeit slowly, the actual procollagen assembly and
folding machinery in the cell are complex involving interplay between the collagen
α-chains and a temporally and spatially regulated panel of molecular chaperones
and post-translational processing enzymes. Collagen assembly and processing has
been recently reviewed (Ishikawa and Bachinger 2013). Because of the strict
requirements for correct collagen folding, it is unsurprising that mutations in the
C-terminal domains that compromise initial chain assembly, or mutations in the
triple helix that interrupt the essential Gly-X-Y triplet repeat sequence and impact
triple helix formation, or mutations in the folding machinery itself result in many
collagenopathies (Fig. 6.2). While autosomal recessive null mutations in compo-
nents of the collagen assembly complex such as CRTAP, P3H (LEPRE1), cyclophilin
B (PPIB), FKBP65 (FKBP10) and HSP47 (SERPINH1) have been described for
osteogenesis imperfecta, predominantly impacting collagen I, mutations of these or
comparable processing partners have not yet been implicated in cartilage disorders.
As we learn more about tissue-specific and collagen type-specific foldases and
chaperones, it seems certain that these components will be involved in chondrodys-
plasias, and mutations will be found that alter cartilage collagen assembly (Ishikawa
and Bachinger 2013; Ishikawa et al. 2015). In this review we will focus on collagen
structural mutations that predominantly affect cartilage biology.
The most common mutations found in collagens are missense and exon-skipping
mutations that affect the amino acid sequence. The clinical severity depends on how
the mutation impacts collagen assembly, stability and/or interactions and how
important the collagen is for developing and maintaining matrix integrity and
function.
With collagen IX the predominant mutations in COL9A1, COL9A2 and COL9A3
that cause forms of multiple epiphyseal dysplasia (Table 6.2) are exon-skipping
changes that are concentrated in the gene region encoding the N-terminal COL3
domain (Jackson et al. 2012). These mutations can affect mRNA stability but also
disrupt collagen IX heterotrimer assembly and secretion (Briggs and Chapman
2002). Since collagen IX on the collagen II/XI fibril surface interacts with other
ECM networks, these quantitative and qualitative changes could alter cartilage
structure. Importantly, collagen IX associates with matrilin-3 and COMP within the
cell, so that secretion defects in collagen IX caused by misfolding and ER retention
have the potential to also impact the secretion efficiency of these interacting part-
ners (for further details, see Chap. 7).
Heterozygous mutations in COL11A1 or COL11A2, which introduce structural
changes such as in-frame exon deletions or glycine substitutions, disturb the obliga-
tory Gly-X-Y repeat sequence of the helix and cause conditions such as Stickler
Fig. 6.2 Protein consequences of pathogenic structural collagen mutations. Structural mutations
that lead to protein misfolding have a dominant negative effect. Mutations in the C-terminal pro-
peptide compromise collagen chain trimerisation. Misfolded unassembled chains are retro-
translocated to the cytoplasm where they are degraded by proteasomes. This results in protein
deficiency and, if some of the chains are incorporated into collagen trimers and are secreted, fur-
ther deleterious effects on collagen extracellular assembly and matrix function. Mutations in the
triple helical domain, commonly substitutions for the obligatory glycine residues in the Gly-X-Y
collagen sequence or in-frame deletions as a result of exon-skipping mutations, allow initial chain
selection and assembly, but the abnormal helical structure leads to retention in the ER and protein
aggregation. The protein aggregates are degraded by the autophagy/lysosomal system and the
result is a severe protein deficiency and ECM disorganisation
syndrome type 2 and Marshall syndrome (COL11A1) or Stickler syndrome type 3

and Weissenbacher-Zweymuller syndrome (COL11A2) (Table 6.2). Little direct
data is available on how these structural mutations affect collagen XI assembly and
stability, but dysfunction can be inferred from the other more extensively studied
fibrillar collagen mutations, such as collagen II and I, discussed below. While in
general the collagen XI structural mutations are less damaging than those in the
more abundant collagen II, compound heterozygous COL11A1 or COL11A2 muta-
tions can result fibrochondrosis type I and II (Tompson et al. 2010). In this condition
the presence of a null allele in combination with in-frame exon deletions or helix
glycine substitutions results in this very severe, often lethal, chondrodysplasia dem-
onstrating the importance of the collagen XI template for collagen II fibrillogenesis.
The third component of the collagen XI trimer is the α1(II) chain produced by the
COL2A1 gene [and formally called α3(XI)]; thus mutations in COL2A1 have poten-
tial ramifications for collagen XI trimer stoichiometry and integrity.
Collagen II structural mutations cause cartilage disorders of varying severity
from mild to severe and lethal conditions. Collagen II is structurally similar to the
major non-cartilage fibrillar collagen, collagen I, and many of the genotype-
phenotype correlations for the more intensely studied collagen I mutations in dis-
eases such as osteogenesis imperfecta shed light on the molecular pathology of
collagen II. Our discussion will focus on collagen II but will look to examples from
studies on collagen I to provide mechanistic insights. As discussed above COL2A1
mutations that result in haploinsufficiency cause mild phenotypes such as Stickler
syndrome (Fig. 6.1). Likewise COL1A1 haploinsufficiency causes the relatively
mild dominant form of osteogenesis imperfecta. COL2A1 missense mutations
impact cartilage pathology according to their effect on collagen structure, folding,
secretion and ability to form functional fibrils in the ECM. As an overarching con-
cept, mutations that affect the C-propeptide and compromise the initial assembly of
the three proα-chains and mutations that interfere with triple helix formation such
as glycine substitutions in the obligatory Gly-X-Y sequence have serious clinical
consequences, whereas other sequence changes are less severe. A full understand-
ing of the relationship between the mutation type, sequence context and clinical
outcome has not been achieved, but some general concepts are emerging including
important differences in the cellular consequences of helix mutations and those that
affect initial assembly. We will discuss what we know about the different mutation
groups and how they may affect cartilage structure and function.
Numerous non-glycine polymorphisms are found in COL2A1 in apparently nor-
mal individuals (Exome Aggregation Consortium (ExAC)) emphasising that the
helix can accommodate X and Y position changes within the strict helical structure.
These benign missense changes tend to conserve amino acid charge and size and
thus would not be expected to affect helix structure or folding. Helix X or Y position
missense mutations causing chondrodysplasias are rare; however, several substitu-
tions of cysteine for arginine, p.R275C (Williams et al. 1993), p.R565C (Richards
et al. 2000), p.R719C (Hoornaert et al. 2006), p.R904C (Hoornaert et al. 2006) and
p.R989C (Chan et al. 1993) cause disorders that range from severe but non-lethal
forms of spondyloepiphyseal dysplasia congenita through to Stickler syndrome and
precocious osteoarthritis. The position of the mutation in the helix does not predict
severity suggesting that local sequence contexts must be important in determining
the phenotype. This is supported by normal sequence data (60,000 unrelated indi-
viduals; Exome Aggregation Consortium) which includes individuals with
p.R1058C and p.R584C helix sequence variants (Exome Aggregation Consortium
(ExAC)). The p.R1058C variant occurs in three individuals, highly suggestive that
the arginine to cysteine change is tolerated in this region of the helix despite the fact
that the cysteine, which does not normally occur in the helix, would be predicted to
interfere with helix structure and introduce internal disulphide bonding into the col-
lagen II heterotrimer.
Collagen II glycine to cysteine changes have been studied in patient samples to a
limited extent, but more systematically in transfected cell model systems which
reveal a complex pattern of how these mutations affect collagen II. Several muta-
tions (p.R275C, p.R719C) occur in regions of the helix that are thermolabile and are
accommodated in the helix without reducing helix thermal stability (Steplewski
et al. 2004b). These mutations do not induce apoptosis in vitro (Hintze et al. 2008)
and, consistent with only a small impact on collagen structure, they produce mild
clinical phenotypes (Arnold and Fertala 2013). On the other hand, p.R989C is in a
thermostable helix region and this substitution reduces helix thermal stability. The
p.R989C mutation, and a comparable p.R1147C mutation found in a spondyloep-
iphyseal mouse model, cause chondrocyte apoptosis in vitro and in vivo and result
in a more severe phenotype (Hintze et al. 2008). It has been suggested (Arnold and
Fertala 2013) that the reduced thermostability in vitro may indicate that these muta-
tions produce more misfolding at physiological temperatures than p.R275C and
p.R719C. Importantly, p.R989C and p.R1147C also induce a cellular endoplasmic
reticulum stress response, discussed in the next section. In contrast the p.R275C and
p.R719C mutants do not appear to induce such cellular stress (Arnold and Fertala
2013). In addition to the cellular stress caused by p.R989C and p.R1147C muta-
tions, culminating in apoptosis, p.R989C interacts with fibronectin inside the cell
and extracellularly, and this binding likely inhibits collagen II fibril formation (Ito
et al. 2005). Since collagen and fibronectin fibrillogenesis in vivo requires both
proteins (Velling et al. 2002), strong fibronectin binding to p.R989C mutant colla-
gen II could also be responsible for the observed disorganised fibronectin matrix
and could potentially change the organisation of other extracellular matrix compo-
nents. By contrast, the p.R275C mutant binds only very weakly to fibronectin and
rapidly dissociates (Ito et al. 2005), and fibronectin deposited in the extracellular
matrix of cells expressing this mutant has a normal fibrillar structure. The arginine
to cysteine mutations do not obviously affect collagen II secretion (Ito et al. 2005),
and the abnormal collagen likely impacts fibril assembly and structure (Steplewski
et al. 2004a). These data emphasise the potential of both intracellular and extracel-
lular impacts to contribute to the final functional phenotype of the cartilage.
The most common and most studied mutations in the collagen helix are hetero-
zygous missense mutations that substitute other amino acids for glycine in the heli-
cal Gly-X-Y sequence. Approximately 100 of these substitution mutations in
COL2A1 have been reported in a range of severe chondrodysplasias including
several forms of spondyloepiphyseal dysplasia, Kneist dysplasia, achondrogenesis

type II and hypochondrogenesis (Kannu et al. 2012). Only one glycine substitution
(p.G940R) has been reported in normal individuals (Exome Aggregation Consortium
(ExAC)). With this single exception, the case for glycine helix-disrupting mutations
as the major cause of cartilage collagenopathies is overwhelming, mirroring the
predominance of COL1A1 glycine mutations causing osteogenesis imperfecta.
Aspects of the molecular pathology of helix glycine mutations are very clear and
can be generalised. Studies on mutant collagen II parallel the wealth of data on
mutant collagen I and demonstrate that helical glycine substitutions can delay helix
formation and destabilise the helix, and as a result, the mutant collagen trimers have
increased post-translational modifications (Bonaventure et al. 1995). As in collagen
I, not all collagen II glycine mutations reduce thermostability and this likely reflects
the mutation position within thermostable or thermolabile regions (Steplewski et al.
2004b; Ito et al. 2005). The disturbance to helix folding leads to intracellular reten-
tion of the mutant-containing molecules (Chan et al. 1995). Because collagen II is a
homotrimer of three chains, 7/8 of the trimers will contain at least one mutant chain,
so that the effect of heterozygous mutant expression has much more impact than it
would if collagen chains did not form multimers. Intracellular retention of the
mutant-containing trimers targets them for degradation by cellular quality control
processes (Chan et al. 1995). While the extent of degradation may be influenced by
the specific mutation, the extreme pathology that can result from this degradation
was dramatically demonstrated in two lethal chondrodysplasia patients, one with
hypochondrogenesis (p.G1113C [legacy mutation G913C] (Mundlos et al. 1996))
and one with achondrogenesis type II (p.G996S [legacy mutation G796S] (Chan
et al. 1995)) where the mutations lead to complete degradation, and collagen II in
the poorly formed cartilages is replaced with collagens I and III.
Mutations in the collagen II C-propeptide cause severe chondrodyplasias includ-
ing spondyloepiphyseal dyspalsia, achondrogenesis II-hypochondrogenesis and
spondyloperipheral dysplasia, Torrance type (Zankl et al. 2004, 2005; Nishimura
et al. 2004). While there are no biochemical studies to directly determine the effect
of the mutations on procollagen assembly, it is assumed based on mutations in this
domain in collagen I (Lamande et al. 1995; Bateman et al. 1989) and collagen X
(Bateman et al. 2005) that these compromise or prevent initial procollagen assem-
bly (Fig. 6.2). Heterozygous mutations in COL10A1 provide the most complete data
set on how mutations in the C-terminal trimerisation domains result in failed colla-
gen folding. To date 22 COL10A1 missense mutations have been reported in
metaphyseal chondrodysplasia Schmid-type patients (Table 6.2) (Bateman et al.
2005). These mutations cluster in a restricted region of exon 3 coding for the col-
lagen X NC1 domain which is critical for the initial association of the chains into
the trimeric collagen X structure. While crystal structures of this trimerisation
domain (Bogin et al. 2002) predicted that mutations disrupting the hydrophobic
core would exclude chains from assembly, in vitro and mouse studies have shown
that disease-causing mutations are not restricted to the hydrophobic core sequence,
and other mutations in the NC1 sequence from codon 555 to 677 impair collagen X
trimerisation (Bateman et al. 2005). The unassembled collagen X chains are retained
intracellularly and degraded (Wilson et al. 2002) suggesting that the reduced extra-
cellular collagen X, like that caused by nonsense-mediated decay of mRNAs con-
taining premature termination mutations, was the cause of the metaphyseal
chondrodysplasia Schmid-type phenotype. However, this is not the case and a much
more interesting scenario has emerged – the unassembled collagen X chains retained
in the endoplasmic reticulum trigger a cellular stress response, the unfolded protein
response (UPR) – and this is a major downstream effect that causes the pathology.
The UPR, and how it applies to collagen X and other collagens, is discussed in the
next section. The importance of the trimerisation domain and the UPR in COL10A1
pathology is highlighted by the almost complete absence of disease-causing
COL10A1 missense mutations outside (i.e. more N-terminal) the NCI domain.
Unlike other collagens, where glycine mutations are the major pathological change,
in COL10A1 only one glycine substitution in the triple helix has been reported in a
patient, p.G288R (Park et al. 2015). The ExAc database (Exome Aggregation
Consortium (ExAC)) reveals 70 glycine substitutions in COL10A1 in normal indi-
viduals, including several close to p.G288, casting some doubt on whether this is the
disease-causing mutation. Two mutations in the N-terminal signal peptide cleavage
site (Ikegawa et al. 1997; Bateman et al. 2004) are predicted to prevent release of the
mutant chains into the endoplasmic reticulum and result in haploinsufficiency
(Chan et al. 2001).
6.8 ndoplasmic Reticulum Stress in Cartilage

E
Collagenopathies
The endoplasmic reticulum (ER) imposes stringent quality control on protein fold-
ing, such that only folded functional proteins leave the ER (van Anken and Braakman
2005). The cellular response to misfolded proteins is called the unfolded protein
response (UPR) [recently reviewed (Wang and Kaufman 2016)]. Activation of the
UPR via canonical ER stress sensors, IRE1α, PERK and ATF6, results in a cascade
of adaptive responses in a coordinated attempt to reduce the load of misfolded pro-
teins (Hetz et al. 2011) (Fig. 6.3). These adaptive responses include attenuation of
translation, degradation of misfolded proteins by proteasomal ER-associated degra-
dation (ERAD) and autophagy. Initial UPR activation is cytoprotective, offering the
cells an opportunity to return to protein-folding homeostasis. With increased dura-
tion of unfolded protein load, the balance in activities of the three pathways changes,
and prolonged ER stress has deleterious effects for the cells such as apoptosis.
It is now clear that collagen misfolding mutations (as with other ECM gene
mutations, e.g. matrilin-3, COMP) can induce a cellular UPR with consequences on
tissue pathology (reviewed (Bateman et al. 2009; Boot-Handford and Briggs 2010;
Patterson and Dealy 2014)). The most compelling data so far comes from studies on
collagen X misfolding mutations in metaphyseal chondrodysplasia, Schmid type. In
vitro studies show that the mutant collagen X misfolding results in retention of the
mutant chains in the ER, aberrant disulphide-bonded dimer formation and degrada-
tion by ERAD, a key proteasomal degradation pathway activated by the UPR
Fig. 6.3 The UPR signalling pathways. The UPR has three parallel branches activated by
canonical UPR sensors embedded in the ER membrane, IRE1α, ATF6p90 and PERK. The
molecular chaperone BiP is thought to bind constitutively to the sensors and render them inac-
tive. Misfolded proteins in the ER bind to BiP, sequestering it away from the sensors and trig-
gering their signalling pathways. IRE1α and PERK homodimerize and autophosphorylate and
this activates XBP1 splicing and eIF2α phosphorylation. XBP1s produces a transcription factor
that stimulates the synthesis of chaperones and foldases to increase the protein-folding capacity.
Phosphorylated eIF2α inhibits general protein translation to reduce protein load and at the same
time promotes translation of the transcription factor ATF4 which then increases production of
apoptosis genes. Release of BiP from ATF6p90 allows the sensor to move to the Golgi where it
is cleaved. The active cytosolic fragment ATF6p50 enters the nucleus and stimulates transcrip-
tion of genes with an ER stress-responsive element
(Wilson et al. 2002, 2005). In vitro expression studies (Wilson et al. 2005) and
mouse models (Tsang et al. 2007; Ho et al. 2007) where Col10a1 trimerisation
domain mutants were expressed in growth plate cartilage showed that the misfolded
collagen X elicits an UPR where all three canonical arms IRE1α, ATF6 and PERK
are activated, indicated by Xbp1 splicing, ATF6 cleavage and eIF2α phosphoryla-
tion (Rajpar et al. 2009; Cameron et al. 2011). UPR activation resulted in upregula-
tion of downstream UPR targets, including molecular chaperones, foldases,
ER-associated degradation machinery and CHOP (Tsang et al. 2007; Rajpar et al.
2009; Cameron et al. 2011). While chondrocyte apoptosis was increased from
around 2 weeks of age (Cameron et al. 2015), increased apoptosis was not detected
earlier in development (Tsang et al. 2007; Rajpar et al. 2009; Cameron et al. 2011).
A key and unexpected consequence of the UPR were downstream effects on termi-
nal differentiation, with the persistence of a proliferative chondrocyte-like expres-
sion profile in ER-stressed Schmid chondrocytes. It was postulated that CEBPβ
dysregulation was a key component of this effect on the progression of chondro-
cytes from the proliferative to hypertrophic state (Cameron et al. 2011). A similar
block in terminal differentiation was reported in another mouse model of collagen
X misfolding, with a similar UPR upregulation (Tsang et al. 2007), although in this
case it was proposed that the re-expression of proliferative chondrocyte genes was
an adaptive response to the UPR; by downregulating collagen X expression and
reverting to a less mature phenotype, the cells survive, although at a significant cost
to their normal function. Whatever the precise mechanism of altered differentiation
caused by the misfolded collagen X and the UPR is, the pathological impact of the
collagen X-dependent UPR is disruption to cartilage maturation, remodelling, vas-
cularisation and mineralisation, leading to cartilage hypertrophic zone expansion
and dwarfism. These studies raise important questions about the relative impact of
the two consequences of collagen X misfolding, the UPR and the quantitative and
possible qualitative impact of structurally abnormal collagen X in the cartilage
ECM (Fig. 6.4). For collagen X the consequences of the UPR alone are sufficient to
cause the growth plate pathology. This was demonstrated by expressing a UPR-
inducing protein, a mutant thyroglobulin, specifically in growth plate cartilage
where it is normally not expressed and has therefore no role in cartilage structure
and function (Rajpar et al. 2009). The mutant misfolded thyroglobulin induced an
UPR in the growth plate cartilage of the transgenic mouse, resulting in the same
metaphyseal chondrodysplasia phenotype as was caused by Col10a1 missense
mutations. These data unequivocally show that for collagen X misfolding muta-
tions, the UPR is the key component of the molecular pathology.
The nature of the UPR and its role in the pathology caused by other cartilage
collagen types is not yet resolved, but overall it seems that aspects of the UPR may
be involved although the mutation specificity of the response remains to be defined.
Collagen X is like virtually all other collagens in having a C-terminal domain that
is involved in initial proα-chain selection and trimerisation; thus, it is expected that
mutations in other collagens in these domains will also impact folding and cause a
UPR which may contribute to the pathology. COL2A1 missense and other struc-
tural mutations in the C-propeptide cause a range of chondrodysplasias including
spondyloepiphyseal dysplasia (Mortier et al. 2000), spondyloperipheral dysplasia

(Zankl et al. 2004), platyspondylic skeletal dysplasia Torrance type (Zankl et al.
2005; Nishimura et al. 2004), avascular necrosis of the femoral head (Kannu et al.
2011) and vitreoretinopathy with phalangeal epiphyseal dysplasia (Richards et al.
2002). The functional effects of these different mutations on assembly and how
this can explain the clinical phenotypic differences have not been determined.
However, mouse models with Col2a1 p.S1386P, p.Y1391S and p.D1469A
C-propeptide mutations produce chondrodysplasias with the hallmarks of the
canonical unfolded protein response including protein retention in the ER (Esapa
et al. 2012; Furuichi et al. 2011), an expanded ER (Esapa et al. 2012; Furuichi et al.
2011; Kimura et al. 2015), increased mRNA expression of ER stress markers
Grp94 and Chop (Kimura et al. 2015; Furuichi et al. 2011) and chondrocyte apop-
tosis (Kimura et al. 2015). While this strongly suggests that the canonical UPR is
activated by collagen II C-propeptide mutations, there is also supporting evidence
from COL1A1 C-propeptide mutations in osteogenesis imperfecta (OI). In a mouse
model of OI caused by a frameshift mutation in the C-propeptide of collagen I,
there was ER retention of the mutant collagen in heterozygous mice, increased BiP
and the collagen-specific chaperone Hsp47, increased Chop with consequent cas-
pase-12 and caspase-3 activation and apoptosis of osteoblasts both in vitro and
in vivo (Lisse et al. 2008). The engagement of a canonical UPR with COL1A1
trimerisation mutations in osteogenesis imperfecta is supported by early biochemi-
cal studies demonstrating BiP binding and proteasomal degradation in patient
fibroblasts and transfected cells (Lamande et al. 1995; Fitzgerald et al. 1999;
Chessler and Byers 1993).
While there appears to be involvement of a canonical UPR downstream of col-
lagen II mutations that affect initial assembly, it is not yet known how much this
contributes to the clinical phenotype, in addition to the reduced collagen secretion
due to retention and degradation. Given that the phenotypes for heterozygous
C-propeptide mutations are generally more severe than null allele mutations, it is
reasonable to assume the UPR may contribute to the pathology in many instances.
However, the consequences may well depend on whether the mutation completely
prevents assembly or allows partial assembly and has further dominant negative
effects on the collagen II population.
Fig. 6.4 Cellular and extracellular consequences of collagen mutations. Reduced protein synthe-
sis caused by heterozygous premature stop codon mutations and mRNA decay or null alleles
results in a protein deficiency in the ECM and relatively mild clinical phenotypes. Structural muta-
tions have dominant negative effects. Misfolded proteins are partially or completely retained in the
ER and, depending on the folding defect, are degraded by either ERAD/proteasomes or autophagy.
If abnormal proteins are secreted, they have a further deleterious effect on ECM structure and func-
tion. Misfolded proteins can also induce the cellular unfolded protein response, which if unre-
solved can lead to changes in gene expression and differentiation and eventually apoptosis. The
relative impact of extracellular and intracellular consequences for different mutations in different
collagen types has not been fully resolved
The most common collagen II (and other collagen) mutations are helix-disrupting
mutations such as glycine substitutions. Given that glycine mutations result in intracel-
lular retention and degradation, it would seem logical that some form of ER stress
would result which may contribute to the pathology. In collagen II there is good evi-
dence of an UPR resulting from disruptions in the helix caused by p.G1182A (achon-
drogenesis type II), p.G450R (hypochondrogenesis) and an exon 41 skipping mutation
(achondrogenesis type II) (Okada et al. 2015). Patient fibroblasts were directly con-
verted into chondrocyte-like cells in vitro by transducing cells with c-MYC, KLF4 and
SOX9. In these cells there was increased BiP, GRP94 and CHOP and increased XBP1
splicing, ATF6 cleavage, eIF2α phosphorylation and apoptosis, indicating activation of
the UPR. Treating the mutant chondrocytes with TMAO (trimethylamine N-oxide),
which is a chemical chaperone that can rescue protein-folding defects, reduced the
elevated BiP and CHOP and reduced apoptosis (Okada et al. 2015). Similarly, BiP
upregulation, ER stress, UPR activation and apoptosis are evident in the Col2a1
p.G1170S-mutated mouse chondrodysplasia model (Liang et al. 2014). While these
data provide strong evidence for a canonical UPR for these mutations, other data dis-
cussed below suggests that the cellular response to collagens containing helix muta-
tions may be more complex and dependent on additional factors such as the mutation
context. One key to this may be if the helix disruption is in a region that is thermostable
or thermolabile (Steplewski et al. 2004a) as discussed above for arginine to cysteine
mutations where only those mutations that reduce collagen helix thermal stability
result in an UPR (Chung et al. 2009). In support of this hypothesis, the Col2a1
p.G1053E mutation does not reduce helix thermal stability or elicit apoptosis (Hintze
et al. 2008); however, the thermal stability of only a relatively few collagen II helix
mutations has been determined, so correlating the destabilising effect of the mutation
with an UPR is not yet possible and so far has only been clearly demonstrated with the
arginine to cysteine mutations (Arnold and Fertala 2013). It is of interest that studies on
the helix destabilising p.R989C and p.R1147C mutations demonstrated that the chemi-
cal chaperone TMAO stabilised the mutant collagen and increased mutant secretion
(Gawron et al. 2010), presumably as a consequence of reducing the UPR.
For collagen I, the only studies to look at BiP binding to helix-mutant collagen I
failed to show any interaction (Lamande et al. 1995) that would be indicative of
helix-mutant collagen I triggering a conventional ER stress response. Recent studies
on a mouse model of OI with a Col1a2 p.G610C mutation also failed to show BiP
upregulation despite intracellular retention of the mutant collagen (Mirigian et al.
2016). However, consistent with other studies, autophagy was upregulated, and
there was clearly a “stress” response that resulted in osteoblast malfunction. Further
studies are required to determine how collagen I and collagen II helix mutations
engage with the osteoblast/chondrocyte quality control systems and how this may
depend on the mutation context and effect on helix structure.
Whatever the precise nature of the UPR is, and what downstream pathological path-
ways may be affected, one key question in understanding the molecular pathology and
envisaging new therapeutic approaches is how do the chondrocytes degrade the mutant
collagens. With mutations that prevent assembly, such as C-propeptide collagen muta-
tions discussed above, it seems clear from inhibitor studies in several cell model
systems that the unassembled procollagen chains are retro-translocated to the cyto-
plasm and degraded by the proteasome system (Lamande et al. 1995; Fitzgerald et al.
1999; Ishida et al. 2009) (Fig. 6.3). This ERAD is one of the well-characterised cellular
responses directed by the UPR, a targeted response to reduce the mutant protein load.
In the case of collagen I with mutations in the helix that allow initial assembly of the
procollagen trimer but affect downstream helix formation and stability, the partially
assembled molecules are not retro-translocated for proteasomal breakdown and form
aggregates in the ER. These aggregates are degraded by the autophagosome-lysosomal
system (Ishida et al. 2009; Mirigian et al. 2016), and although a stress response results
in mouse osteoblasts expressing Col1a1 p.G610C mutant chains, it is an unconven-
tional stress response that does not involve BiP upregulation or XbpI splicing (Mirigian
et al. 2016). Importantly, stimulating autophagy with rapamycin or nutrient deprivation
prevented accumulation of the misfolded collagen I, eliminated the ER distention and
restored collagen I extracellular matrix deposition (Mirigian et al. 2016). While these
studies suggest potential therapeutic strategies for collagen I mutations, further studies
on collagen II mutants are required to understand the cellular responses and degrada-
tion pathways downstream of different mutations.
6.9 Therapeutically Targeting the UPR
In a number of protein-folding diseases, therapeutic approaches that target protein

misfolding at multiple levels are being deployed (Cao and Kaufman 2013). In gen-
eral, these approaches attempt to restore proteostasis by stimulating degradation of
the misfolded protein, or correcting the mutant protein folding, or act downstream
of protein folding to manipulate different UPR signalling components. While many
questions remain to be answered about how collagen misfolding mutations interact
with the cellular unfolded protein response machinery, these approaches are a prom-
ising avenue to explore with collagen mutations.
Studies with the chemical chaperone, TMAO, have shown in vitro that the mutant
collagen II helix is stabilised (Gawron et al. 2010), and secretion is increased in cell
disease models (Okada et al. 2015). Studies with another chemical chaperone
sodium phenylbutyrate on cells expressing mutant collagen IV have shown compa-
rable rescue of misfolding and secretion (Murray et al. 2014). However, in cartilage
in vivo, the possible effect of rescuing mutant collagen secretion on extracellular
matrix formation and stability must be considered. In this context the altered inter-
action of collagen II p.R989C with fibronectin and the ability of the mutant-“kinked”
collagen to impact both collagen and fibronectin fibrillogenesis (Steplewski et al.
2004a) suggest caution in this approach until details of both the intracellular and
extracellular consequences of the mutant collagen are clarified experimentally.
Increasing breakdown of the mutant misfolded intracellular collagens by stimu-
lating the proteasomal and/or autophagosome-lysosome system is another promis-
ing approach. While this has not been tested with cartilage collagen misfolding
mutations, rapamycin treatment of osteoblasts isolated from a mouse model of OI
with a Col1a2 p.G610C mutation rescued the altered osteoblast differentiation and
apoptosis that contributed to the pathology (Mirigian et al. 2016). While stimulating
mutant degradation will reduce ER stress, it will also reduce mutant collagen secre-
tion, thus overcoming any possible dominant negative effects that secreted mutant
could have on the ECM. However, clearly with this approach, there will be an over-
all reduction in the total collagen deposited in the matrix. While 50 % of the normal
level of collagen II caused by heterozygous null alleles leads to a relatively mild
Stickler phenotype, a much greater reduction might result from stimulating break-
down depending on how many of the mutant and normal collagen II heterotrimers
are degraded. This is likely to be mutation specific and it will be important to experi-
mentally determine the ECM impacts of this approach.
A third therapeutic approach that is being explored in several diseases including
cancer, neurodegenerative disorders, metabolic diseases, heart ischaemia, inflam-
mation and liver disease is targeting the UPR sensing and downstream signalling
pathways (Maly and Papa 2014; Rivas et al. 2015; Jiang et al. 2015; Hetz et al.
2013). Both IRE1α and PERK have druggable enzyme activities and are the main
focus for drug development. Small molecules that modulate PERK signalling and
the IRE1α-XBP1 arm are generating a lot of interest, and some are already FDA
approved or in preclinical trials for cancer (Maly and Papa 2014; Jiang et al. 2015;
Hetz et al. 2013). Natural compounds that upregulate or downregulate ATF6 expres-
sion have also been identified and promise therapeutic potential but their mode of
action is not currently known (Rivas et al. 2015).
The downstream cellular and extracellular consequences of different collagen
mutations are clearly complex and much work remains to be done before we have a
sophisticated and detailed understanding. A major challenge to advancing effective
therapies for the collagen chondrodysplasias that appropriately target the different
signalling branches of the UPR will be developing accurate human in vitro disease
models that can be used to fully define the molecular pathology and used for high-
throughput drug screening. A powerful emerging approach is using patient-derived
induced pluripotent stem cells (iPSCs) and differentiating them into chondrocytes
(Okada et al. 2015; Tsumaki et al. 2015) to produce in vitro cartilage. The utility of
this approach was recently demonstrated in elegant experiments that showed statins
could rescue the poor chondrogenic differentiation of FGFR3 mutant iPSCs and then
went on to show that lovastatin treatment of Fgfr3 mutant mice significantly improved
their bone growth (Yamashita et al. 2014). While these approaches offer hope for
treating genetic chondrodysplasias once thought to be intractable, we need to go for-
ward with a systematic approach that assesses the consequences of manipulating the
UPR on global cellular pathways as well as the organisation and structure of the extra-
cellular matrix.
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Pseudoachondroplasia and Multiple
Epiphyseal Dysplasia: Molecular 7
Genetics, Disease Mechanisms
and Therapeutic Targets
Michael D. Briggs, Peter Bell, and Katarzyna A. Piróg
Abstract
Genetic skeletal diseases (GSDs) are a diverse and complex group of over 450
rare diseases that affect the development and homoeostasis of the skeleton.
Although individually rare, as a group of related genetic skeletal diseases, they
have an overall prevalence of at least 1 per 4000 children, which extrapolates to
a minimum of 225,000 people in the European Union, and this extensive burden
in pain and disability leads to poor quality of life and high healthcare costs.
Dominant-negative (qualitative) defects in numerous cartilage structural pro-
teins result in a broad range of GSDs, and this chapter will focus on a disease
spectrum resulting from mutations in the glycoproteins, cartilage oligomeric
matrix protein (COMP), type IX collagen and matrilin-3, which together cause a
continuum of phenotypes that are amongst the most common of the autosomal
dominant GSDs.
Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal
dysplasia (MED) define a disease spectrum typified by varying degrees of short-
limbed dwarfism, joint pain with stiffness and early-onset osteoarthritis (OA). The
generation and deep phenotyping of a range of genetic cell and mouse models of
the PSACH and MED disease spectrum has allowed the disease mechanisms to be
characterised in detail. Furthermore, the generation of novel phenocopies to model
specific disease mechanisms has confirmed the importance of endoplasmic reticu-
lum stress, reduced chondrocyte proliferation and increased dysregulated apopto-
sis as key indicators of growth plate dysplasia and eventually reduced bone growth.
Lastly, new insight into disease-related musculoskeletal complications such as
myopathy, ligamentous laxity and tendinopathy has been gained through the analy-
sis of mouse models of the PSACH and MED disease spectrum.
M.D. Briggs (*) • P. Bell • K.A. Piróg

Institute of Genetic Medicine, Newcastle University, International Centre for Life,
Central Parkway, Newcastle upon Tyne NE1 3BZ, UK
e-mail: michael.briggs@newcastle.ac.uk; Peter.Bell3@newcastle.ac.uk;
Katarzyna.Pirog@newcastle.ac.uk

136 M.D. Briggs et al.
7.1 Endochondral Ossification and the Skeletal Dysplasias
All long bones in the skeleton grow by endochondral ossification (EO), a com-
plex process in which chondrocytes differentiate forming a growth plate (GP),
which subsequently results in a remodelling and mineralisation of the cartilage
anlagen (Karsenty 2003; Provot and Schipani 2005). Chondrocytes within the
GP undergo a coordinated and highly regulated process of cell proliferation,
maturation and hypertrophy, at which stage osteoblasts begin to replace the car-
tilage matrix with the trabecular bone (Fig. 7.1) (Ortega et al. 2004; Buckwalter
et al. 1986). At each stage of this process, the chondrocytes synthesise and secrete
specific proteins that are incorporated into the ECM extracellular matrix biology.
Chondrocyte proliferation and hypertrophy in the growth plate are vital for cor-
rect long bone growth, whilst apoptosis of terminal hypertrophic chondrocytes
plays a critical role in the transition from chondrogenesis to osteogenesis (Cowell
et al. 1987). Reduced chondrocyte proliferation, decreased hypertrophy and
increased/dysregulated apoptosis are therefore likely to have a profound effect
on EO and the rate of long bone growth.
Disruption to EO leads to delayed and irregular bone formation and results in
a heterogeneous group of diseases known as the skeletal dysplasias (Superti-
Furga et al. 2001). There are over 300 unique skeletal dysplasia phenotypes,
which range in severity from mild to lethal conditions and have an overall preva-
lence of at least 1/4000 (International nomenclature and classification of the
osteochondrodysplasias (1997). International Working Group on Constitutional
Diseases of Bone 1998). Many of these individual phenotypes have been grouped
Resting Zone
Proliferative Zone
Pre-hypertrophic Zone
Hypertrophic Zone
Vascular Invasion Front
Fig. 7.1 Hip radiograph of a child with pseudoachondroplasia (left) demonstrating a small and
irregular secondary centre of ossification (epiphyses) and a 3-week-old mouse growth plate high-
lighting the individual zones
7 Pseudoachondroplasia and Multiple Epiphyseal Dysplasia: Molecular Genetics 137
into ‘bone dysplasia families’ on the basis of similar clinical and radiographic
features (Spranger 1988) and are postulated to share common disease mecha-
nisms. One such bone dysplasia family is the pseudoachondroplasia (PSACH)
and multiple epiphyseal dysplasia (MED) group of diseases, which are a collec-
tion of various phenotypes that are characterised by varying degrees of dispro-
portionate short stature resulting from epimetaphyseal dysplasia (Briggs and
Chapman 2002; Bateman et al. 2005).
7.2 he Pseudoachondroplasia and Multiple Epiphyseal

T
Dysplasia Disease Group
PSACH and MED are members of the same bone dysplasia family and were pos-
tulated to share a common disease pathophysiology based on clinical and radio-
graphic similarities (Spranger 1988; Briggs and Chapman 2002). This family
comprises a broad spectrum of clinically related diseases that manifest with vary-
ing degrees of short stature, joint laxity and early-onset osteoarthritis (Table 7.1)
(Fairbank 1947; Barrie et al. 1958; Rimoin et al. 1994). In addition, there are allelic
conditions and/or phenotypic outliers of the well-established PSACH and MED
disease spectrum that share some common clinical, radiographic and genetic char-
acteristics. These diseases include recessive spondyloepimetaphyseal dysplasia,
matrilin-3 related (rSEMD, matrilin-3), pseudo-pseudoachondroplasia (pPSACH),
Meyers dysplasia, Beukes hip dysplasia (BHD), bilateral Perthes disease (BPD)
and dominant osteochondritis dissecans (OCD) (Table 7.1). In addition, mutations
in aggrecan can cause phenotypes such as dominant osteochondritis dissecans
(with short stature and early-onset osteoarthritis) and idiopathic short stature (with
advanced bone age early-onset osteoarthritis) which may be reminiscent of mild
MED (Gibson and Briggs in press). Indeed, the carrier of a recessive ACAN muta-
tion in a single family with recessive spondyloepimetaphyseal dysplasia aggrecan
related (rSEMD, aggrecan) shows some clinical evidence of a mild MED-like phe-
notype (Tompson et al. 2009).
7.3 seudoachondroplasia and Multiple Epiphyseal

P
Dysplasia (PSACH-MED): Known Disease Genes
and Their Relative Frequency
PSACH results exclusively from mutations in the gene-encoding cartilage

oligomeric matrix protein (COMP) (Briggs et al. 1995; Hecht et al. 1995).
Although several studies have failed to identify COMP mutations in a small
proportion of clinically confirmed cases of PSACH, no alternative gene has yet
been identified (Jackson et al. 2012). In addition, a form of pseudo-pseudoa-
chondroplasia has been described that does not result from a COMP mutation,
but the causative molecular defect also remains unresolved in this single family
(Spranger et al. 2005).
Table 7.1 Phenotypes comprising the pseudoachondroplasia and multiple epiphyseal dysplasia
disease spectrum, allelic disorders and phenotypic outliers
Genetic
Gene Protein Phenotype symbol OMIN ORPHA
COMP Cartilage PSACH PSACH 177170 ORPHA750
oligomeric MED EDM1 132400 ORPHA93308
matrix
protein
Unknown Not known pPSACH
MATN3 Matrilin-3 Epiphyseal EDM5 607078 ORPHA93311
dysplasia, multiple, (BHMD)
5 (microepiphyseal
dysplasia, bilateral
hereditary)
rSEMD matrilin-3 608728 ORPHA156728
related
OA susceptibility 2 OS2 140600
COL9A1 Type IX Epiphyseal dysplasia, EDM6 614135 ORPHA166002
collagen multiple, 6
COL9A2 Epiphyseal dysplasia, EDM2 600204 ORPHA166002
multiple, 2
COL9A3 Epiphyseal EDM3 600969 ORPHA166002
dysplasia, multiple,
3 (with myopathy
included)
SLC26A2 Solute Epiphyseal dysplasia, EDM4 226900 ORPHA93307
carrier multiple, 4
family 26
(anion
exchanger),
Member 2
COL2A1 Type II MED variants that ORPHA166011
collagen have clinical and ORPHA2380
radiographic overlap
ORPHA93279
with SED
congenital, ORPHA86820
including EDMMD
ACAN Aggrecan dOCD OD 165800 ORPHA251262
rSEMD, aggrecan 612813 ORPHA171866
type
Short stature- n/a ORPHA435804
advanced bone
age-early-onset
osteoarthritis
syndrome
SED Kimberley type SEDK 608361 ORPHA93283

Genetic
Gene Protein Phenotype symbol OMIN ORPHA
UFSP2 Ubiquitin- Beukes familial hip BHD 142669 ORPHA2114
fold dysplasia
modifier-1
Each of the confirmed members of the PSACH–MED disease spectrum (PSACH; EMD1-6) is
shown along with other phenotypes that are either allelic or share some phenotypic similarities.
When available the genetic symbol, On-line Mendelian Inheritance in Man (OMIM: http://www.
omim.org) and Orphanet (ORPHA: http://www.orpha.net) reference numbers are shown
Key: PSACH pseudoachondroplasia, MED multiple epiphyseal dysplasia, pPSACH pseudo-
pseudoachondroplasia, rSEMD recessive spondyloepimetaphyseal dysplasia, OA osteoarthritis,
SED spondyloepiphyseal dysplasia, EDMMD epiphyseal dysplasia, multiple with myopia and
conductive deafness, dOCD dominant osteochondritis dissecans, BHMD bilateral hereditary
microepiphyseal dysplasia, BHD Beukes familial hip dysplasia
Autosomal dominant MED is a genetically heterogeneous disease, and autoso-

mal dominant forms can result from mutations in the gene-encoding COMP, matri-
lin-3 (MATN3) (Chapman et al. 2001) and type IX collagen (COL9A1, COL9A2 and
COL9A3) (Muragaki et al. 1996; Czarny-Ratajczak et al. 2001; Bonnemann et al.
2000). More recently, mutations in exon 50 of COL2A1 have been identified in two
families with a MED-like phenotype (Jackson et al. 2012). Finally, a recessive form
of MED can result from mutations in the 26A2 sulphate transporter (SLC26A2)
(Rossi and Superti-Furga 2001). Regardless of the individual genes, the majority of
MED mutations cluster in distinct regions and usually affect conserved residues that
are important for the structure and/or function of the relevant proteins (Briggs and
Chapman 2002).
Taken together mutations in the six known genes can account for between 50 and
70 % of MED depending on ascertainment criteria (Table 7.1) (Itoh et al. 2006;
Zankl et al. 2007; Newton et al. 1994; Jackson et al. 2012). These data suggest that
additional MED-disease loci exist, but their identity has not been determined to
date, and mutations in these unknown genes do not account for the majority of MED
cases as previously suggested (Jakkula et al. 2005). It is interesting to speculate
which other genes might be associated with the development of MED; for example,
one study has excluded matrilin-1 mutations as a cause of MED (Jackson et al.
2012). However, it remains possible that mutations in other structural proteins
expressed in the cartilage growth plate can cause MED, but their identity remains
elusive. The use of next-generation sequencing on small families and cohorts of
patients, who do not have mutations in the known genes, will provide a panel of
novel candidate variants for functional validation.
The clinical presentation of MED caused by type IX collagen gene mutations can
also be associated with osteochondritis dissecans (OCD) and mild myopathy
(Jackson et al. 2010). However, like PSACH and MED caused by COMP mutations,
the myopathy does not appear to be a consistent feature and is poorly documented.
Furthermore, the lack of suitable tissue obtained through surgery has limited our
insight into these secondary consequences of type IX collagen and COMP gene
mutations. The study of relevant animal models of PSACH and MED has provided
new insight into these additional musculoskeletal complications of PSACH and
MED and is discussed elsewhere in this chapter.
7.4 SACH and MED–COMP Related (EDM1: Genetic

P
and Cell-Based Studies
COMP is the fifth member of the thrombospondin (TSP) protein family and is a modu-
lar protein that comprises a coiled-coil oligomerisation domain, four TSP type II
repeats (TSP2), eight TSP type III repeats (TSP3) and a large C-terminal globular
domain (CTD) (Newton et al. 1994). The precise role of COMP in the cartilage ECM
has not been fully determined, but it can interact in vitro with matrilin-3, type II and IX
collagen molecules via the CTD (Rosenberg et al. 1998; Holden et al. 2001; Thur et al.
2001; Mann et al. 2004). COMP has been shown to act as a catalyst in collagen fibril-
logenesis (Halasz et al. 2007) and binds to chondrocytes via interactions with integrins,
suggesting that it also plays a role in cell-matrix signalling (Chen et al. 2005).
Of the many different COMP mutations identified to date, the majority (85 %)
are found within exons 8–14, which encode the Ca2+-binding TSP3 of COMP; how-
ever, key mutations (~15 %) have also been identified within the CTD (Briggs et al.
2014) that appear to cluster in distinct regions. Approximately 50 % of the TSP3
mutations are small in-frame deletions or duplications, whilst the common
p.Asp473del mutation accounts for ~30 % of all PSACH (Briggs et al. 2014).
Over the last 20 years, the effect of COMP mutations on chondrocyte function and
ECM assembly has been studied using three different cell model systems. These have
been (a) a limited microscopic analysis of patient cartilage obtained from iliac crest
biopsy (Hecht et al. 2001; Hecht et al. 2004; Cotterill et al. 2005), (b) the redifferentia-
tion and culture of patient chondrocytes in vitro (Hecht et al. 1998, 2001, 2005) and
(c) the use of cell culture systems employing a variety of chondrocyte and non-chon-
drocyte cells (Chen et al. 2004; Hashimoto et al. 2003; Cotterill et al. 2005; Otten
et al. 2005). These various studies have consistently demonstrated that mutant COMP
harbouring type III mutations is retained within the rough endoplasmic reticulum
(rER) of chondrocytes along with other ECM proteins such as type IX collagen and
matrilin-3 (Hecht et al. 2005). The retained mutant COMP is also associated with a
number of chaperone proteins that are involved in either protein folding or the
unfolded protein response (UPR) (Hecht et al. 2001). Furthermore, several studies
have demonstrated that the retention of mutant COMP eventually results in increased
cell death (Hecht et al. 2004; Hashimoto et al. 2003).
Interestingly, in contrast to the type III mutations, the expression of COMP har-
bouring a CTD mutation (Thr587Arg) has shown that this mutant protein is effi-
ciently secreted in vitro and therefore likely to exert a dominant-negative effect
within the ECM (Schmitz et al. 2006; Spitznagel et al. 2004). However, very little is
known to date about the precise effect of these mutant COMP molecules on colla-
gen fibrillogenesis, ECM assembly and cell-matrix signalling.
7.5 ED–MATN3 Related (EDM5): Genetic and Cell-Based

M
Studies
Matrilin-3 is the third member of a family of four ECM proteins; matrilin-1 and
matrilin-3 are primarily expressed in the cartilage, whilst matrilin-2 and matrilin-4
have a wider pattern of expression in a variety of ECM including nonskeletal tissues
(Wagener et al. 2005). Matrilin-3 consists of a single von Willebrand factor A-like
domain (A-domain), four EGF-like motifs and a coiled-coil oligomerisation domain
(Wagener et al. 1997). Matrilin-3 can form hetero-oligomers with matrilin-1 (Zhang
and Chen 2000; Wu and Eyre 1998) and has been shown to bind to COMP and type
II and type IX collagen in vitro (Budde et al. 2005; Mann et al. 2004) (for further
details see Chap. 3 in volume 1 (Zaucke 2016)).
All of the MED mutations identified in MATN3 are missense mutations, which
primarily affect conserved residues that comprise the single A-domain of matrilin-3.
Of the numerous MATN3 mutations reported to date, most affect residues in the
β-strands that comprise the single β-sheet (Jackson et al. 2004; Cotterill et al. 2005;
Mabuchi et al. 2004; Mostert et al. 2003; Maeda et al. 2005). The exceptions to this
rule are several missense mutations located in the α-helical regions of the A-domain
(Maeda et al. 2005; Mabuchi et al. 2004; Fresquet 2007a). Overall, these data confirm
that mutations, which affect important and/or conserved residues in both the α-helices
and β-sheets of the matrilin-3 A-domain, can cause MED (Jackson et al. 2012).
The effect that A-domain mutations have on the folding and trafficking of matrilin-
3 has been studied in vitro using surrogate cell models. Mutant matrilin-3 is retained
within the rER of cells (Otten et al. 2005; Cotterill et al. 2005), where it exists as an
unfolded intermediate and is associated with ERp72, a chaperone protein known to be
involved in mediating disulphide bond formation (Cotterill et al. 2005). In contrast,
the effect of α-helix mutations on the folding and trafficking of matrilin-3 and ulti-
mately disease mechanism(s) has not been fully determined. An in vitro expression
system, previously used to characterise β-sheet mutations (Cotterill et al. 2005), was
used to study the effect of α-helical mutations on the structure and function of mutant
A-domains (Fresquet 2007a). Interestingly, two of the three α-helical mutations
(p.F105S and pA173D) prevented the secretion of mutant A-domain in vitro, whilst
p.K231N did not prevent the secretion of the matrilin-3 A-domain, nor did it disrupt
the structure of this domain or inhibit its binding to type II or type IX collagen
(Fresquet 2007a). Therefore, despite extensive biochemical analysis, the disease
mechanism of several MATN3 variants remains unresolved.
In addition to the MED mutations in MATN3, two mutations have been identified
in the first EGF-like repeat of matrilin-3 that result in either a recessive form of spon-
dyloepiphyseal dysplasia, matrilin-3 related (rSEMD: Cys304Ser) (Borochowitz
et al. 2004), or confer susceptibility to osteoarthritis (OA: Thr303Met) (Pullig et al.
2007; Min et al. 2006; Stefansson et al. 2003). The biochemical effect of the EGF-
like mutations has been studied in vitro, and whilst the Cys304Ser mutation results
in the retention of some misfolded mutant protein, the Thr303Met mutation allows
the mutant protein to be efficiently secreted and participate in the formation of fila-
mentous networks surrounding the cells (Otten et al. 2005). However, the structural
and functional consequence of this mutated form of matrilin-3 in the ECM has not
been determined, and it is critical therefore to understand these effects in order to
provide insight into its role in OA.
7.6 ED: Type IX Collagen Related (EDM2, EDM3

M
and EDM4): Genetic and Cell-Based Studies
Type IX collagen gene mutations are perhaps the most remarkable of all the MED
mutations reported to date because all but one cause splicing defects that have a
very restricted distribution. These mutations are all postulated to cause in-frame
deletions from an equivalent region of the COL3 domain in the α1(IX), α2(IX) or
α3(IX) chains (Fig. 7.2) (Briggs and Chapman 2002). The one reported exception is
c.104G>A in exon 2 of COL9A3 that causes Gly35Asp in α3(IX) and was identified
in an affected father and son with typical MED.
It is interesting to note that of all the different forms of MED, the least is known about
the disease mechanisms of MED caused by type IX collagen gene mutations. For exam-
ple, it has not been resolved as to whether mutant type IX collagen is retained within the
rER (Bonnemann et al. 2000) or secreted into the ECM (Spayde et al. 2000). Furthermore,
the precise grouping of type IX collagen gene mutations has yet to be fully explained;
however it is possible that these deletions may disrupt important interactions with other
components of the ECM. Indeed it has been shown that the A-domain of matrilin-3
COL9A1 +3
Ex8 C C G G T A A G T ......In8
NC4
NC3 COL2 NC2 COL1 NC1
COL3 α1(IX)
α2(IX)
α3(IX)
−2 −1 −1 +2 +5+6
In2...... T T T C A G Ex3 Ex3 C C G G T G A G T ......In3
↓↓ ↓ ↓ ↓ ↓
TA A C CG
COL9A3 C COL9A2
Fig. 7.2 Schematic of a type IX collagen molecule showing the exon skipping mutations found
in the COL9A1, COL9A2 and COL9A3 genes that cause multiple epiphyseal dysplasia. All exon
skipping results in the in-frame deletion of amino acid residues from the equivalent region of the
COL3 domain. Key: NC non-collagenous, COL collagenous, Ex exon, In intron
interacts with the COL3 domain of type IX collagen and that this interaction is abolished
when type IX collagen harbours an MED mutation (Fresquet 2007b). These studies
have provided the first biochemical insight into why MED mutations might cluster in
the COL3 domain of type IX collagen and provide some rationale for the non-allelic
genetic heterogeneity of these forms of MED (EDM2, EDM3, EDM4). The Gly35Asp
substitution affects the third Gly-Xxx-Yyy repeat of the α3(IX) chain, which is situated
amongst a region of contiguous Gly-Pro-Pro triplets suggesting that this area may be
important for the stability of the collagen IX triple helix.
7.7 etermining the Mechanistic Link Between a Mutant

D
Gene Product and Disturbed Endochondral Ossification
Through the Use of Targeted Mouse Models of PSACH
and MED
It is understandable that in vitro studies have been unable to determine exactly how
the expression of a mutant gene product results in disturbed endochondral ossifica-
tion and reduced bone growth in vivo. Unfortunately, the use of knockout technology
has also proven to be unsuitable since both the Comp and Matn3 knockout mice have
no overt skeletal phenotype (Ko et al. 2004; Svensson et al. 2002). Therefore, to
determine the disease mechanisms that underpin the pathophysiology of PSACH–
MED in vivo, homologous recombination and Cre-lox technology were used to gen-
erate a series of mouse lines that genetically modelled phenotypes within the full
PSACH and MED disease spectrum. These mice provided a valuable resource for
describing key pathological findings, identifying and studying disease mechanisms
in detail and as preclinical models for the validation of therapeutic targets.
7.7.1 evere PSACH Resulting from Asp469del in the Type III

S
Repeat Region of COMP
A knock-in mouse model of typical severe PSACH was generated with the
Asp469del mutation in the TBSP3 repeat region of COMP (Table 7.2). Mutant ani-
mals were normal at birth but grow slower than their wild-type littermates, and by
9 weeks of age, they had short-limbed dwarfism. In the growth plates of mutant
mice, the chondrocyte columns were sparse and poorly organised, whilst mutant
COMP was retained within the rER of cells and elicited a novel cell stress response.
Ultimately chondrocyte proliferation was significantly reduced, whilst apoptosis
was both increased and spatially dysregulated in the mutant growth plates.
7.7.2 ild PSACH/MED Resulting from a Thr585Met Missense

M
Mutation in the CTD of COMP
A knock-in mouse model of mild PSACH was generated with the Thr585Met in the
C-terminal domain of COMP (Table 7.2) (Pirog-Garcia et al. 2007). Mutant animals
were normal at birth but grow slower than their wild-type littermates and developed
Table 7.2 Mouse models of the PSACH–MED disease spectrum and novel phenocopies to model
ER in the cartilage growth plate
Experimental approach
Disease Gene Mutation taken Promoter
PSACH COMP Asp469del Homologous recombination Native
(knock in) mouse
PSACH-MED COMP Thr585Met Homologous recombination Native
(knock in) mouse
MED MATN3 Val194Asp Homologous recombination Native
(knock in) mouse
Chondrodysplasia TG Rdw (G2320R) Transgenic overexpressing Col2a1
phenocopy
Chondrodysplasia TG Cog (L2293P) Transgenic overexpressing Col2a1
phenocopy
Key: COMP cartilage oligomeric matrix protein, MATN3 matrilin-3, PSACH pseudoachondropla-
sia, MED multiple epiphyseal dysplasia, Tg thyroglobulin, Rdw congenital dwarfism mutation,
Cog congenital goitre mutation
mild short-limbed dwarfism (Pirog-Garcia et al. 2007). In the growth plates of

mutant mice, the chondrocyte columns were sparse and poorly organised. Mutant
COMP was secreted into the extracellular matrix, but its localisation was disrupted
along with the distribution of several COMP-binding proteins (Pirog-Garcia et al.
2007). Although mutant COMP was not retained within the ER, there was a mild
UPR/cell stress response, and chondrocyte proliferation was significantly reduced,
whilst apoptosis was both increased and spatially dysregulated (Pirog-Garcia et al.
2007). Overall, these data suggest a mutation in the CTD of COMP which exerts a
dominant-negative effect on both intra- and extracellular processes.
7.7.3 ED Resulting from a Val194Asp Missense Mutation

M
in the A-domain of Matrilin-3
A knock-in mouse model with a Val194Asp mutation in the β-sheet of the A-domain of
matrilin-3 was generated by homologous recombination (Table 7.2). Mice that are homo-
zygous for the mutation developed a progressive growth plate dysplasia and short-limbed
dwarfism that was consistent in severity with the relevant human phenotype (Leighton
et al. 2007). Mutant matrilin-3 was retained within the ER of chondrocytes (Figs. 7.3 and
7.4) and associated with an UPR characterised by the upregulation of classical early
markers of the UPR such as binding immunoglobulin protein (BiP) and calreticulin
(Leighton et al. 2007). Eventually, there was reduced proliferation and spatially dysregu-
lated apoptosis of chondrocytes in the cartilage growth plate (Leighton et al. 2007).
Fig. 7.4 Electron micrograph showing enlarged ER in mutant chondrocytes (a) compared to wild
type (b). Scale bars represent 8 μm
Fig. 7.3 The organisation

of the growth plate in a
V194D Matn3 mutant
mouse (bottom) is
disrupted by 3 weeks of
age and shows
mislocalisation of
matrilin-3 compared to
wild type (top). Mutant
matrilin-3 (brown staining)
is retained within the cell
and not secreted into the
extracellular matrix
7.8 ommon Disease Mechanisms in the PSACH and MED

C
Disease Spectrum
Studies to date had demonstrated that whilst there are clear differences in the traf-
ficking of mutant forms of COMP and matrilin-3, the expression of all three mutant
proteins resulted in ER stress that had downstream consequences in terms of chon-
drocyte proliferation and apoptosis (Table 7.3) (Briggs et al. 2015a, b). It had been
hypothesised that reduced proliferation and increased spatially dysregulated apop-
tosis of chondrocytes was the most likely cause of disrupted linear bone growth and
could therefore be defined as ‘core disease mechanisms’ in the PSACH and MED
bone dysplasia family.
Therefore, to further delineate the relative contributions of (1) mutant protein
retention vs. disturbed cartilage ECM and (2) reduced chondrocyte proliferation
vs. increased apoptosis, to growth plate dysplasia and reduced bone growth, a
novel transgenic approach was adopted. In these experiments two different forms
of mutant thyroglobulin (Tg) were expressed in the cartilage under the Col2a1
promoter (Table 7.3). Tg is a 660-kDa homodimeric protein produced exclusively
by the thyroid gland and is the major secretory glycoprotein produced by thyro-
cytes. Cog (congenital goitre mutation) and Rdw (congenital dwarfism) are natu-
rally occurring thyroglobulin mutations in mouse and rat, respectively. The Cog
mutation causes ER stress in thyrocytes, whilst Rdw leads to increased cell death.
Expression of both of these mutant proteins in the cartilage produces a chondro-
dysplasia phenotype resulting from ER stress that was independent of an endog-
enous cartilage protein Gualeni 2013 and Kung 2015. Significantly, neither
mutation caused increased chondrocyte apoptosis in the growth plate, but both
Table 7.3 Key pathological and quantitative measures of disease severity in various knock-in
mouse models of PSACH–MED and novel ER stress phenocopies
Mutant
Age of Cell protein ER Stress pathway
Mouse model onset proliferation Apoptosis retention stress activated
CompD469del ~6 weeks Decreased Increased and Yes Yes Novel (APR/
spatially EOR)
dysregulated
CompT585M ~9 weeks Decreased Increased and Slight Mild Mild UPR
spatially
dysregulated
Matn3V194D ~2 weeks Decreased Increased and Yes Yes UPR
spatially
dysregulated
Col2-TgRdw At birth Decreased No Yes Yes Novel (APR/
differences EOR)
Col2-Tgcog ~3 weeks Decreased No Yes Yes UPR
differences
EOR ER overload response, APR aggregated protein response, UPR unfolded protein response
mutations did result in a significant reduction in chondrocyte proliferation,

thereby confirming this as the likely mechanism of reduced long bone growth
(Table 7.3).
7.9 he Induction of Genotype-Specific Stress Pathways

T
in Growth Plate Chondrocytes
The retention and/or delayed processing and trafficking of mutant structural pro-
teins causes ER stress in PSACH and MED cell and mouse models that has been
well documented. In addition to the accumulation of misfolded mutant proteins, ER
stress can also be induced by a variety of causes including viral infection, chronic
inflammation, hypoxia and ischemia.
Although mutant forms of COMP, matrilin-3 and thyroglobulin all induce ER
stress, the specific downstream genetic pathways that are activated appear to be
genotype specific (Table 7.3). For example, the expression of the Val194Asp
mutant form of matrilin-3 induces classical UPR characterised by the upregulation
of BiP (a sentinel marker of UPR) and a plethora of other chaperones, foldases and
protein disulphide isomerases (Leighton et al. 2007). To a certain extent, the
Thr585Met mutant form of COMP also induces mild UPR due to the delayed
secretion of the mutant protein. In contrast, the Asp469del mutant form of COMP
induces novel stress pathways that are characterised by changes in the expression
of genes implicated in oxidative stress, cell cycle regulation and apoptosis (Suleman
et al. 2012). This disease signature is similar to other UPR-independent pathways
that are activated by the aggregation of mutant proteins as a result of toxic gain of
function. These novel stress pathways have been termed either an aggregated pro-
tein response (APR) or an ER overload response (EOR). For example, the serpi-
nopathies are due to the aggregation of insoluble mutant α1-antitrypsin that triggers
EOR, which is independent of the UPR and involves activation of nuclear factor-
kB signalling. Interestingly, the two different mutant forms of thyroglobulin (Cog
and Rdw) induced different stress pathways when expressed in chondrocytes
in vivo. The Cog mutation induced a canonical UPR characterised by increased
BiP expression, phosphorylation of eIF2α and splicing of Xbp1 (Kung et al. 2015).
In contrast, the Rdw mutation did not induce transcriptional activation of a classi-
cal UPR but a downregulation in the expression of genes involved in cell prolifera-
tion (Gualeni et al. 2013).
7.10 Genetic Modifiers of Disease Severity
Patients with PSACH and MED present with a high degree of inter- and/or intra-
familial variability with regard to disease severity and phenotypic features. Various
studies have suggested that this variability is most likely due to the influence of
genetic modifiers that can influence the penetrance, expressivity and severity of the
primary disease-causing mutation. These modifiers might directly influence the
structure and/or function of the mutant gene product or act to modify the cell’s
response to its expression. For example, a non-synonymous polymorphism in
MATN3 found in a family with MED, although in itself not disease causing, may act
as a modifier of disease severity. The affected individuals, described by Jackson and
colleagues (Jackson et al. 2004), carried a disease mutation but with an additional
change in the matrilin-3 (V245M) that was subsequently shown to influence the
trafficking of matrilin-3 in vitro.
Genetic modifiers of PSACH and MED severity might also affect the cell’s
response to the expression of mutant protein. For example, increasing the relative
proportion of a C57BL6/J background in the COMP Thr585Met mouse model
resulted in a more severe chondrodysplasia phenotype (Pirog et al. 2014). Studies in
mouse models of other genetic conditions such as Treacher Collins syndrome
(TCS), Pelizaeus–Merzbacher disease (PMD) and spastic paraplegia type 2 (SPG2)
have shown that the C57BL6/J background in particular has a heightened reaction
to a misfolded protein stimulus, specifically showing increased levels of pro-
apoptotic protein CHOP. Indeed, abnormal chondrocyte apoptosis in the growth
plates was further increased in the Thr585Met COMP on an enriched C57BL6/J
background (Pirog et al. 2014).
Understanding further the role of genetic modifiers in PSACH and MED patho-
biology is of utmost importance for the discovery and tailoring of future therapies.
Recent developments in genomic technologies as well as advancements in systems
biology will enable the discovery of these modifiers in the future.
7.11 Soft Tissue Complications in PSACH/MED
Matrilin-3 is expressed almost exclusively in the cartilage; however, COMP and

type IX collagen are also present at significant levels in the tendon, ligament and
skeletal muscle. It is therefore not surprising that a subset of PSACH and MED
patients presents with a variety of musculoskeletal complications such as mild
myopathy and joint laxity. Ligamentous abnormalities associated with PSACH and
MED disease spectrum include differences in collagen fibre diameters and mutant
protein retention within the tenocytes of affected individuals.
However, the mild myopathy proved more difficult to elucidate, with variable
phenotypic presentation and sporadic recording of the clinical case reports. The in-
depth investigation of the knock-in mouse models of the PSACH and MED disease
spectrum (T585M COMP, D469del COMP and V194D Matn3) finally revealed that
mild myopathy associated with these disease spectra derives from structural abnor-
malities in fibrous tissues at the myotendinous and perimysial junctions, which led
to changes in biomechanical properties of these tissues. This correlation was
observed for both COMP genotypes (T585M and D469del) but not for the V194D
Matn3 model MED. This finding possibly explains the milder clinical manifestation
of MED caused by MATN3 mutations and provides important information for the
clinical management.
7.12 Conclusions and Future Perspectives
Extensive studies on both the knock-in PSACH and MED mouse models and trans-
genic ‘ER stress phenocopies’ confirmed that ER stress in growth plate chondro-
cytes, induced by the retention and/or processing of mutant proteins, results in
decreased chondrocyte proliferation and increased apoptosis in the growth plate that
ultimately causes short-limbed dwarfism. Therefore, a therapeutic approach to tar-
get the ER stress is an attractive proposal that is independent of genome editing and
manipulation of the cartilage ECM. Such chemical chaperones are already in use for
some rER stress-related conditions such as type II diabetes and cystic fibrosis and
may provide a suitable treatment for a broad range of human connective tissue
diseases.
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Integrin-Mediated Interactions
in Cartilage Physiology 8
and Pathophysiology
Attila Aszódi
Abstract
The development and homeostasis of cartilaginous tissues are regulated by
diverse microenvironmental cues including integrin-mediated interactions
between chondrocytes and the extracellular matrix (ECM). Integrins are mem-
brane receptors responsible for bi-directional communication between the cells
and the surrounding by transmitting physicochemical signals through adhesion
complexes. In addition, integrins are involved in sensing mechanical stress sig-
nals generated by the ECM and transduce them into the cell interior converting
physical stimuli to biochemical signaling. Integrin-activated signaling cascades
modulate various chondrocyte functions and play important roles in cartilage
morphogenesis, homeostasis, and repair. This chapter will summarize and dis-
cuss the role of integrins in the physiology and pathophysiology of the growth
plate and articular cartilage.
8.1 Introduction
In vertebrates, the skeletal system derives from mesodermal progenitors through a

complex morphogenetic process laying down bone, cartilage, and ligaments/ten-
dons, components of the skeleton which all together provide body support and
enable movement. Endochondral bones of the axial and appendicular skeleton
develop through cartilaginous intermediates which intensively grow until the end of
puberty owing to the coordinated proliferation and differentiation activities of chon-
drocytes residing in a specialized structure called growth plate. During
A. Aszódi, PhD
Laboratory of Experimental Surgery and Regenerative Medicine, Clinic for General, Trauma
and Reconstruction Surgery, Ludwig-Maximilians-University,
Nussbaumstrasse 20, D-80336 Munich, Germany
e-mail: attila.aszodi@med.uni-muenchen.de

156 A. Aszódi
morphogenesis, the transient cartilaginous templates are gradually replaced by

bone, except at the articulating surfaces where cartilage exists permanently as artic-
ular cartilage that enables load distribution and frictionless movements of synovial
joints. Skeletal tissues are adopted for specific mechanical demands through distinct
organization of their hierarchical composite material consisting of a network of col-
lagen fibrils embedded into a heterogeneous mixture of extracellular matrix (ECM)
molecules. In the hyaline cartilage, the fibrillar meshwork composed of mixed
fibrils of collagen types II, IX, and XI provides tensile strength, whereas the osmotic
swelling pressure generated by the hydrated proteoglycans resists compressive
forces. The composition of cartilaginous matrices is reflected in the synthetic activi-
ties of chondrocytes and is maintained with the help of carefully balanced anabolic
and catabolic processes. Changes in ECM structure, composition, and chondrocyte
metabolic activities, either in the growth plate or in the articular cartilage, caused by
aging, environmental, or genetic factors, could lead to cartilage-associated skeletal
pathologies such as chondrodysplasias and osteoarthritis (for further details, see
Chaps. 1, 6, and 7). Injuries are another challenge of the cartilaginous tissues (see
also Chap. 2), especially in the articular cartilage which has a very limited intrinsic
healing capacity. Differentiation, function, and repair of cartilaginous tissues cru-
cially depend on the continuous interactions between cells and the immediate peri-
cellular compartment of the ECM. Integrins are the major class of adhesion and
signaling receptors on the surface of chondrocytes, and their role for numerous
aspects of skeletal tissue properties is increasingly recognized. In this chapter, we
will focus on integrin expression and function in the cartilaginous growth plate and
the articular surface and emphasize the importance of integrin signaling in response
to chemical and mechanical stress signals.
8.2 Integrins
Integrins are heterodimeric, type I transmembrane glycoproteins composed of non-

covalently associated α and β subunits (Legate et al. 2009). Each subunits contains
a large extracellular domain, a single transmebrane α helix, and a usually short
cytoplasmic domain (Wegener and Campbell 2008). In mammals, 18 α and 8 β
subunits have been identified, which can combine into 24 different heterodimers
with specific or partially overlapping binding capacity to ECM components (Fig.
8.1a). Integrins can be classified into four main categories based on the molecular
properties of integrin-ligand interactions (Humphries et al. 2006; Campbell and
Humphries 2011). The subunits α5, α8, αv, and αIIb in combination with the β1 or
β3 subunits interact with the arginine-glycine-aspartate (RGD) tripeptide motif
found in a large number of ECM molecules including fibronectin, tenascin, and
vitronectin. Eight heterodimers including the four members of the β2 subfamily
bind to an RGD-related LDV or LDV-homologous site. Four α integrins (α1, α2,
α10, and 11), which dimerize with the β1 subunit, are collagen-binding receptors
carrying an “inserted” or “αI domain.” This inserted domain, which is present in five
8 Integrin-Mediated Interactions in Cartilage Physiology and Pathophysiology 157
Lam
a RGD b Col
αIIb α1Col β1
β5 β6 Fn β2
α2Col
β3 β8 α4Fn α β β4
α5Fn β5
α5 αV α8 α6Lam
α11 α10Col
α3 Laminin
Collagen
α10 αvFn
α2 α6 β4
β1 extracellular
α1 α7
α7 α4 LDV intracellular
focal adhesion
β7 actin
αE integrin signaling cascades
αL αD
proliferation survival metabolism migration
β2
differentiation polarity
αX αM chondrocyte response
Fig. 8.1 Integrins (a) In mammals, 18 α and 8 β subunits can combine into 24 heterodimers.
Based on the predominant binding specificity, integrin-ligand interactions are classified into four
main classes. (b) Integrin subunits expressed on normal and osteoarthritic (in red) chondrocytes.
Chondrocyte integrins interact with the major proteins of the cartilage extracellular matrix (ECM)
including type II collagen (Col), fibronectin (Fn) and laminin (Lam). Integrins link the ECM to the
actin cytoskeleton and control chondrocyte behavior through signaling cascades initiated at the
focal adhesion sites
additional α subunits, carries metal ion-dependent adhesive sites essential for diva-
lent cation-dependent ligand binding and shows homology to von Willebrand factor
A domains. Finally, integrins α3β1, α6β1, α7β1, and α6β4 are selective laminin-
binding heterodimers without αI-domain.
Following a series of intracellular events referred as inside-out signaling, integ-
rins are activated and able to bind ECM molecules. Integrin-ligand interactions
mediate cell adhesion to the ECM and are pivotal for development, tissue remodel-
ing, tissue repair processes, host defense, and hemostasis. Integrins link the extra-
cellular microenvironment to the intracellular actin cytoskeleton providing the
mechanical basis for anchorage, cell shape determination, force transmission, and
migration (Fig. 8.1b). Integrins transmit extracellularly generated chemical and
mechanical signals into the cell interior in complex process called outside-in signal-
ing via focal adhesion sites. These focal sites by which the cells attach to the ECM
are multiprotein complexes associated with the integrin cytoplasmic tails and are
composed of numerous signaling, adaptor, and actin-binding molecules (Geiger
et al. 2009). The multiple signaling cascades induced by integrin engagement
include phosphorylation by kinases (e.g., focal adhesion kinase (FAK), Src family
tyrosine kinases, mitogen-activated protein (MAP) kinases), calcium influx, activa-
tion of Rho GTPases, and cytoskeletal rearrangements which all together regulate
158 A. Aszódi
various aspects of cell behavior including proliferation, survival, and differentiation

(Legate et al. 2009). In addition, integrins cooperate and share common signaling
pathways with growth factor receptors to generate a fine-tuned cellular response for
extracellular cues. At least 12 integrin receptors contain the β1 subunit (Fig. 8.1a)
and form the largest integrin subfamily. Ablation of the β1 integrin gene in mice
leads to peri-implantation lethality (Fassler and Meyer 1995; Bouvard et al. 2001),
which demonstrates the essential role of β1 integrins in early embryonic
development.
8.3 Integrin Function in Cartilage Morphogenesis
Most elements of the mammalian skeleton are generated through a cartilaginous

template by the multistep developmental process of endochondral bone forma-
tion (Yeung Tsang et al. 2014; Lefebvre and Bhattaram 2010). The initial carti-
laginous molds of the future axial and appendicular skeleton are formed by the
condensation and chondrogenic differentiation of the committed mesenchymal
cells. The molds, which are surrounded by the perichondrium, consist of imma-
ture, proliferative chondroblasts which synthesize an extracellular matrix (ECM)
composed of type II collagen fibrils, the large proteoglycan aggrecan, and vari-
ous small proteoglycans and non-collagenous glycoproteins. Next, centrally
located cells exit from the cell cycle (maturation), increase in cell volume, and
became hypertrophic chondrocytes followed by the differentiation of perichon-
drial osteoprogenitor cells into osteoblasts, which produce osteoid to form a
bony collar around the shaft (diaphysis) of the bone. Chondrocyte hypertrophy
triggers ECM mineralization, blood vessel invasion, and the recruitment of chon-
droclasts/osteoclasts, which gradually degrade the cartilage matrix, and osteo-
blasts, which replace the cartilage scaffold by bone. The program of proliferation,
maturation, hypertrophy, and remodeling takes place within a specialized struc-
ture, called growth plate (for further details, see Chap. 4 in volume 1 (Aszódi
2016)). Growth plate chondrocytes arrange into horizontal zones of resting, pro-
liferative, prehypertrophic, and hypertrophic chondrocytes according to the met-
abolic and/or differentiation stages. Vertically, proliferative chondrocytes form
columnar stacks in which the flattened cells are oriented with right angle to the
longitudinal axis of the growing bone. Proliferation, matrix production, and
hypertrophy of growth plate chondrocytes are the major factors contributing for
proper and timely elongation of endochondral bones. Chondrodysplasias, a
diverse group of genetic disorders with primary effect on cartilage development
and growth, commonly result in short stature (dwarfism) and, occasionally,
lethality (Krakow and Rimoin 2010; Krakow 2015; Warman et al. 2011) (for
details, see Chaps. 6 and 7). The coordinated process of endochondral bone for-
mation is under the control of multiple molecular mechanisms involving tran-
scription and growth factors, ECM proteins, and integrin-mediated cell-matrix
interactions (Docheva et al. 2014; Aszodi et al. 2000).
8.3.1 I ntegrin Expression on Fetal and Growth Plate

Chondrocytes
Several studies investigating cartilages in the developing mammalian limb have

demonstrated that chondrocytes express various integrin receptors for cartilage
ECM proteins including α1β1, α2β1, and α10β1 (for collagen II); α5β1, αvβ3, and
αvβ5 (for fibronectin); and α6β1 (for laminin). Fluorescence-activated cell sorter
(FACS) analysis of isolated human fetal knee chondrocytes has showed the surface
expression of α2, α5, α6, αv, and β1 integrins, whereas only the α5, αv, and β1 sub-
units were detected on intact tissue sections by immunohistochemistry (Durr et al.
1993). In a more detailed immunohistochemical study, α5, α6, αv, and β1 integrins
were found throughout the human fetal growth plate, whereas α1, α2, and α3 were
absent in growth plate chondrocytes (Salter et al. 1995). In adolescent human
growth plate, immunohistochemistry indicated the presence of α6, αv, and β1 sub-
units in all growth plate zones; the α1, α2, and α5 subunits in the proliferative and
hypertrophic zones; and the β5 subunit in the hypertrophic zone (Hausler et al.
2002). The integrin subunit α10, which was isolated using its capacity to bind col-
lagen type II (Camper et al. 1998; Lundgren-Akerlund and Aszodi 2014), is pre-
dominantly present in collagen II expressing human tissues at the mRNA level
(Gullberg and Lundgren-Åkerlund 2002).
The spatially variable expression pattern of integrin subunits was also revealed in
the mouse cartilage. Integrin β1 was found throughout the cartilage anlage at embry-
onic day 18.5 (just before birth), while α2, α3, and α5 subunits were predominantly
expressed in the proliferative zone (Ma et al. 1998). The distribution of the three
collagen-binding integrins α1β1, α2β1, and α10β1 was further analyzed by immu-
nohistochemistry in the developing mouse limb at various embryonic and postnatal
stages (Camper et al. 2001). The integrin subunit α10 co-localized with collagen II
in the initial cartilaginous molds at embryonic day 11.5. The integrin subunit α10
was the predominantly expressing collagen-binding integrin in epiphyseal and
growth plate cartilage until birth, while α1 was detected only in the articular surface
and the hypertrophic zone. Integrin α2β1 was undetectable in any region of the
developing limb cartilage. At 4 weeks after birth, α10 was strongly, and α1β1 was
weakly detectable in growth plate chondrocytes.
The aforementioned experiments showed good agreement between the dynamic
localization of integrins on chondrocytes and the deposition of their respective ligands
into the cartilage ECM. Laminin and fibronectin are weakly and/or zone-specifically
present in the growth plate cartilage, while collagen II is being the most abundant
throughout the growth plate (Durr et al. 1993, 1996; Hausler et al. 2002; Lee et al.
1997; Salter et al. 1995; Camper et al. 2001). The importance of collagen II in the
structural and functional properties of the developing cartilage and the prominent
expression of integrins on the surface of chondrocytes thus clearly imply that cell-
matrix interactions mediated by integrin heterodimers are important for cartilage dif-
ferentiation and chondrocyte function in the growth plate. Furthermore, they indicate
the most critical roles for the β1 and the collagen-binding α10 integrin subunits.
160 A. Aszódi
8.3.2 Integrin Function: In Vitro Studies
The functional role of integrins in cartilage differentiation was intensively studied

in micromass culture of limb-bud mesenchymal cells, which mimics several steps
of developmental chondrogenesis in vitro. Administration of β1 integrin-blocking
antibodies to mouse micromass culture prevented the formation of cartilaginous
nodules indicating a pivotal role of β1 integrins in chondrogenic differentiation of
skeletogenic mesenchymal progenitor cells (Shakibaei 1998; Bang et al. 2000). β1
integrin-mediated tyrosine phosphorylation of FAK and fibronectin were required
for precartilaginous condensation and chondrogenic differentiation of chick mesen-
chymal cells (Bang et al. 2000). Another study has demonstrated that matrix metal-
loprotease-2 (MMP-2) activity regulated by microRNA-488 impairs chondrogenic
cell condensation by disrupting matrix adhesion sites and downregulating FAK-β1
integrin interaction (Jin et al. 2007). On the other hand, mouse fibroblasts lacking
FAK could form cartilage nodules, and pharmacological inhibition of FAK/Scr sig-
naling induces the expression of chondrogenic genes suggesting that FAK signaling
may suppress chondrogenesis by regulating the shift from cell-ECM to cell-cell
interactions (Pala et al. 2008). Collectively, these observations indicate that focal
adhesion complexes containing fibronectin, β1 integrins, and FAK/Scr are impor-
tant for the transition of mesenchymal precursors to chondrocytes in vitro.
Studies using chick whole sternum and isolated sternum chondrocytes revealed
that inhibition of β1, α1, and α2 integrins by blocking antibodies results in growth
retardation, increased apoptosis, and disorganization of the actin cytoskeleton indi-
cating important regulatory roles of integrins for chondrocyte behavior (Hirsch
et al. 1997). Further in vitro studies have demonstrated that proliferation of rabbit
growth plate chondrocytes requires α5β1 and fibronectin (Enomoto-Iwamoto et al.
1997), whereas survival of chicken chondrocytes in suspension culture is mediated
via β1 integrin-collagen interactions (Cao et al. 1999). The importance of integrins
in hypertrophic chondrocyte differentiation was demonstrated by experiments
showing that transglutaminase-induced hypertrophy of cultured articular cartilage
chondrocytes requires signaling through α1β1 and α5β1 integrins (Johnson et al.
2008), and that β1 integrin-blocking antibodies impaired the deposition of the
hypertrophic chondrocyte marker collagen X in chick sternal organ culture (Hirsch
et al. 1997). Interaction of annexin V, a collagen II receptor and cytoplasmic signal-
ing protein, with β5 integrin cytoplasmic tail in hypertrophic chondrocytes induced
apotosis of cultured growth plate chondrocytes, while siRNA-mediated silencing of
annexin V increased chondrocyte survival (Wang and Kirsch 2006).
Cultured primary chondrocytes rapidly lose their chondrogenic phenotype on
plastic monolayer characterized by changes in cell geometry (from spread to
rounded), actin cytoskeleton organization (from cortical, sub-membrane localiza-
tion to stress fiber formation throughout the cell), and gene expression (e.g., from
cartilage-specific collagen II and aggrecan expression to fibroblast-specific collagen
I and fibronectin expression). A unique and sensitive marker for monitoring chon-
drocyte phenotypic changes during cell culture expansion is the relative ratio of the
α10 and α11 integrin subunits (Gouttenoire et al. 2010). Long-term culturing and
TGF-β1 treatment result in chondrocyte dedifferentiation associated with decreased

α10 integrin and increased α11 integrin expression levels. BMP-2 supplementation,
however, stabilizes the chondrogenic phenotype accompanied by high levels of α10
expression (Gouttenoire et al. 2010). Interestingly, chondrocytes cultured on plastic
monolayer coated with type II collagen keep their rounded shape and express col-
lagen II suggesting that collagen-binding integrins may stabilize the chondrogenic
phenotype (Shakibaei et al. 1997). In line with this hypothesis, the formation of
chondrocyte aggregates with a cartilage-like pericellular matrix environment in sus-
pension culture requires α10β1 integrin-collagen II interaction (Gigout et al. 2008).
8.3.3 I n Vivo Studies with Knockout Mice: The Essential Role

of β1 Integrins in Cartilage Development
In order to investigate the in vivo function of β1 integrins during endochondral bone

formation, the floxed β1 integrin gene (β1fl/fl) was conditionally inactivated in the
entire cartilaginous skeleton using a transgene which drives expression of the cre
recombinase in differentiated chondrocytes under the control of the mouse collagen
II promoter (Col2a1cre) (Sakai et al. 2001). β1fl/fl-Col2a1cre mice display general
chondrodysplasia during embryogenesis leading to lethality shortly after birth due
to breathing difficulties (Aszodi et al. 2003). To overcome lethality, the β1 integrin
gene was also deleted in limb-bud mesenchymal progenitor cells using the Prx1cre
transgene (Raducanu et al. 2009; Logan et al. 2002). The β1fl/fl-Prx1cre mice sur-
vive after birth, but mesenchymal limb tissues such as the cartilage, tendon, and
ligaments or the synovium completely or partially lack β1 integrins (Raducanu et al.
2009). The β1fl/fl-Prx1cre mutant mice show similar embryonic limb phenotype
characterized by reduced growth of the cartilaginous skeletal elements as the β1fl/fl-
Col2a1cre mice but develop postnatal abnormalities of synovial joint tissues (see
later). Importantly, β1 integrin deficiency in mesenchymal progenitors does not
influence chondrogenic differentiation implicating that β1 integrin-mediated cell-
matrix interactions and signaling processes are not essential for early stages of
endochondral bone formation (Raducanu et al. 2009). Supporting this conclusion,
homozygous deletion of the floxed β1 integrin alleles at the time of mesenchymal
condensation by the Twist2-cre transgene allows cartilage formation but severely
impairs later stages of skeletal development (Shekaran et al. 2014).
The most dramatic phenotype of mice lacking β1 integrins in chondrocytes and
hence lacking chondrocyte adhesion to collagen II, laminin, and partially fibronec-
tin was observed in the growth plate (Fig. 8.2) (Aszodi et al. 2003). The normal
columnar growth plate is a polarized structure characterized by specifically shaped
and oriented cells in the proliferative zone (for details, see Chap. 4 in volume I
(Aszódi 2016)). Proliferative chondrocytes display a strongly flattened, anisotropic
geometry and align along the mediolateral (ML) axis of the growth plate, thus per-
pendicular to the proximo-distal (PD) direction of the longitudinal growth. The
mitotic figures in these elongated chondrocytes lie also perpendicular to the PD
axis, and cell divisions occur parallel with the columns (Dodds 1930). The two
162 A. Aszódi
Normal chondrocyte-ECM Loss of chondrocyte-ECM

adhesion adhesion
Lam
Col II
Lam
Col II Fn
Fn
α β1 α β1
organized disorganized
collagen network collagen fibrils
flattened chondrocytes rounded chondrocytes
elongated columns lack of columns
Fgfr3 Fgfr3
FAK
cortical actin ring disrupted actin cytoskeleton Fgf signaling
Fgf signaling Fgfr3↑
ERK 1/2 ERK 1/2 ↓

cyclin D ↓
p16↑ p21↑
Apoptosis Cell cycle progression Apoptosis ↑ G1/S transition ↓
Fig. 8.2 The role of β1 integrins in the growth plate cartilage. Adhesion through multiple αβ1
heterodimers to the ECM proteins collagen II (Col II), fibronectin (Fn), and laminin (Lam) regu-
lates the shape, oriented cell division, and intercalation movements of proliferative chondrocytes
resulting in the formation of elongated growth plate columns (left panel). Signaling pathways
including FAK and ERK1/2 activation control chondrocyte survival and, together with fibroblast
growth factor (Fgf) signaling, cell cycle progression in the proliferative zone. When β1 integrin is
ablated in chondrocytes (right panel), the loss of matrix attachment mediated via αβ1 integrins
leads to cell rounding, impaired cytokinesis, diminished rotation movements, and the complete
lack of the columnar structure of the growth plate. In the absence of β1 integrins, the link between
the ECM and the actin cytoskeleton is disrupted leading to disorganized collagen (extracellularly)
and actin (intracellularly) networks. Furthermore, ERK1/2 activation and Fgf receptor 3 (Fgfr3)
mRNA expression are perturbed resulting in increased apoptosis and impaired G1/S transition due
to abnormal expression of cyclin D and the cell cycle inhibitors p16 and p21
mediolaterally oriented, semicircular daughter cells subsequently undergo gradual

flattening and extensive rotational or gliding movements around each other to
arrange into the longitudinal column. Polarized cell migration and ML intercalation
is characteristic for convergent extension, an essential morphogenetic process
resulting in tissue narrowing along the ML axis and lengthening along the PD axis.
This mechanism was completely disrupted in β1 integrin-deficient growth plates
resulting in shortened and broadened long bones. The mutant chondrocytes in the
proliferative zone were rounded up, failed to rotate, and showed random orientation
of the mitotic spindle and the cleavage plain (Aszodi et al. 2003 and Aszodi, 2016
unpublished). Although an increasing body of evidence suggests that β1 integrins
are actively involved in spindle and cell division positioning in various tissues
including the skin (Lechler and Fuchs 2005) and mammary epithelium (Taddei
et al. 2008), the mechanisms by which β1 integrins governs mitotic spindle orienta-
tion and gliding movements in cartilage are still not fully understood. In contrast to
other cell types, normal proliferative chondrocytes do not round up during mitosis
but remain flattened throughout the cell cycle. For such elongated cells Hertwig’s
laws can be applied postulating that (1) spindles are formed in the longest axis of the
cells and (2) the division plane is always perpendicular to the long axis (reviewed in
Shah 2010). In this model, simple geometric constraints provide the necessary cue
for orientation of the mitotic spindle and direction of cell division. β1-null chondro-
cytes do not adhere to collagen II and laminin, and only partially to fibronectin
(Aszodi et al. 2003), therefore tempting to speculate that such significant loss of cell
anchorage to the cartilage ECM causes the shape change (rounding) of chondro-
cytes in the proliferative zone of the mutant growth plate. Consequently, the isotro-
pic β1-deficient chondrocytes lack geometric and adhesive cues which guide
oriented cell division and rotational movements resulting in disorganized growth
plate.
In addition to the crucial role of β1 integrins for spindle and division positioning,
β1fl/fl-Col2a1cre mice exhibited further cellular and ECM defects (Aszodi et al.
2003). Mutant chondrocytes were frequently binucleated and displayed actin abnor-
malities demonstrating that disrupted cell-ECM interactions may impair adhesion-
generated pulling forces and actomyosin ring constriction at the cleavage furrow,
which are required to complete cytokinesis. β1-null chondrocytes also showed
abnormal G1/S transition caused by reduced cyclin D level, increased nuclear trans-
location of signal transducer, and activator of transcription 1 (Stat1) and Stat5a, and
elevated expression of the cell cycle inhibitors p16 and p21. The defective cell cycle
progression was associated with significantly elevated expression of fibroblast
growth factor receptor 3 (Fgfr3) mRNA in proliferative growth plate chondrocytes
suggesting a crosstalk of β1 integrin to Fgf signaling. Since FGFR3 activation muta-
tions in chondrocytes activate Stat1/Stat5 and upregulate cell cycle inhibitors result-
ing in skeletal dysplasias both in human and in mice (Li et al. 1999; Su et al. 1997),
it is likely that β1 integrin-Fgf signaling pathways are cooperating in the regulation
of chondrocyte proliferation. Furthermore, an increased apoptosis rate of growth
plate chondrocytes was observed in the mutant mice. These abnormalities were
accompanied with reduced activation of FAK and Erk1/Erk2 suggesting that altered
integrin signaling is likely contributing to the proliferation and survival defects.
Indeed, cartilage-specific deletion of the gene encoding integrin-linked kinase (Ilk),
an effector molecule interacting with the cytoplasmic domain of β1/β3 integrins,
leads to similar but milder growth plate abnormalities including reduced prolifera-
tion, disorganization of the cytoskeleton, and chondrocyte shape change (Grashoff
et al. 2003; Terpstra et al. 2003).
Finally, the β1fl/fl-Col2a1cre cartilage has abnormal ultrastructure characterized
by thick and disordered collagen fibrils indicating that β1 integrins on chondrocytes
are important for proper assembly of the collagenous fibrillar network. In fibroblast,
a preformed fibronectin matrix is essential for the formation of collagen fibrillar
network, and the collagen-binding α2β1 and α11β1 integrins strongly enhance this
process (Velling et al. 2002). It has been shown that mice lacking fibronectin in
cartilage have normal growth plate structure (Aszodi et al. 2003) predicting that one
or more collagen-binding β1 integrins directly modulate the polymerization of
164 A. Aszódi
collagen II and/or incorporation of the fibrils into the meshwork. In accordance, β1

integrins were shown to play a role in the arrangement of the ECM around chondro-
cytes by anchoring and bending type II collagen fibrils (Lee and Loeser 1999). The
absence of β1 integrins on the cell surface and the ECM changes had profound
effect on the diffusion and biomechanical properties of the cartilage (Bougault et al.
2013). Rheological measurements on intact costal cartilage isolated from 17.5-day-
old embryos revealed increased compressive stiffness of β1fl/fl-Col2a1cre samples
likely due to high cellular rigidity induced by uncoupling the cytoskeleton-integrin
interaction. In contrast, the diffusivity of macromolecular fluorescent solutes was
greatly reduced in close vicinity of the cell membrane indicating that the lack of β1
integrin-ECM interactions influences the organization and/or composition of the
pericellular matrix compartment.
8.3.4 I n Vivo Studies with Knockout Mice: Mild Role of Integrin

α Subunits in Cartilage Morphogenesis
In contrast to the severe cartilage phenotype of the mice lacking all β1 integrins in
chondrocytes, most mutant strains deficient for a particular α/β1 heterodimer such
as α1β1, α2β1, or α6β1 do not show defects in endochondral bone formation
(Bouvard et al. 2001). However, the α10 integrin-deficient mice partially recapitu-
late the growth plate abnormalities observed in the β1fl/fl-Col2a1cre and in β1fl/fl-
Prx1cre mice. Constitutive deletion of the gene encoding α10 (Itga10) results in a
mild, nonlethal chondrodysplasia characterized by mild chondrocyte shape change
and disorganization of the growth plate (Bengtsson et al. 2005). Furthermore, the
lack of α10 on chondrocytes impairs proliferation via the modulation of Stat1/
Stat5a/p16 pathway and leads to reduced collagen fibrillar density in the cartilage
matrix as seen in the β1 null growth plate. These findings imply that although α10β1
integrin is the most critical collagen-binding integrin, which modulate chondrocyte
function, the absence of multiple β1 integrins is necessary for the most severe carti-
lage defects indicating partial compensation among β1 integrin-containing het-
erodimers. Interestingly, double knockout mice lacking both α2β1 and α11β1
integrin develop dwarfism which is, however, caused by decreased production of
insulin-like growth factor-1 in the liver and does not result from growth plate dys-
function (Blumbach et al. 2012).
Heritable bone diseases are frequently caused by mutations in genes coding for
ECM structural proteins or molecules associated with extracellular signaling
(Briggs et al. 2015; Tosi and Warman 2015), suggesting the importance of cell-
matrix interactions in proper development and normal function of the cartilaginous
skeleton. However, no mutations in genes encoding integrin subunits have so far
been identified in human chondrodysplasias (Warman et al. 2011). Therefore, inter-
esting to note that a truncating mutation in the canine ITGA10 gene has been recently
correlated with the disproportionate short-limbed dwarfism phenotype of two breeds
of Nordic hunting dogs (Kyostila et al. 2013). The autosomal recessive chondrodys-
plasia of Norwegian Elkhound and Karelian Bear Dog breeds is characterized by
irregular and disorganized growth plates (Kyostila et al. 2013; Bingel and Sande
1982) and is associated with a nonsense mutation in exon 16 of ITGA10 causing the
complete lack of α10 protein in the cartilage of the affected dogs. As the canine and
human ITGA10 genes are highly homologous, these findings suggest that a similar,
naturally occurring loss-of-function mutation in human ITG10 is a potential candi-
date for chondrodysplasias with so far unidentified genetic background.
Recently, the in vivo role of the fibronectin-binding α5β1 integrin in endochon-
dral bone formation was analyzed using an ex utero manipulation system (Inoue
et al. 2014). Injection of RGDS (Arg-Gly-Asp-Ser) peptides or anti-α5β1 integrin
antibody into the upper part of the forearms of 15.5-day-old embryos resulted in
shortened humerus, just one day after the surgical manipulation. Decreased BrdU
incorporation, apoptosis, and lower ratios of collagen X/collagen II RNA and pro-
tein were observed in the cartilage of the micro-manipulated embryos implicating
that α5ββ1 integrin-mediated cell-matrix interactions through the RGD sequence
may regulate proliferation, survival, and differentiation of growth plate
chondrocytes.
8.3.5 I ntegrin-Associated Signaling Pathways Involved

in Endochondral Bone Growth
Filamin B is an intracellular scaffolding protein that mediates signaling between

cell membrane receptors and the actin cytoskeleton (Nakamura et al. 2011).
Mutations in the gene coding for filamin B (FLNB) result in a range of human skel-
etal disorders. Loss-of-function mutations (null alleles) lead to recessive spondylo-
carpotarsal syndrome (SCT) characterized by short stature and bone fusions
(Krakow et al. 2004). Autosomal dominant (gain of function) mutations cause a
range of skeletal dysplasias including boomerang dysplasia and atelosteogenesis I
and III (Bicknell et al. 2005; Farrington-Rock et al. 2006). A mouse model lacking
Flnb mimics the human characteristic of SCT (Farrington-Rock et al. 2008) and
exhibits reduced phosphorylation of β1 integrin at the residue Ser785 associated
with reduced chondrocyte adhesion to ECM ligands, premature hypertrophic dif-
ferentiation, and abnormal organization of the collagen fibrillar network in the car-
tilage (Lu et al. 2007; Hu et al. 2014). The interaction between filamin B and β1
integrin in cartilage also modulates chondrocyte proliferation through the regulation
of Cdk1/Cyclin B, an effector complex required for G2/M progression of the cell
cycle. Mechanistically, the Pi3k/Akt pathway is activated downstream of Flnb-β1
integrin to promote endochondral bone growth (Hu et al. 2014). Thus, disruption of
the ECM-β1 integrin-filamin B axis may contribute to skeletal dysplasias both in
mouse and human.
The CCN matricellular proteins are a group of extracellular modular molecules
which bind to ECM components, integrins, and growth hormone receptors to regu-
late diverse cellular processes (Krupska et al. 2015). All the six members (CCN1-6)
of the family are expressed in the developing cartilage (Kawaki et al. 2008), and
CCN2/connective tissue growth factor (CTGF) deficiency in mice results in severe,
166 A. Aszódi
perinatal lethal chondrodysplasia accompanied by diverse growth plate abnormali-

ties including reduced chondrocyte proliferation, delayed hypertrophic differentia-
tion, and impaired ECM production (Hall-Glenn et al. 2013; Ivkovic et al. 2003).
Mutant chondrocytes exhibit enlarged endoplasmatic reticulum in vivo, a typical
sign of cellular stress, and upregulate the stress responsive genes BiP and Chop in
three-dimensional alginate culture upon chemically induced stress (Hall-Glenn
et al. 2013). CCN2/CTGF is localized pericellularly, where it interacts with α5β1
integrin to modulate chondrocyte proliferation and ECM production (Nishida et al.
2007). In stress situation, binding of the C-terminal part of CCN2/CTGF to α5β1
decreases Chop mRNA levels, while α5 integrin-blocking antibodies prevent the
antistress role of CCN2/CTGF in cultured chondrocytes (Hall-Glenn et al. 2013).
These findings suggest that the protective role of CCN2/CTGF on chondrocytes
under stress conditions could be, at least partially, mediated through α5β1 integrin.
Lysophosphatidic acid (LPA), small glycerophospholipid produced from lyso-
phosphatidylcholine by the extracellular enzyme autotaxin (ATX), modulates
diverse cellular functions in various tissues through the activation of LPA receptors
(LPA1–6) (Aikawa et al. 2015). A recent study has demonstrated through genetic
studies in zebrafish and mice that the ATX-LPA signaling axis is pivotal for proper
cartilage formation (Nishioka et al. 2016). LPA1 knockout and ATXflox/− mice with
significantly reduced ATX level in the serum show skeletal growth retardation asso-
ciated with chondrocyte misalignment and proliferation deficit. Mechanistically, the
ATX-LPA1 axis enhances proliferation by promoting S-phase entry of the cell cycle
through activation of β1 integrin signaling and subsequent deposition the pericel-
lular fibronectin matrix. The ATX-LPA1-induced, integrin-dependent fibronectin
assembly may promote chondrocyte attachment to the ECM and support prolifera-
tion, both processes required for proper cartilage morphogenesis.
8.4 Synovial Joint and Articular Cartilage
Synovial joints unite different types of tissues to ensure load transmission within
skeletal structures and coordinate their movements. The opposing bones are covered
with articular cartilage (AC) and are separated by a joint cavity enclosed in a cap-
sule linking the skeletal elements. The capsule is lined by a synovial membrane
secreting synovial fluids, which lubricates the AC surface and provides nutrition to
the components of joints.
Synovial joints develop through two main processes called joint specification
and joint morphogenesis (Pacifici et al. 2005) (for details, see also Chap. 7 in
Volume 1 (Wang et al. 2016)). In the step of joint specification, skeletogenic MSCs
expressing the Sox trio are directed to the articular fate under the control of TGF-
beta receptor 2 (Spagnoli et al. 2007), canonical Wnt (e.g., Wnt4 and Wnt9a/14)
(Hartmann and Tabin 2001; Guo et al. 2004) and growth differentiation factor-5
(Gdf5) (Settle et al. 2003) signaling cascades, resulting in the formation of pre-
sumptive joint regions called interzones. Subsequently, a cavity forms in the inter-
zone between the articulating skeletal elements driven by a not fully understood
mechanism involving high-level synthesis of hyaluronan (Matsumoto et al. 2009),

specific ECM composition (Pacifici et al. 1993; Choocheep et al. 2010), and skeletal
movement (Kahn et al. 2009). During joint morphogenesis, specific articular cells
differentiate and form the joint tissues. The continued expression of the Sox trio is
pivotal for articular chondrocyte differentiation, while it seems that Wnt/beta-
catenin signaling prevents chondrocyte differentiation at sites not destined for artic-
ular cartilage such as the synovial lining and ligaments.
8.4.1 Integrins and Joint Morphogenesis
The function of integrin-mediated cell-cell or cell-matrix interactions in joint deter-

mination and morphogenesis is relatively poorly investigated. Garciadiego-Cázares
and colleagues reported that α5 integrin is downregulated in the interzones of devel-
oping mouse fingers suggesting a role of α5β1 integrin for patterning of the hands
(Garciadiego-Cazares et al. 2004). Indeed, by microinjecting anti-β1 and anti-α5
monoclonal antibodies or RGD peptides into the wrist region of embryonic mouse
forelimb induced ectopic joints formation at the distal part of the radius and the
ulna. A narrow gap region expressing interzonal markers such as Gdf5 and Wnt9a/14
but not expressing the prehypertrophic marker Indian hedgehog (Ihh) had formed in
between the proliferative and hypertrophic zones. In contrast, forced expression of
human cDNAs coding for human α5 and β1 integrin subunits in embryonic chick
legs had resulted in joint fusions of the phalanges accompanied by the differentia-
tion of prehypertrophic chondrocytes expressing Ihh. Consequently, the authors
proposed that α5β1 integrin-RGD ligand (fibronectin) interactions are pivotal in cell
fate determination during the development of the appendicular skeleton by dictating
either the differentiation of interzonal cells or prehypertrophic chondrocytes on the
basis of α5β1 expression level in chondrocytes. Although this hypothesis is clearly
attractive, genetic experiments with conditional knockout mice did not support a
mechanistic role for α5β1-mediated matrix attachments in joint morphogenesis. To
date, no in vivo experiments have been performed to investigate the role of α5 inte-
grins in skeletogenesis. However, neither β1fl/fl-Col2cre mice nor β1fl/fl-Prx1cre
mice display ectopic joint formation, and in both models, Ihh is expressed at the
normal level in the prehypertrophic zone (Aszodi et al. 2003; Raducanu et al. 2009).
The lack of any obvious phenotype in joint formation in the absence of β1 integrins
on skeletogenic MSCs, perichondrial cells, and chondrocytes suggests that antibody
perturbation might trigger nonspecific and/or nonphysiological responses (e.g.,
apoptosis), which cannot be seen upon genetic ablation.
8.4.2 Integrins in the Articular Cartilage
Articular cartilage (AC) is a permanent, specialized type of hyaline cartilage that

functions as a load bearing tissue and can resist shearing forces and friction. The
AC, similar to the growth plate, is characterized by a high degree of anisotropy and
168 A. Aszódi
is divided into four zones with distinct organization of the matrix and cells: superfi-
cial (or tangential), intermediate (or transitional), deep (or radial), and calcified
(Poole et al. 2001). The superficial zone is the thin, outermost layer of the AC,
which contains elongated cells and densely packed collagen fibrils arranged parallel
to the surface. This zone has low aggrecan content, but it is enriched in small pro-
teoglycans such as decorin and biglycan (Poole et al. 1996). The intermediate zone
contains individual spherical chondrocytes and a relatively disorganized collagen
network, whereas in the deep zone, the cells tend to organize into columns with
radially oriented collagen fibril bundles between them. The pericellular compart-
ment of these zones has high collagen VI, decorin, and perlecan content, while
aggrecan is more abundant in the territorial and interterritorial regions. The calcified
zone is separated from the upper zones by the tidemark, and its main function is to
anchor the AC to the subchondral bone. The hypertrophic chondrocyte marker col-
lagen X is expressed in this zone (Gannon et al. 1991; Hoyland et al. 1991; von der
Mark et al. 1992), but unlike in the growth plate, the calcified matrix normally
resists vascular invasion and resorption. Cartilage homeostasis is carefully balanced
by metabolic and catabolic pathways to maintain ECM structure and function
throughout the articular cartilage zones. Mechanical overloading, age-associated
changes in chondrocyte function, and disturbed cytokine activities may all initiate
ECM degradation and contribute to the onset and progression of articular cartilage
deterioration resulting in osteoarthritis (for further details, see also Chaps. 1 and 2).
The upregulation of matrix-degrading enzymes such as matrix metalloproteinases
(MMPs) and aggrecanases belonging to the ADAMTS (a disintegrin and metallo-
proteinase with thrombospondin motifs) family causes the degradation of the col-
lagenous and proteoglycan networks which in turn weakens the biomechanical
function of the articular cartilage (for details, see Chaps. 3 and 4). Matrix turnover
in normal and osteoarthritic articular cartilage occurs more rapidly in the pericel-
lular compartment than in the interterritorial area (Wu et al. 2002) suggesting a role
of cell surface receptors like integrins in the control of matrix remodeling.
Normal articular cartilage chondrocytes expose various integrins on their sur-
faces with the most prominent expression of α1β1, α2β1, and α10β1 (for collagens);
α5β1, αvβ3,and αvβ5 (for fibronectin); and α6b1 (for laminin) (Durr et al. 1993;
Salter et al. 1995; Camper et al. 2001; Woods et al. 1994). In vitro studies have
demonstrated that human articular cartilage chondrocytes predominantly use β1
integrins, including α5β1 and αvβ5 integrins, for attachment to the cartilage extra-
cellular matrix (Kurtis et al. 2003). In articular cartilage, chondrocytes interact with
a dense collagen network in an environment which has low oxygen tension and
exposed to mechanical stimuli. Bovine articular chondrocytes cultured in three-
dimensional collagen sponges express high levels of collagen II, aggrecan, and inte-
grin α10 mRNAs (Cortial et al. 2006). Chondrocytes cultured in alginate beads
respond to hypoxia by elevating the expression of β1 integrin mRNA (Grimshaw
and Mason 2001). As a response for mechanical loading, articular cartilage chon-
drocytes elevate the expression levels of α5 subunit in bovine explants (Lucchinetti
et al. 2004) and the expression of α5β1 integrin in monolayer culture (Wright et al.
1997). α5β1-ECM interaction was shown to promote survival of AC chondrocytes
cultured on fibronectin-coated plastic surface or in alginate-suspension culture

under serum-free conditions (Pulai et al. 2002). Blocking antibodies against the α5
subunit also inhibited insulin-like growth factor I-mediated chondrocyte survival
emphasizing the role of α5β1 integrin-growth factor signaling crosstalk in prevent-
ing chondrocyte death (Pulai et al. 2002). Overexpression and siRNA-mediated
silencing studies revealed that β1 integrins regulate articular chondrocyte prolifera-
tion and survival, at least partially, by modulating the expression of the β1 integrin
downstream target molecule G-protein-coupled receptor kinase interacting pro-
tein-1 (GIT1) (Zhang et al. 2015). Collectively, these results imply that integrins are
important sensors and mediators of chemical and mechanical signals of the extra-
cellular matrix which regulate numerous physiological functions of articular carti-
lage chondrocytes.
8.4.3 he Role of Integrins in Mechanotransduction of Articular

T
Cartilage Chondrocytes
Biomechanical factors play a pivotal role in the normal function and health of syno-
vial joints. Under normal circumstances, physiological mechanical loading results
in a variety of environmental alterations including ECM and chondrocyte deformity,
changes in fluid flow, ion concentrations, and hydrostatic and osmotic pressures
which in turn lead to controlled anabolic changes that maintain the integrity of the
articular cartilage. In contrast, nonphysiological mechanical stress, due to overload-
ing or injury, is promoting cartilage catabolism and predisposes to OA. Upon stim-
uli, chondrocytes transmit mechanical signals through membrane associates called
mechanotransducers such as stretch-activated ion channels, receptor tyrosine
kinases, the hyaluronan receptor CD44, and integrins (Millward-Sadler and Salter
2004; Bader et al. 2011; Vincent 2013). Under physiological loading, integrins are
activated on the surface of isolated chondrocytes leading to recruitment of adaptor
proteins in focal adhesion complexes and to lateral interactions with growth factor
receptors and ion channels (Millward-Sadler and Salter 2004). Induction of the inte-
grin mechanotransduction pathway by intermittent hydrostatic pressure in
monolayer-cultured chondrocytes is accompanied by α5β1 integrin- and protein
kinase C (PKC)-dependent membrane hypopolarization and the secretion of the
anabolic cytokine interleukin-4 (IL-4), which in turn reduces the expression of
MMP-3 and increases aggrecan synthesis and glycosaminoglycan (GAG) produc-
tion (Millward-Sadler et al. 1999, 2000; Wright et al. 1997; Holledge et al. 2008).
Mechanical load elevates the surface expression of the α5 subunit on chondrocytes
in bovine articular cartilage explants, which may suggest the existence of a positive
feedback loop in the regulation of integrin-associated mechanotransduction pro-
cesses (Lucchinetti et al. 2004). The application of dynamic cyclic compression on
human chondrocytes in the presence of TGF-β3 also promotes proliferation and
proteoglycan synthesis, which can be reversed by addition of the α5β1 ligand
GRGDSP to the system (Chowdhury et al. 2004). Mechanotransduction via α5β1 is
modulated through multiple integrin-associated pathways including activation of
170 A. Aszódi
the focal adhesion proteins FAK and paxillin (Lee et al. 2000), assembly of PKCα-
RACK1-β1 integrin complexes (Lee et al. 2002), and the interaction of α5β1 with
the integrin-associated protein (IAP) CD47 (Orazizadeh et al. 2008). In addition to
α5β1, normal human articular chondrocytes also respond for cyclic pressure-
induced strain by the activation of αvβ5 and αvβ3 integrin-mediated signaling, since
blocking αvβ5 or αvβ3 abolishes mechanical stimulation-induced GAG and proteo-
glycan synthesis (Holledge et al. 2008; Chai et al. 2010). Similar to monolayer-
cultured human chondrocytes, IL-4 expression is upregulated in three-dimensionally
embedded, mechanically stimulated rat chondrocytes and regulates matrix synthesis
through the activation of the p38 MAPK signaling pathway (Shioji et al. 2014).
Further experiments with rat chondrocytes exposed to periodic mechanical stress
revealed the β1 integrin-mediated activation of the FAK-ERK1/2 signaling pathway
indicating a complex role of MAP kinases downstream of integrins in chondrocyte
mechanotransduction (Liang et al. 2013).
In contrast to the protective role of physiological loading, nonphysiological
mechanical stress disrupts the actin cytoskeleton via α5β1-mediated signaling that
activates MAP kinases and NF-kappa B. Those, in turn, result in elevation of nitric
oxide, prostaglandin E2, reactive oxygen species, proteases, and cytokines, which
mediate articular cartilage degradation (Bader et al. 2011). Furthermore, impact
injury-induced chondrocyte death was also found to be dependent on cell-matrix
interactions and cytoskeletal rearrangements which require integrin activation and
FAK signaling (Jang et al. 2014) (further details, see Chap. 2).
Compression of cartilage leads to pressurization and exudation of the interstitial
water, while the retained negatively charged GAGs attract positive counterions
resulting in changes in osmolarity and osmotic pressure in the vicinity of the chon-
drocyte surface. Chondrocytes respond for osmotic stress by transient increase of
intracellular Ca2+, mostly mediated by the nonspecific cation channel, transient
receptor potential vanilloid-4 (TRPV4) (McNulty et al. 2015; Clark et al. 2010). A
recent study has revealed that α1 integrin subunit-deficient chondrocytes exhibit
impaired intracellular calcium transient in response to osmotic stress and the TRPV4
agonist GSK1016790A (Jablonski et al. 2014). These observations implicate that
integrin α1β1 may play important, adhesion-independent role in the osmotransduc-
tion of chondrocytes.
8.4.4 Integrins and Osteoarthritis
Articular cartilage is highly prone to injury- and age-associated pathological dete-

rioration. Once damaged, the AC is unable to initiate an efficient regenerative pro-
cess owing to the loss of mitotic activity of adult AC chondrocytes, decreased
responsiveness to anabolic growth factors, and synthesis of less functional matrix
molecules. Osteoarthritis (OA) is a common degenerative disorder of the synovial
joints that not only affects the articular cartilage but also involves changes in sub-
chondral bone, synovium, ligaments, and muscle. OA is a complex disorder influ-
enced by both environmental and genetic factors. Although the precise etiology is
largely unknown, it is generally accepted that uncontrolled metabolism of skeletal

tissues is critical for the pathophysiology of this disabling condition. In primary
OA, the normal turnover of ECM molecules produced by chondrocytes is disturbed,
and the equilibrium between anabolic and catabolic processes is shifted toward pro-
teolytic activities, which eventually results in progressive, age-dependent articular
cartilage destruction (Goldring and Goldring 2007) (for overview, see also Chap. 1).
In addition, congenital abnormalities of the joints, as a typical result of impaired
development of endochondral bones, are often associated with early-onset degen-
eration and loss of the articular cartilage (secondary OA). Physiological ECM
remodeling of the articular cartilage occurs in a spatially and temporally controlled
fashion and involves the activities of both proteinases and proteinase inhibitors,
which are tightly regulated at multiple levels. The regulatory mechanisms are only
partially understood and include, in addition to growth factor and cytokine signaling
pathways, chemical and mechanical signals modulated by matrix-matrix and cell-
matrix interaction (Knudson and Loeser 2002; Loeser 2014). There is a growing
consensus that changes of ECM composition, e.g., by proteolytic degradation of
matrix constituents, or alterations of the biomechanical microenvironment of chon-
drocytes caused by chronic stress or injury significantly increase the risk of OA
through the perturbation of integrin-mediated signaling. The strongest evidence for
the participation of integrins in OA has been emerged from experiments studying
integrin expression and ECM fragments-induced signaling processes. In osteoar-
thritic monkey articular cartilage, increased immunostaining of α1, α3, and α5β1
integrins was reported compared with normal cartilage (Loeser et al. 1995). In
human OA samples, the expression of β1 integrin was inversely correlated with the
severity of the lesions suggesting that β1 integrin-mediated-ECM adhesion is
decreasing with OA progression (Lapadula et al. 1998). In contrast, osteoarthritic
human femoral head cartilage expressed the integrin subunits β2, α2, and α4 which
were not observed in normal AC cartilage (Ostergaard et al. 1998).
Subsequently, numerous reports have shown that fibronectin (Fn) and collagen II
degradation fragments are present in synovial fluid and cartilage of OA patients as
a result of increased catabolic activity, and these fragments can further mediate joint
destruction through the activation of integrin signaling which in turn elevates the
gene expression of catabolic cytokines, matrix metalloproteinases (MMPs), and
catabolic mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) (reviewed
by (Sofat 2009; Yasuda 2006; Loeser 2014) (Fig. 8.3). Treatments of cultured rabbit
synovial fibroblasts with the central 110–120 kDa cell-binding Fn fragment (Fn-f)
carrying the RGD sequence or ligation of α5β1 integrin with a monoclonal antibody
were shown to stimulate MMP-1 and MMP-3 expression (Werb et al. 1989).
Similarly, the central Fn-f and α5β1/α2β1 activating antibodies were shown to
upregulate MMP-13 synthesis accompanied by elevated expression of various inter-
leukins (ILs) including IL-1β, IL-6, IL7, and IL-8 and the growth-related oncogene
β (Groβ) in cultured human articular cartilage chondrocytes (Forsyth et al. 2002;
Loeser et al. 2003; Pulai et al. 2005; Long et al. 2008). In addition to central Fn-f,
the amino-terminal heparin binding 29 kDa fragment, which can interact with
α5β1 in a non-RGD-dependent manner (Dzamba et al. 1994; Hocking et al. 1998),
172 A. Aszódi
intact Fn intact type II collagen network

Catabolic signals
Fn fragments (mechanical stress,
fragmented/modified/monomeric
(120 kDa, 50 kDa, 29 kDa) pro-inflammatory collagen type II
α5 β1 cytokines) α col β1 fibrillar collagen type I
PYK2 PKCδ PYK2
Src
Rac1
Col If
ROS
Col IItelo Col IIm
p38
p38 ERK 1/2 JNK ERK 1/2 JNK
NFκB
NFκB AP-1
MMP13 MMP13 MMP3 MMP1 ADAMTS-5
MMP14 MMP13 MMP3 MMP1 IL1 IL6 IL8

MMP13 MMP3 MMP1 MMP10 ADAMTS-5
IL1 IL6 IL7 IL8 TNFa GRO iNOS COX2
Elevation of catabolic gene expression
Fig. 8.3 Integrins-associated signaling pathways induced in articular cartilage chondrocytes by

cartilage degradation fragments. Fibronectin (Fn) fragmentation products signals through α5β1
integrin to induce catabolic gene expression. Collagen-binding integrin heterodimers (such as
α1β1 and α2β1) might activate alternative intracellular pathways for catabolic response to fibrillar
type I collagen (Col If), type II collagen telopeptides (Col IItelo), or monomeric collagen type II
(Col IIm)
and the gelatin-binding 45–50 kDa fragment are also chondrolytic and likely inter-
fere with the native Fn/α5β1 signaling pathway (Homandberg and Hui 1994;
Homandberg et al. 2002a, b). The 29 kDa Fn-f in micromolar concentration stimu-
lated the expression of MMP-3 and the catabolic cytokines tumor necrosis factor α
(TNFα), IL1 and IL-6; and neutralizing antibodies against these cytokines were able
to reduce MMP-3 release in human articular cartilage explants (Homandberg et al.
1997). It has been reported that synthetic RGD peptides regulate the expression of
MMP-3 via an IL-1 autocrine regulatory loop in cultured rabbit articular chondro-
cytes (Arner and Tortorella 1995); however, IL-1 is not required for the synthesis of
MMP-13 induced by the 120 kDa Fn-f but did increase the activation of MMP-13 in
human articular cartilage chondrocytes (Forsyth et al. 2002). The 45 kDa fragment,
besides increasing MMP-3 and MMP-13 synthesis, was shown to elevate the levels
of TEGE, an aggrecan degradation neoepitope generated by ADAMTS-4 and
ADAMTS-5 (see also Chap. 3), independently of IL-1 in porcine articular cartilage
chondrocytes (Stanton et al. 2002). Mechanistically, the stimulation of α5β1 integ-
rin with ligation antibodies or Fn-fs activates protein kinase C (PKC)-delta, which
in turn phosphorylates the proline-rich tyrosine kinase-2 (PYK2) resulting in activa-
tion of MAPK (Erk-1/2, p38, and JNK) signaling cascades, increased phosphoryla-
tion of c-jun subunit of the AP-1 transcription factor, and the activation of the NFkB
signaling (Loeser et al. 2003; Pulai et al. 2005; Ding et al. 2008, 2009).
Fibronectin fragment-induced upregulation of MMPs requires endogenous
production of reactive oxygen species (ROS) (Del Carlo et al. 2007; Ding et al.
2009), the active form of the small GTPase Rac1 (Long et al. 2013), and Src, a
focal adhesion tyrosine kinase activating FAK and PYK2 (Ding et al. 2009). In
human chondrocytes, Src activity is apparently under the regulation of redox
signaling-associated oxidative posttranslational modification, which mediates
S-sulfenylation of Scr and required for Fn-f-induced MMP-13 production (Wood
et al. 2016). In bovine cartilage explants, Fn-fs increased NO release and induc-
ible NO synthase (iNOS) protein and mRNA levels, and iNOS inhibitors blocked
proteoglycan depletion and repaired damaged cartilage (Pichika and Homandberg
2004). The physiological relevance of Fn-fs in cartilage destruction was evi-
denced by studies demonstrating that injection of Fn-fs into knee joint dramati-
cally increases proteoglycan loss of rabbit articular cartilage (Homandberg et al.
1993). In pathological situations such as joint injury caused by mechanical
impact, cartilage degradation is associated with increased Fn-f levels, the activa-
tion of p38 and ERK1/ERK2 MAP kinases, and the upregulation of MMP-3,
MMP-13, TNF-α, and ADAMTS-5 (Ding et al. 2010, 2014). All together these
observations implicate that mechanical or catabolic stress-induced fibronectin
fragmentation converges with integrin and MAP kinase signaling in the control
of matrix destruction in osteoarthritis.
Collagen II degradation fragments and synthetic N- and C-terminal telopeptides
(NT and CT) have also been shown to upregulate MMPs and ADAMTS-5 in cul-
tured articular cartilage chondrocytes or in cartilage explants implicating an impor-
tant role of these collagen-derived degradation products in matrix homeostasis
(Jennings et al. 2001; Fichter et al. 2006; Guo et al. 2009). Both NT and CT stimu-
late MMP-1, MMP-3, and MMP-13 expression and proteoglycan depletion in
bovine cartilage explants at lesser extent and do not induce IL-1ß and TNFα expres-
sion compared with the 29 kDa Fn fragment (Guo et al. 2009). In contrast, mono-
meric, non-fibrillar type II collagen dose-dependently upregulates not only MMP-1,
-3, -13, and -14 but also the expression of the catabolic cytokines IL-1ß, IL-6, and
IL-8 in primary human articular chondrocytes, which may lead to collagen frag-
mentation and further MMPs and cytokines production (Klatt et al. 2009). Similar
to Fn fragments, the expression of these matrix destructive molecules required the
activation of the p38 MAPK and NFkB signaling pathways (Klatt et al. 2009).
Collagen fragments and peptides likely interact with collagen-binding integrins on
chondrocytes, since the CB10 cyanogen bromide fragment of type II collagen binds
to α2β1 integrin (Tuckwell et al. 1994). It is important to note, however, that chon-
drocytes might utilize alternative non-integrin receptors for the modulation of
174 A. Aszódi
collagen fragment-induced cartilage metabolism such as annexin V (Lucic et al.

2003) and discoidin domain receptor II (DDR2) (Xu et al. 2010).
The exact mechanism by which ECM degradation fragments accelerate cartilage
catabolism is unclear. The most popular explanation supposes that FN and collagen
degradation products disturb or even disrupt normal integrin clustering induced by
native FN or collagen II, which in turn alters physiological outside-in signaling and
cartilage metabolisms (Peters et al. 2002). However, beside degradation fragments,
fibrillar collagen itself is able to induce MMP expression upon binding to α1β1
integrin. Collagen I is upregulated in OA cartilage (Adam and Deyl 1983; Miosge
et al. 2004) and in the chondrogenic cell line MC615, and it stimulates the expres-
sion of MMP-13 via the activation of ERK and JNK MAP kinases. In the presence
of α1 or β1 blocking antibodies, the induction of MMP-13 expression was inhibited
(Ronziere et al. 2005). Furthermore, a recent report has suggested that collagen II
fibrils modified by the lipid peroxidation end-product 4-hydroxynonenal, which is
upregulated in synovial fluid of OA patients, induce cell death and catabolic and
inflammatory responses of human AC chondrocytes, presumably through perturba-
tion of α1β1 signaling (El-Bikai et al. 2010).
8.4.5 enetic Models: The In Vivo Role of Integrins

G
in the Articular Cartilage
Although the expression studies and in vitro experiments highlight the importance
of integrin signaling in OA and suggest that inhibition of integrin pathways might
be a potential approach to ameliorate ECM and ECM fragment-mediated articular
cartilage destruction, the in vivo role of integrins in OA pathogenesis has just begun
to be elucidated. Most knockout mouse lines lacking a particular α subunit, except
the α1 integrin-null mice, develop an apparently normal AC without reported abnor-
malities. The collagen-binding α1β1 integrin normally is expressed at low level in
the deep zone of healthy mouse AC, but it is significantly upregulated in the middle
and upper zones of early osteoarthritic cartilage suggesting that this integrin het-
erodimer supports cartilage remodeling (Zemmyo et al. 2003). Knee joints of
α1-null mice exhibit proteoglycan loss, cartilage erosion accompanied by increased
MMP-2 and MMP-3 expression, and synovial hyperplasia in younger animals than
wild-type controls (Zemmyo et al. 2003). It has been shown that microRNA-101
and the long noncoding RNA HOTTIP (Hoxa transcript at the distal tip) regulate the
expression of α1 integrin during chondrogenic differentiation of mesenchymal stem
cells and in osteoarthritic chondrocytes (Kim et al. 2013). Lentiviral-mediated over-
expression of miR-101 or α1 integrin in the joint ameliorates osteoarthritis induced
by surgical destabilization of the medial meniscus (DMM) in mice. Precocious
development of knee OA was also observed in mice lacking the membrane-anchored
disintegrin and metalloproteinase ADAM15 (Bohm et al. 2005). ADAM15 carries a
RGD motif and has multiple pericellular functions including the modulation of
outside-in signaling in chondrocyte-matrix interactions (Bohm et al. 2009), which
in turn enhances chondrocyte adhesion to collagen II and promotes AC
homeostasis. In contrast to α1 integrin, α2 integrin deficiency does not cause spon-

taneous OA but suppresses cartilage erosion and synovial inflammation associated
with reduced MMP-3 expression in mouse models of rheumatoid arthritis (Peters
et al. 2012). The ablation of the gene coding for the articular chondrocyte-abundant,
collagen-binding α10 integrin in mice results no reported pathology of the articular
cartilage up to 1 year of age (Bengtsson et al. 2005).
Similarly to the growth plate, β1 integrins are critical regulators of chondrocyte
phenotype in the articular cartilage as well. β1fl/fl-Prx1cre mice display severe
abnormalities of AC chondrocytes including rounding of the superficial zone cells,
loss of columnar arrangement in the deep zone, reduced proliferation, increased cell
death, frequent bi-nucleation, and disrupted actin cytoskeleton (Raducanu et al.
2009). These observations extend the view that in vivo β1 integrins are pivotal for
maintaining the normal shape, organization, proliferation, and survival of chondro-
cytes independent of their location in the cartilaginous skeleton. Furthermore, β1
integrin deficiency of AC chondrocytes leads to prominent ECM defects including
disorganized collagen II network in the interterritorial compartment and reduced
pericellular deposition of collagen VI indicating that β1 integrins are important
organizers throughout the ECM. Importantly, the ablation of β1 integrins differen-
tially affects chondrocyte maturation in the growth plate and the articular cartilage.
While in β1fl/fl-Col2cre and β1fl/fl-Prx1cre mice the hypertrophic differentiation of
growth plate and epiphyseal chondrocytes is delayed, the AC in the β1fl/fl-Prx1cre
mice exhibits reduced ratio of the noncalcified and calcified zones and contains col-
lagen X expressing chondrocytes above the tidemark, suggesting accelerated hyper-
trophic differentiation (Aszodi et al. 2003; Raducanu et al. 2009). This may imply
that β1 integrins have a spatially restricted, tissue context-dependent role in chon-
drocyte maturation.
Despite the osteoarthritis-like structural abnormalities, β1fl/fl-Prx1cre mice
exhibit no obvious articular cartilage erosion or elevated expression of matrix-
degrading proteases compared with controls (Raducanu et al. 2009). In contrast to
the previous in vitro experiments, no evidence was found for disturbed activation of
MAP kinases in mutant AC and in hip explants upon stimulation with a 40 kDA
cell-binding FN fragment suggesting that the lack of β1 integrins might be effi-
ciently compensated via signaling through β3/β5 integrins and/or growth factor
receptors. In addition to the growth plate and articular cartilage phenotypes, β1fl/fl-
Prx1cre mice develop ectopic cartilage and bone in various limb tissues including
tendons, synovial ligaments, and vessels (Raducanu et al. 2009). These pathologies,
in turn, result in significant immobilization of the legs and high mortality rate
between 8 and 12 months of age due to vessel rupture. Since the onset and progres-
sion of OA are influenced by biomechanical factors, the articular cartilage of the
β1fl/fl-Prx1cre mice mechanically might be less exposed providing a possible expla-
nation for the lack of cartilage deterioration.
The secreted melanoma inhibiting activity/cartilage-derived retinoic acid-
sensitive protein (MIA/CD-RAP)-deficient mice partially recapitulate the AC
abnormalities seen in the β1fl/fl-Prx1cre mice. MIA/CD-RAP is expressed in chon-
drocytes (Bosserhoff et al. 1997; Dietz and Sandell 1996), and its absence in mice
176 A. Aszódi
leads to mild ultrastructural abnormalities of the collagen fibrils in the developing

growth plate cartilage (Moser et al. 2002). In 3-month-old mice, MIA/CD-RAP-
deficient articular cartilage shows disturbed columnar arrangement of the chondro-
cytes and increased height of the calcified zone (Schubert et al. 2010). Based on
in vitro studies with cultured chondrocytes, it was proposed that MIA/CD-RAP
physically binds to and inhibits α5β1 integrin leading to reduced ERK signaling,
stabilization of the chondrocyte phenotype, and delay of hypertrophy. This finding
is consistent with a recent study demonstrating correlation between ERK activa-
tion and hypertrophic differentiation of human AC chondrocytes (Prasadam et al.
2010). Furthermore, an antibody activating α5β1 integrin/ERK signaling (Forsyth
et al. 2002) was shown to abolish the negative effect of MIA/CD-RAP on the
expression of the osteogenic marker osteocalcin in cultured AC chondrocytes
(Schubert et al. 2010).
Taken together, in vitro and loss-of-function genetic studies in mice strongly
suggest that integrins are important for proper differentiation and spatial organiza-
tion of AC chondrocytes. Since the ablation of both β1 integrin and the α5β1
integrin-inhibitory MIA/CD-RAP results in similar phenotype, the exact nature of
integrin-mediated signaling pathways on articular chondrocyte hypertrophy remains
to be elucidated.
8.5 Articular Cartilage Repair
Human articular cartilage has a limited intrinsic capacity to repair due to low prolif-
eration of chondrocytes, inefficient regeneration potential of resident chondro-
progenitors, and the lack of vasculature to deliver mesenchymal stem cells (MSCs)
into the damaged tissue. Current orthopedic therapeutic strategies include marrow
stimulation-based, osteochondral transfer, and cell-based techniques (Richter 2009)
with various advantages and disadvantages of each. Adult MSCs derived from the
bone marrow or adipose tissues, for example, have better proliferative activity com-
pared to autologous chondrocytes in vitro but require specific culture conditions to
differentiate into chondrocytes. Cell-based regeneration of the articular cartilage
requires the induction and stabilization of the chondrogenic phenotype, and the piv-
otal role of integrins in these processes is increasingly recognized.
In vitro, MSC differentiation into the chondrogenic, osteogenic, or adipogenic
lineages can be modulated via growth factors, cell-matrix interactions, three-
dimensional (3D) microenvironment, cytoskeletal tension, or mechanical stimula-
tion (McBeath et al. 2004; Engler et al. 2006; Woods et al. 2007; Kelly and Jacobs
2010). Collagen II promotes chondrogenesis of bone marrow-derived MSCs
(BMSCs) and adipose-derived MSCs (ASCs) in hydrogels (Lu et al. 2010;
Bosnakovski et al. 2006) by inducing cell rounding through β1 integrin-mediated
RhoA/Rock signaling (Lu et al. 2010). Culturing of human BMSCs in pellet together
with type II collagen fibrils requires β1 and α2 integrins for chondrocyte differentia-
tion and the expression of cartilaginous markers (Chang et al. 2007). In contrast,
RGD peptides block chondrogenesis of BMSCs by inducing cell spreading through
αvβ3 integrin and actin cytoskeletal reorganization (Connelly et al. 2007, 2008).
Stem cell commitment toward the chondrogenic lineage can be further modulated
through ECM remodeling. MT1-MMP (MMP-14), a membrane-bound pericellular
collagenase, activates β1 integrin-Rho GTPase signaling cascade in mesenchymal
progenitors and triggers the nuclear translocation of YAP/TAZ transcriptional
coactivators (Tang et al. 2013). An elegant study using MT1-MMP-deficient MSCs
and mice lacking MT1-MMP in skeletogenic MSCs has demonstrated that in the
absence of MT1-MMP, MSCs preferentially differentiate toward the chondrogenic
and adipogenic lineages. Changing the internal stiffness of the hydrogels and exter-
nal mechanical loading were also shown also influence MSC chondrogenesis
(Huang et al. 2004; Pelaez et al. 2009; Meyer et al. 2011), which involves integrin-
mediated cell-matrix interactions (Steward et al. 2012, 2014).
Many further studies have demonstrated the potential of integrin signaling mod-
ulation in articular cartilage repair and regeneration. The activation of FAK is
increasing upon exogenous stimulation of alginate beads with collagen II, whereas
FAK knockdown reduces chondrocyte redifferentiation in the same culture system
(Kim and Lee 2009). MIA/CDRAP-deficient mice exhibit increased regeneration
potential of the AC in a surgically induced OA model due to enhanced proliferation
of the chondrocytes (Schmid et al. 2010). In contrast, the lack of tenascin C, a spe-
cific ligand for some β1 and β3 integrins, delays the repair of cartilage defects in
mice (Okamura et al. 2010). Magnesium, a divalent cation required for activation of
β1 integrins, enhances the adherence and cartilaginous differentiation of human
synovial MSCs in a rabbit osteochondral defect model (Shimaya et al. 2010).
Human BMSCs expanded in monolayer express both α10β1 and α11β1 integ-
rins, and during chondrogenic differentiation in pellet culture, the expression of
α10β1 is increasing, while the expression of α11β1 is decreasing. Simple adminis-
tration of FGF-2 to the monolayer culture medium elevates the levels of α10β1
integrin and the chondrogenic marker Sox9, which in turn results in better chondro-
genic differentiation potential of MSCs (Varas et al. 2007). This observation raises
the possibility that α10β1 is a potent and unique biomarker for quality assurance of
MSCs used for cartilage repair.
8.6 Perspectives
Chondrocyte integrins have indispensable roles in cartilage development, skeletal

growth, and articular cartilage function. Acting as signaling hubs, integrins sense
chemical and mechanical stimuli arising from the cartilage extracellular matrix,
transmit them into the chondrocytes, and orchestrate a variety of cellular responses
important for differentiation, survival, proliferation, polarity, matrix production,
and assembly. Integrating and connecting ECM cues with the actin cytoskeleton
through signaling pathways are the essential function of integrins to maintain nor-
mal physiology of cartilaginous tissues. The complexity of integrin signaling in the
control of chondrocyte behavior is increasingly recognized through in vitro and
in vivo models, and drawing a valid conclusion for integrin functions from the
178 A. Aszódi
expanding data obtained from two- or three-dimensional culture systems and

genetic animal models is a great challenge of the future research. Careful morpho-
logic evaluation of mice carrying ablation for integrin subunits in combination with
cutting-edge proteomics, genomics, and metabolomics techniques held a promise to
dissect more precisely the role of integrin heterodimers and their associated signal-
ing cascades in cartilage physiology. Furthermore, disturbed integrin-mediated
adhesion or altered signaling in response to nonphysiological ECM signals is asso-
ciated with pathological situations such as chondrodysplasia and osteoarthritis.
Overwhelming body of evidence supports the view that integrin-mediated signaling
play pivotal roles in matrix destruction, and interfering with the way how chondro-
cytes sense the environment through integrins may open novel paths of therapeutic
approaches to slow down osteoarthritis or correct growth abnormalities. Finally,
modulating integrin expression and/or signaling during chondrogenic differentia-
tion of stem cells could help to design better tissue engineering constructs for cell-
based regeneration of the damaged articular cartilage.
Acknowledgment The author is supported by the DFG grants AS 150/11-1 (FOR2407) and AS
150/7-1.
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The Sensory and Sympathetic Nervous
System in Cartilage Physiology 9
and Pathophysiology
Susanne Grässel, Rainer H. Straub, and Zsuzsa Jenei-Lanzl
Abstract
The peripheral nervous system is critically involved in metabolism of joint tissue
and intervertebral disks (IVD). Nerve fibers of sympathetic and sensory origin
innervate synovial tissue and subchondral bone of diarthrodial joints. In patho-
physiological situations as in osteoarthritis (OA), rheumatoid arthritis (RA), and
IVD degeneration, innervation patterns of sympathetic and sensory nerve fibers
are partly altered in joint tissue and IVD.
Various resident cell types of the musculoskeletal system express receptors
for sensory and sympathetic neurotransmitters allowing response to neuronal
stimuli. Among them are mesenchymal stem cells, synovial fibroblasts, bone
cells, and different types of chondrocytes, which express distinct subtypes of
adrenoceptors, receptors for vasoactive intestinal peptide (VIP), for substance P
(SP), and calcitonin gene-related peptide (CGRP). Some of these cell types even
synthesize neuropeptides such as SP, and they are positive for tyrosine hydroxy-
lase (TH), the rate limiting enzyme for biosynthesis of catecholamines. During
S. Grässel (*)
Experimental Orthopaedics, Department of Orthopaedic Surgery, University of Regensburg,
ZMB/BioPark 1, Am Biopark, 93053 Regensburg, Germany
e-mail: susanne.graessel@ukr.de
R.H. Straub
Laboratory of Experimental Rheumatology and Neuroendocrine Immunology, Department of
Internal Medicine I, University of Regensburg,
BioPark 1, Am Biopark 9, 93053 Regensburg, Germany
e-mail: rainer.straub@ukr.de
Z. Jenei-Lanzl
Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Orthopaedic University Hospital
Friedrichsheim gGMBH Johann Wolfgang Goethe-University, Marienburgstr. 2,
60528 Frankfurt a.M., Germany
e-mail: zsuzsa.jenei-lanzl@friedrichsheim.de

192 S. Grässel et al.
endochondral ossification in embryonic limb development, sensory and sympa-

thetic neurotransmitters modulate osteo-chondrogenic differentiation of mesen-
chymal progenitor cells, vascularization, and matrix differentiation indicating a
distinct role in skeletal growth and possible limb regeneration processes. In
adults, sensory and sympathetic neurotransmitters are involved in pathology of
inflammatory diseases as rheumatoid arthritis which manifests mainly in joints.
In addition, they might play a role in pathogenesis of a priori degenerative joint
disorders, as osteoarthritis and intervertebral disk degeneration.
Altogether it became evident that sensory and sympathetic neurotransmitters
have crucial trophic effects which are critical for proper limb formation during
embryonic skeletal growth. In adults, they modulate articular cartilage,
subchondral bone and synovial tissue homeostasis, and physiological and patho-
physiological conditions, in addition to their classical neurological features.
9.1 Introduction
Clinical observations demonstrated the importance of the peripheral nervous system

for maintaining body homeostasis and mediating organogenesis and tissue repair.
On one hand, disorders of nerves (central or peripheral) can have substantial influ-
ence on bone health, repair, and regeneration. On the other hand, dramatic altera-
tions in density and distribution of sympathetic and sensory nerve fibers are reported
in some musculoskeletal pathophysiologies. Changes in the density of sympathetic
nerve fibers, which are characterized by tyrosine hydroxylase and/or neuropeptide
Y (NPY), in synovial tissue contribute to rheumatoid arthritis (RA) (Dirmeier et al.
2008). Loss of sensory joint innervation during aging is suggested to accelerate
degenerative cartilage alterations, which adds to development of spontaneous osteo-
arthritis (OA) in mice (Salo et al. 2002). Nerve fibers of sympathetic and sensory
origin innervate frequently the trabecular bone, periosteum, and fracture callus
(Hukkanen et al. 1992; Madsen et al. 1998). They are involved in controlling vascu-
larization and matrix differentiation during endochondral ossification in embryonic
limb development (Gajda et al. 2000) indicating a distinct role in modulating skel-
etal growth and limb formation processes.
Sensory nerves contain in general four different nociceptive neuropeptide fami-
lies: the tachykinins (Zhang et al. 2000), calcitonin-gene-related peptides (CGRP),
glutamine, and galanin. Effects of all tachykinins are mediated by three neurokinin
receptors with varying affinities for the individual ligands (Maggi 1995) (Table 9.1).
Classically, tachykinin substance P (SP) is known as a mediator of nociception and
of inflammation (Hartung and Toyka 1983). CGRP is encoded together with calci-
tonin and is generated by alternative splicing (Rosenfeld et al. 1983) and signals
through a complex family of receptor proteins (Poyner et al. 2002) (Table 9.1). The
most important neurotransmitter of the catecholaminergic sympathetic nervous sys-
tem is norepinephrine (NE), which signals through α- and ß-adrenoceptors (AR)
depending on concentration (Molinoff 1984) (Table 9.1; Fig. 9.1). However,
9 The Peripheral Nervous System in Cartilage Physiology and Pathophysiology 193
Table 9.1 Mammalian sensory and sympathetic neurotransmitters and their receptors
Sensory Receptor (highest Neuropeptide Neuropeptide receptor
neurotransmitters affinity) genes genes
Substance P (SP) Neurokinin (NK)1 Tachykinin (TAC) Tachykinin receptor
receptor 1 or PPT-A or (TACR)1
PPT-I
Neurokinin A (NKA) NK2 receptor TAC1 TACR2
Alternatively spliced
forms are neuropeptides
(NP) K and NPγ
Neurokinin B (NKB) NK3 receptor TAC3 or PPT-B TACR3
or PPT-II
Hemokinin 1 (HK1) NK1 receptor, TAC4 or PPT-C TACR1
alternatively spliced hemokinin (HK)-1
forms are endokinins receptor?
(EK) A, B, C, D
Alpha-calcitonin- CL-receptor (CRLR)/ CALCA CALCR/RAMP1
related peptide receptor activity
(αCGRP) modifying protein
(RAMP-1)
Beta-calcitonin-related CRLR/RAMP-1 CALCB CALCR/RAMP1
peptide (ßCGRP)
Sympathetic Receptors Neuropeptide Neurotransmitter
neurotransmitters genes receptor genes
Catecholaminergic
Norepinephrine (NE) or ≤10−8M: alpha-1a-, ADRA1A, ADRA1B,
noradrenaline (NA) b-, d-adrenoceptors ADRA1D
≤10−8M: alpha-2a-, ADRA2A, ADRA2B,
b-, c-adrenoceptors ADRA2C
≥10−7M: ß1-, ß2-, ADRB1, ADRB2,
ß3-adrenoceptors ADRB3
Peptidergic
Vasoactive intestinal VIP-1, VIP-2, and VIP VIPR1, VIPR2
peptide (VIP) VIP/PACAP receptors
Neuropeptide Y (NPY) NPY1-, NPY NPY1R, NPY2R,
2-, and 5- receptor NPY5R, PPYR1
NPY4-receptor or
pancreatic-
polypeptide
receptor-1 (PPR1)
Modified from Grassel (2014)
Information on glutamine and galanin were not included
sympathetic nerves release not only NE but also different co-transmitters such as
adenosine triphosphate (ATP), NPY, vasoactive intestinal peptide (VIP), and nitric
oxide (Pongratz and Straub 2014; Straub 2015). In addition, neurotransmitters can
be locally converted to differentially active neurotransmitters when respective con-
verting enzymes are present in the tissue. Relevant for cartilage (patho)physiology
nerve terminal
βAR
TH+ cell
αAR αAR
βAR-dominant αAR-dominant
-EC50, β2AR
10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 [M]
concentration of catecholamine
dependent on distance from source
Fig. 9.1 Concentration-dependent influence of norepinephrine on ß- and α-adrenergic receptors.

At high concentrations of 10−5 to 10−6 mol/l, norepinephrine (NE) binds to ß- and α-adrenergic
receptors, which typically elicits an increase in cyclic AMP with anti-inflammatory effects (e.g.,
TNF inhibition). At low concentration of 10−8 to 10−9 mol/l, NE inhibits formation of cyclic AMP
which is a pro-inflammatory signal. Thus, nerve fiber density and nerve terminal distance to target
cells plays an important role in neuroimmuno-modulation. EC50 of NE is 10−7 mol/l
is the conversion of co-released ATP to adenosine by ectonucleotidases (CD39,

CD73) located on the surface of cells (Romio et al. 2011; Van Linden and Eltzschig
2007). NPY is one of the most abundant peptides in the central (CNS) and periph-
eral (PNS) nervous systems and other peripheral tissue. NPY receptors are identical
for all members of the NPY family and belong to class A G-protein-coupled recep-
tors. Results from several studies suggest that the NPY system is linked to the aging
process (for review, see Botelho and Cavadas 2015) (Table 9.1). VIP belongs to a
family of structurally related peptides including secretin, glucagon, gastric inhibi-
tory peptide, growth hormone-releasing factor, and PACAP and signals through
three different subtypes of VIP receptors (reviewed in Lerner and Persson 2008)
(Table 9.1).
A further important point to understand the effects of sympathetic neurotrans-
mitters is that they do not have 0-1 effects. NE, for instance, binds to α2-AR below
the concentration of 10−7 mol/l and decreases cAMP via inhibitory G-protein (Gi)
signaling, whereas at concentrations higher than 10−7 to 10−8 mol/l, NE binds to
β-AR leading to cAMP increase via stimulatory G-protein (Gs) signaling (Fig. 9.1).
This phenomenon explains why these neurotransmitters show U-shaped and bell-
shaped dose-response curves (reviewed in detail in Straub 2015).
Unlike other musculoskeletal connective tissue as bone, periosteum, and
synovium, cartilage does not contain blood vessels and is not innervated by nerve
fibers indicating that cartilage for some reason might be a hostile environment for
spreading of nerve fibers. However, despite lack of nervous innervation, cartilage
metabolism is modulated and influenced by neurotransmitters released either from
nerve fibers located in neighboring tissue or directly from chondrocytes (for review
see Grassel 2014).
This chapter will review recent literature describing effects of sensory and sym-
pathetic nerve fibers and their neurotransmitters on joint physiology and pathophys-
iology including articular cartilage, growth cartilage, intervertebral disk cartilage,
synovium, and subchondral bone.
9.2 ensory and Sympathetic Nerve Fibers in Cartilage

S
Physiology
9.2.1 Synovial Joints
There is some evidence that sensory nerve fibers may come in contact with those
chondrocytes located in growth cartilage and at the surface of articular cartilage. In
rat knee joints, CGRP-positive fibers, which originate from the periosteum and near
insertion regions of muscle and tendons, penetrate for up to 25 μm into the outer
layer of articular and meniscal cartilage. These fibers are located between single
chondrocytes indicating a local effector function. However, there are subpopula-
tions of SP-positive axons in perichondrium and periosteum which for unknown
reasons do not innervate the cartilage (Schwab and Funk 1998). CGRP- and
SP-positive nerve fibers precede the development of cartilage canals, which are
formed during skeletal growth shortly after birth. These fibers were detected when
penetrating into canals of growth cartilage in the epiphysis of young rats, thereby,
coming into close contact to chondrocytes (Edoff et al. 2000; Hara-Irie et al. 1996).
The presence of cartilage canals carrying sensory nerve fibers precedes the develop-
ment of the secondary ossification center, and it is speculated that these nerve fibers
modulate the formation of synovial joints through trophic effects (Oliva et al. 2005).
These observations imply important functions of sensory nerve fibers for regulat-
ing chondrogenic differentiation during limb growth in embryonic development.
Along this line, Edoff et al. reported that articular chondrocytes respond specifically
to application of CGRP by increasing cAMP level (Edoff et al. 2000; Edoff and
Hildebrand 2003). They assume that dorsal root ganglion (DRG) neurons, which
project to growth cartilage, may influence chondrocyte differentiation via CGRP. It
is described that increased levels of cAMP as response to CGRP suppresses termi-
nal differentiation of chondrocytes and matrix mineralization (Jikko et al. 1996).
This makes it likely that locally released CGRP can delay chondrocyte hypertrophy
and subsequent terminal differentiation through modulating cAMP level (Fig. 9.2).
Chondrogenic differentiation
Endochondral ossification
Proliferation and
Chondrogenesis Ossification
terminal differentiation
Cartilage
Mesenchymal cell Bone
template
Mesenchymal stem Osteochondro- Resting Proliferative Hypertrophic Osteocyte

cell progenitor chondrocyte chondrocyte chondrocyte
Collagen I, III; Sox9 Collagen IIA, N- Collagen IIA, IX, Collagen IIB, IX, XI; Collagen X, MMp- Osteocalcin, Runx2
Cadherin; Sox9 XI; Sox9 Sox9, Runx2 13; Runx2
2?
NK1Rb2 NK1R b2
a1/
b2
CR
NK1R LR b2
Chondrogenic differentiation (3)
b2
Chondrogenic differentiation (3)
NE, Iso,
Terminal differentiation (8, 9)

Proliferation (1, 2)
SP NE NE SP Pro NE
differentiation (5, 6)
Chondrogenic
Adhesion (4)
CGRP Pro, Iso

Adhesion (4)
Proliferation (4) SP NE
Proliferation (7)
differentiation (7, 10)

Hypertrophic
Apoptosis (4)
Fig. 9.2 Role of sensory and sympathetic neurotransmitters and their receptors in chondrogenic
differentiation. Sensory (SP and CGRP) and sympathetic (NE) neurotransmitters and antagonists/
agonists (isoproterenol, propranolol) affect chondrogenic differentiation and metabolisms of chon-
droprogenitor cells and bone marrow-derived stem cells (BMSC). These neurotropic effects are
mediated through specific receptors for sensory neuropeptides, NK1R and CRLR/RAMP-1, and
mainly the sympathetic ß2-AR. A line with an arrow indicates stimulation and a line with a bar
indicates inhibition. The red (green) nerve ending represents sensory (sympathetic) nerve fibers
(Modified from Grassel 2014). SP substance P, CGRP calcitonin gene-related peptide, NE norepi-
nephrine, Iso isoproterenol, Pro propranolol, Nk1 neurokinin 1 receptor, CRLR CGRP receptor, ß2
ß2-adrenoceptor. Numbers indicate references according to bibliography: 1 Fortier and Nixon
(1997), 2 Goto et al. (2007), 3 Jenei-Lanzl et al. (2014), 4 Opolka et al. (2012), 5 Mitchell et al.
(2011), 6 Takahata et al. (2009), 7 Lai and Mitchell (2008), 8 Edoff et al. (2000), 9 Edoff and
Hildebrand (2003), 10 Mauro et al. (2010)
Similarly, to sensory nerve fibers, sympathetic nerve fibers are abundantly pres-
ent in subchondral bone marrow and synovial joint tissue (Maestroni 2000; Pongratz
and Straub 2013) (Fig. 9.3). Under physiological conditions, sympathetic nerve
fibers control the blood flow in adult joint vessels via vasoconstriction. Ferrell and
colleagues described that these effects are mediated mainly through α1-AR but not
through β-AR (Ferrell and Khoshbaten 1990, 1989). Since hyaline cartilage is an
aneural tissue, there are very few studies that addressed the potential link between
sympathetic nerve fibers or neurotransmitters and cartilage or chondrocytes,
respectively.
The only existing report in this context observed type C nerve fibers (absence of
myelin or Schwann cells) with sensory or sympathetic function in cartilage canals
in human fetal epiphyseal uncalcified cartilage (from 22nd gestational week to
13 months after birth), which serves as bone template during endochondral ossifica-
tion (Strange-Vognsen and Laursen 1997). These nerve fibers may come into close
healthy OA RA
bone
loss of
sensory fiber sympathetic
sprouting fibers
osteophytes
primary
synovitis/
meniscus pannus
cartilage secondary
cartilage
destruction synovitis
bone and cartilage

synovium erosion
bone marrow
sensory nerve fiber mesenchymal progenitor cell synovial fibroblast B-lymphocyte

and neuroransmitter
sympathetic nerve fiber chondrocyte TH+ synovial fibroblast TH+ B-lymphocyte

and neurotransmitter
TH+ chondrocyte macrophage T-lymphocyte
meniscus cell TH+ macrophage TH+ T-lymphocyte
TH+ meniscus cell osteoclast dendritic cell
Fig. 9.3 Neuronal changes from healthy conditions to osteoarthritic (OA) and rheumatoid arthritic
(RA) joints. In the healthy situation, nerve fiber densities of sensory and sympathetic nerve fibers
are similar, and the ratio of nerve fiber density is 1:1. This situation is more or less the same in OA,
but it markedly changes in RA, where sensory nerve fibers sprout into the tissue while sympathetic
nerve fibers get lost. The nerve fiber ratio (sensory: sympathetic) changes from 1:1 to 10:1. This is
accompanied by a profound thickening of the synovial tissue, called pannus formation. Since the
loss of nerve fibers is specific for the sympathetic branch, it is hypothesized that specific repellent
factors of sympathetic nerve fibers play a major role
contact to chondrocytes and regulate the developmental process of long bone for-
mation as described for sensory nerves (see above) (Fig. 9.2).
For other components of the adult skeletal system, i.e., bone, the importance of
the sympathetic nervous system has been shown. Best characterized are the effects
of leptin-induced activation of sympathetic nerve fibers, which leads to changes in
bone formation and remodeling (Elefteriou and Karsenty 2004; Elefteriou et al.
2005; Takeda et al. 2002). These studies focused mainly on β2-adrenergic induction
of bone loss through upregulation of RANKL by osteoblasts and activation of
osteoclasts.
So far, no data exist for sympathetic neurotransmitter concentrations in perma-
nent cartilage or growth cartilage during skeletal growth. One can speculate that
these neurotransmitters contribute to or work against the effects of sensory neu-
rotransmitters via regulation of intracellular cAMP levels. In rabbit growth plate
chondrocyte cultures, dibutyryl cAMP (membrane permeable analog of cyclic
AMP) abrogated the increase in chondrocyte size and the expression of hypertro-
phic markers such as alkaline phosphatase (ALP) and collagen X, affirming that
cAMP plays a crucial role in suppressing terminal differentiation of chondrocytes
and cartilage-matrix calcification during skeletal development (Jikko et al. 1996).
Furthermore, Lai et al. observed that β2-AR-dependent stimulation of adenylyl
cyclase by isoproterenol treatment leads to increased chondrocyte proliferation and
decreased expression of hypertrophy markers such as collagen X in murine embry-

onic growth plate chondrocytes (Lai and Mitchell 2008). Although this study uses a
non-selective β-AR agonist and does not provide information on growth plate sym-
pathetic neurotransmitter influence in vivo, the data clearly show that the sympa-
thetic nervous system probably is an important regulator of embryonic cartilage
development (Fig. 9.2).
Sympathetic neurotransmitters secreted from adult joint tissue, e.g., subchondral
bone marrow or synovial tissue, might influence chondrocyte physiology. Since a
while, it is known that sympathetic nerve fibers are present in these joint structures
(Maestroni 2000; Pongratz and Straub 2013) (Fig. 9.3). In synovial fluid of trau-
matic knee injury patients, norepinephrine was detected at a concentration between
10−8 and 10−9 mol/l (Jenei-Lanzl et al. 2014), but levels in cartilage tissue are not
known.
The only report on the effect of chronic immobilization stress on cartilage and
subchondral bone in murine temporomandibular joints demonstrated increased sub-
chondral bone NE levels and concomitantly promoted condylar subchondral bone
loss and cartilage degradation (Jiao et al. 2015). These authors concluded that
increased activation of the sympathetic nervous system by stress might be a factor
that is involved in degenerative changes of condylar subchondral bone.
A further illuminating point in the context of cartilage physiology and sympa-
thetic neurotransmitters is the role of local anesthetics for diagnostic and trauma-
therapeutic purposes (Rao et al. 2014). Rao and colleagues showed that more than
60 % of human chondrocytes in culture were apoptotic after addition of 1:1,000
epinephrine (~5 μM, thus β-AR-mediated) for only 90 min. Similarly, epinephrine
caused a significant increase in cell death of synovial fibroblasts resulting in a higher
release of matrix metalloproteinases (MMPs) and subsequent damage of the proxi-
mate cartilage extracellular matrix (ECM) (Braun et al. 2013).
Taken together, it is abundantly clear that despite the a priori aneural nature of
cartilage tissue, the sensory and sympathetic nervous system influence cartilage
physiology by releasing neurotransmitters from surrounding innervated joint tissue
or by influencing cells of surrounding tissue which then in turn synthesize factors,
i.e., growth factors, cytokines, proteases, etc., to influence chondrocyte function.
9.2.2 Intervertebral Disks
It is common knowledge that the normal nucleus pulposus (NP), the inner annular
zones, and the cartilaginous endplate (CED) are devoid of nerves. Anatomical and
experimental studies during the last decades have provided many details of lumbar
spine innervation (see review Edgar 2007; Raj 2008 and Chap. 10). The normal
intervertebral disk (IVD) is considered as an organ that is poorly innervated sup-
plied only by sensory and sympathetic perivascular nerve fibers. Most of the studies
performed in different animal species, including human, have demonstrated that
nerve fibers in IVDs are found mostly in the periphery of the annulus fibrosus (AF)
and are typically very fine in diameter presumably being C-fibers. The sensory
fibers that innervate the IVD are mainly nociceptive and, to a lesser extent, proprio-
ceptive. In the human healthy IVD, nerve fibers which innervate the outer layers of
the AF are positive for SP, CGRP, VIP, and NPY, among others (see review Garcia-
Cosamalon et al. 2010), whereas the NP is not innervated. The IVD is primarily
innervated by small DRG neurons, which are classified into peptidergic nerve
growth factor (NGF)-dependent and non-peptidergic glial cell line-derived neuro-
trophic factor (GDNF)-dependent neurons that are functionally different (Stucky
and Lewin 1999). The NGF-dependent neurons – positive for SP and CGRP – are
critical to hyperalgesic responses induced by inflammation (Mantyh et al. 1997),
whereas the GNDF-dependent neurons are important in neuropathic pain (Malmberg
et al. 1997). A number of repulsive factors that exist in the healthy IVD presumably
prevent nerve and endothelial cell ingrowth. These factors are aggrecan (Johnson
et al. 2006), chondromodulin (Takao et al. 2000), and the semaphorins (Tolofari
et al. 2010).
Growth factors such as NGF, brain-derived neurotrophic factor (BNDF), NT3,
and NT4/5, which are members of the neurotrophin (NT) family, and their signaling
receptors (NTRs) have been detected in healthy IVDs in innervated regions and also
in regions lacking sensory or sympathetic innervation (Gruber et al. 2008). Upon
binding to their receptors (TRKs A, B, C), NTs initiate a variety of signals that lead
to regulation of cell proliferation, differentiation, and survival. NGF and its receptor
TrkA are expressed in AF cells and BNDF and its receptor TrkB are found predomi-
nantly in the outer AF region and to some extend in the NP cells.
Notably, NTs produced in the IVD can be retrogradely transported to DGR sen-
sory neuron bodies where they regulate expression of SP and CGRP. In addition, the
expression of NTs and NTRs in AF and NP cells suggests autocrine and paracrine
roles in regulating the physiology of IVD (see review Garcia-Cosamalon et al.
2010). NTs also play a role in inflammatory responses and in pain transmission. In
particular, NGF can act as a cytokine (Levi-Montalcini et al. 1996) influencing
development, differentiation, chemotaxis, and mediator release of inflammatory
cells, as well as fibroblast activation, through complex networks that involve other
pro-inflammatory cytokines (Bonini et al. 2003). Furthermore, NTs play key roles
in immunocompetent cells by participating in inflammatory responses (Tessarollo
1998; Sariola 2001; Vega et al. 2003) and in sensing pain, because NT signaling
increases expression of pain-related peptides.
9.3 ensory and Sympathetic Nerve Fibers in Cartilage

S
Pathophysiology
9.3.1 Osteoarthritis
9.3.1.1 Sensory Nerve Fibers and Neuropeptides

Suri et al. have localized both sensory (SP and CGRP positive) and sympathetic
nerve fibers (NPY positive) in similar distributions to the articular cartilage in
human tibiofemoral osteoarthritis (OA) (Suri et al. 2007) (Figs. 9.3 and 9.4). These
Normal OA
Femur
Cracks
Synovial membrane
Erosions
Meniscus
Articular cartilage
Osteophyte
Calcified cartilage
Osteochondral junction
Subchondral bone
Tibia
Sensory nerve fibers
Sensory neuropeptides
Fig. 9.4 Proposed alteration of sensory nervous innervation in osteoarthritis. It is discussed that
there is an increase of sensory nerve fiber innervation which reaches into the calcified cartilage
zone from the subchondral bone or which comes into close contact to the articular cartilage.
Contrary, synovial sensory innervation decreases in an early OA stage and presumably also in later
OA stages. In addition, concentration of sensory neuropeptides in synovial fluid increases with
increasing OA severity
nerve fibers were present within vascular channels in both mild and severe OA. For
the pathogenesis of OA, it is described that fine unmyelinated nerves grow into joint
structures through vascular channels mainly from subchondral bone breaching
through the tidemark.
The exclusively perivascular localization of nerves within the articular cartilage
implies that vascularization is the driving force behind its innervation. Notably, vas-
cularization of the non-calcified cartilage was found throughout a wide range of
histological OA stages and was not restricted to end-stage OA. Possibly, scoring of
nervous innervation and vascularization of cartilage might be exploited as a mea-
sure for severity of degradative changes in OA pathogenesis. The authors suggest
that vascularization and accompanying innervation of the articular cartilage is a
potential source of pain in patients with knee OA. They and others have also shown
sympathetic and perivascular sensory innervation of tibial osteophytes, latter local-
ized to the base of osteophytes (Suri et al. 2007; Wojtys et al. 1990). Sensory inner-
vation of osteophytes may explain why radiological grading of osteophytosis is
associated with reported pain severity (Fig. 9.4).
However, despite the clinical importance of pain in OA, pain mechanisms are
poorly understood. It is unclear which structures in the joint give rise to OA pain,
and the nature of OA pain (nociceptive versus neuropathic) is a matter of ongoing
discussion (Schaible 2012). A tissue compartment, which can be a source of pain in
OA, is the osteochondral junction. Interestingly, angiogenesis seems to predominate
in both early and late OA, although, besides angiogenic factors, also anti-angiogenic
factors have been shown to be upregulated in both OA stages as observed in rats
(Mapp et al. 2008; Mapp and Walsh 2012).
During pathogenesis of OA, osteochondral angiogenesis was increased associ-
ated with increased NGF expression in subchondral spaces, vascular channels, and
by chondrocytes themselves (Walsh et al. 2010). This NGF immunoreactivity was
co-localized with CGRP-immunoreactive nerve fibers in the same vascular chan-
nels. Increased NGF production may thus contribute to OA pain, both structurally
(increased aberrant innervation at the osteochondral junction) and through periph-
eral sensitization. This was confirmed in studies with human OA material showing
that increased vascular penetration and density of perivascular CGRP-positive sen-
sory nerve profiles in the meniscus is a potential source of pain in knee OA and
leads in addition to further inflammation and tissue damage (Ashraf et al. 2011a, b).
In a murine OA model, it was suggested that in some soft joint tissue, CGRP- and
SP-positive nerve fibers disappeared 5 weeks after induction of OA with intra-
articular injection of collagenase (Buma et al. 1992). This observation was con-
firmed by a study of Murakami et al. who report that the densities of PGP 9.5- and
CGRP-positive nerve fibers in the synovium were drastically decreased 1 week after
the collagenase injection. However, by week 4, the density of PGP 9.5- and CGRP-
positive nerve fibers had recovered to 84 % and 79 %, respectively, of their normal
levels. Despite the poor correlation between the synovitis score and the density of
CGRP- or SP-positive nerve fibers in the synovium, the ossification rate of chondro-
phytes in chondro/osteophyte lesions correlated strongly with the density of CGRP-
positive nerve fibers (Murakami et al. 2015). This quite aggressive method of OA
induction leads to profound degenerative alterations in synovial joint tissue (among
other soft tissue) after a few weeks. Contrary, some studies of human OA have
reported an increase in SP- and CGRP-positive nerve fibers in the synovial tissue of
knee joints (Saito 1992; Saito and Koshino 2000; Saxler et al. 2007), whereas others
have described a decrease in these nerve fibers in the synovium (Gronblad et al.
1988). Because most of these reports are from studies of the synovium in chronic
OA, there is little information about the association between the pathological
changes in the knee and changes in these nociceptive fiber innervation patterns dur-
ing the acute phase of the disease. The mechanisms that possibly destroy the normal
vascular and neural network in early or late OA stages are not identified yet, and,
similarly, it is unclear if nerves are destroyed as a consequence of OA or if patho-
genesis of OA is facilitated due to nerve disappearance.
A study by Salo et al. indicated that loss of SP- and CGRP-positive innervation
from the whole joint always preceded histological changes of cartilage degenera-
tion (Salo et al. 2002). They used a mouse model, where mice usually developed
a mild form of OA, but surgical ablation of joint innervation caused the develop-
ment of severe patellofemoral OA. Their findings would be consistent with the
hypothesis that an age-related loss of sensory joint innervation may contribute to
the development of OA. However, in human synovial tissue of late-stage OA
patients, undergoing knee joint replacement surgery, the density of SP-positive

sensory nerve fibers was markedly lower as compared to RA patients and trauma
controls (Weidler et al. 2005) (Fig. 9.3). Whether sensory nerve fibers are lost,
remain unaltered, increase or change tissue distribution as a prerequisite of OA
pathogenesis in humans remains to be determined. Notably, CGRP and SP syno-
vial fluid levels are increased in developmental dysplasia of the hip. CGRP con-
centration in synovial fluid is increased in knee OA patients and clearly correlated
with increased Kellgren-Lawrence scores (indicating OA severity) (Dong et al.
2015; Wang et al. 2015; Li et al. 2015). Further insight into these mechanisms and
relations will require reproducible OA-animal models resembling slow proceed-
ing pathogenesis of human OA and enabling longitudinal studies from early onset
of the disease to late stages.
9.3.1.2 Sympathetic Nerve Fibers and Neurotransmitters

The presence of specific neurotransmitter receptors is a prerequisite for neurotrans-
mitter action. In healthy human cartilage, dopaminergic and β-adrenergic receptors
have been detected and, interestingly, the expression of these receptors was signifi-
cantly increased in OA cartilage (Vignon et al. 1990) suggesting the contribution of
the sympathetic nervous system to OA manifestation.
Recent studies on human chondroprogenitor cell chondrogenesis demonstrated
that the sympathetic neurotransmitter NE might influence the self-regeneration
capacity of articular cartilage via β2-AR signaling. The deposition of cartilage-
specific collagen II was significantly inhibited and concomitantly, collagen X
expressed in hypertrophic chondrocytes, was significantly increased after NE treat-
ment (Jenei-Lanzl et al. 2014). Thus, NE might play a pivotal role in the develop-
ment and manifestation of OA. However, the exact effects of sympathetic
neurotransmitters on cartilage tissue homeostasis, on its capacity for spontaneous
regeneration, and, thus, on OA development are largely unclear.
Another interesting finding in long-standing OA is a higher density of sympa-
thetic nerve fibers in synovial tissue in relation to sensory nerve fibers when com-
pared to trauma controls (Fig. 9.3). This phenomenon might lead to unfavorable
elevated levels of sympathetic neurotransmitters in relation to growth-promoting
sensory neurotransmitters (Weidler et al. 2005).
NPY, a co-transmitter of NE, is increased in OA synovial fluid, which is inter-
preted as a sign of secondary inflammation. Wang et al. observed a direct correlation
between NPY concentration in synovial fluid of OA patients and pain indicating
that NPY may act as a putative regulator of pain transmission and perception in OA
(Wang et al. 2014). High NPY concentrations lead to further degradation of carti-
lage, because NPY potentiates inflammation by modulating key immune cell func-
tions via Y1/Y2 receptors (Dimitrijevic et al. 2008). However, higher levels of NPY
might also demonstrate that the generally higher release of sympathetic neurotrans-
mitters, and NE signaling via α2-AR can induce pain.
Contradictory to presence of sympathetic nerve fibers in OA synovium and car-
tilage channels, OA patients have lower VIP concentration in synovial fluid
compared to trauma patients (Jiang et al. 2006). The reason for this negative asso-
ciation of VIP with progressive joint damage in OA is not known at present (Delgado
et al. 2001). Since VIP has been shown to predominantly possess anti-inflammatory
effects (Jiang et al. 2006), its insufficient expression might contribute to the charac-
teristic secondary inflammation emerging in late OA which is very important to
consider, because over 90 % of OA patients develop synovitis (Roemer et al. 2011).
Since VIP is the neurotransmitter of the cholinergic subtype of sympathetic nerve
fibers, it might also show that this particular sympathetic nerve fiber type decreases
in OA joints.
Of note, sympathetic neurotransmitters have a limited chemical stability, which
is not only caused by converting enzymes but also by degrading enzymes, e.g.,
monoaminooxidase (MAO) and catechol-o-methyltransferase (COMT) for cate-
cholamines (Jiang et al. 2006; Napolitano et al. 1995) or adenosine deaminase
(ADA) for adenosine (Antonioli et al. 2012; Cortes et al. 2015). Taken together,
local availability of neurotransmitters depends on synthesizing, converting, and
degrading enzymes.
Chambers et al. observed an abnormal localization of MAO in murine osteoar-
thritic cartilage, which is normally tightly bound to the outer membrane of mito-
chondria in healthy tissue cells. In OA cartilage, MAO was abnormally located
extending beyond the chondrocytes into the surrounding matrix (Chambers et al.
1992). Under these conditions, catecholamines cannot be metabolized intracellu-
larly, and they accumulate pericellularly and might exhibit unfavorable effects on
chondrocyte function.
COMT, another catecholamine-degrading enzyme, has functional polymor-
phisms linked to different pain levels in OA patients (Stolk et al. 2007). A
single-nucleotide polymorphism results in a valine to methionine mutation at
position 158 (Val158Met). This variant results in three- to fourfold lower
enzyme activity compared to the valine variant. It has been shown to be associ-
ated with higher pain levels and increased radiographic damage of the hip.
Therefore, Stolk and colleagues propose that patients carrying the methionine
allele could be treated more effectively with drugs affecting neurotransmitter
function (Stolk et al. 2007).
Furthermore, it is important to consider adenosine, a co-transmitter of catechol-
amines, in OA pathophysiology. The enzyme ADA converts adenosine to inosine;
thus, ADA deficiency leads to adenosine accumulation. Children with ADA defi-
ciency due to genetic mutation have elevated adenosine levels in extracellular fluids.
They exhibit marked changes in the chondro-osseous tissue of vertebrae, and the
costochondral junction has increased numbers of necrotic chondrocytes and has
large amounts of cellular debris (Mediero and Cronstein 2013). A similar phenom-
enon was observed by Mistry et al. in murine cartilage tissue showing that increased
adenosine concentrations induce chondrocyte apoptosis and, therefore, might play a
role in OA pathogenesis (Mistry et al. 2006). The adenosine effect at high concen-
trations might be induced by elevated intracellular cAMP levels because adenosine
would bind to the A2A-/B-adenosine receptors.
9.3.2 Rheumatoid Arthritis
Although rheumatoid arthritis (RA) is a primary inflammatory systemic disease

with autoimmune characteristics, in contrast to the mainly injury- or wear- and bear-
driven OA, there are some analogies regarding disease outbreak and manifestation.
Similar to OA development, pathogenesis of RA is multifactorial and influenced by
the nervous system. Besides effects of the CNS and endocrine hormones (Del Rey
et al. 2010; Straub et al. 2013) (Straub RH), the peripheral nervous system has been
shown to influence development and progression of RA (Pongratz and Straub 2013;
Levine et al. 1987; Lorton et al. 2013). The first proof of this involvement provided
the observation that patients with hemiplegia develop RA symptoms only in the
non-paralyzed hand, whereas the hemiplegic hand shows no RA-specific signs
(Keyszer et al. 2004). Similar results of hemiplegic patients have been reported
several times. In a recent study, this phenomenon was impressively confirmed in
murine experimental arthritis, where denervation of limbs protected the animals
from the disease (Stangenberg et al. 2014).
The elements of the peripheral nervous system have various functions and
influence the inflammatory response differently. First, the sensory nervous system
transmits pain and danger signals from the periphery to the brain, thereby, acting
mainly pro-inflammatory. This function of the sensory nervous system is called
the efferent function leading to release of local pro-inflammatory SP. Second, the
sympathetic nervous system, which influences blood flow, vascular permeability,
and plays a dual role in inflammation depending on the stage of arthritis (Lubahn
et al. 2004; Harle et al. 2005), is both pro-inflammatory or anti-inflammatory.
Third, the parasympathetic nervous system conveys mainly anti-inflammatory
effects; however, its exact role in arthritis is still unclear (Pongratz and Straub
2013; Schaible and Straub 2014).
As soon as tissue homeostasis is disturbed, the CNS receives this information
from activated peripheral nociceptive sensory nerve fibers or by circulating cyto-
kines released locally due to tissue damage, e.g., IL‑6 (Besedovsky and del Rey
1996; Pongratz and Straub 2013). In turn, the CNS initiates the activation of the
hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic nervous system
both reacting locally at the site of inflammation or in secondary lymphoid organs.
This first neuroendocrine response within hours is pro-inflammatory and leads to
activation of the immune system (Pongratz and Straub 2013).
In addition, sensory (SP and αCGRP) neurotransmitters have strong vasodilatory
properties, and sympathetic neurotransmitters (NE and NPY) are chemotactic, both
leading to the recruitment of immune cells (Ruff et al. 1985; Smith et al. 1993;
Straub et al. 2000; Wheway et al. 2007). In inflamed tissue, peripheral sympathetic
and sensory neurons talk to each other leading to sensitization of sensory afferents
via α2-AR signaling (Niissalo et al. 2002; Chen et al. 1996). In adjuvant-induced
arthritis, β2-agonists and selective blocking of β2-adrenergic receptors and NE
depletion aggravate arthritis symptoms (Niissalo et al. 2002; Lubahn et al. 2004).
This confirms the pro-inflammatory nature of the sympathetic nervous system in an
early immune encounter (Schaible and Straub 2014). This first pro-inflammatory
response of the body is critical in order to fight the target antigen.
During pathogenesis of RA, the original balance between SP-positive sensory
nerve fibers and TH-positive sympathetic nerve fibers is disturbed at the site of
inflammation. Miller et al. observed the loss of TH-positive sympathetic nerve
fibers in chronic inflamed synovial tissue, whereas SP-positive nerve fiber density
increased (Miller et al. 2000) (Fig. 9.3). In contrast to SP-positive fibers, CGRP-
positive nerve fibers are lost from sensory neurons (Imai et al. 1997; Kimberly and
Byers 1988; Dirmeier et al. 2008). SP and CGPR exhibit opposite effects in RA. SP
induces the release of pro-inflammatory factors such as TNF, IL-1, IL-6, IL-8,
IL-12, IFN-γ, and superoxide anion and stimulates chemotaxis of immune cells
(Dirmeier et al. 2008). In contrast, CGRP has anti-inflammatory effects, e.g., the
inhibition of macrophage, monocyte, and human peripheral blood mononuclear
functions. The reasons for the loss of CGRP from sensory nerve fibers are not
known at present; however, it seems to be clear that this preponderance of SP-positive
nerve fibers over CGRP-positive fibers contributes to the maintenance of the pro-
inflammatory situation in RA (Dirmeier et al. 2008).
It has been proposed that the loss of TH-positive nerves is caused by the action
of elevated nerve-repellent factor levels such as semaphorins 3C and 3F, which are
specific for noradrenergic sympathetic nerve fibers (Fassold et al. 2009; Miller
et al. 2004) (for details see Sect. 10.5.2.). The local loss of sympathetic nerve fibers
has been observed also in other inflammatory diseases, e.g., in Crohn’s disease
(Straub et al. 2008) and in chronic Charcot foot (Koeck et al. 2009). Similarly,
sprouting of SP-positive nerve fibers was detected in other inflammatory diseases,
e.g., gastritis (Sipos et al. 2008), in esophagitis (Matthews et al. 2004), and in pso-
riatic skin disease (Naukkarinen et al. 1989) leading to the maintenance of a pro-
inflammatory situation.
Of note, the loss of sympathetic nerves would lead to the local decrease of
neurotransmitter concentration. It is important to consider that besides the first
pro-inflammatory response mentioned above, sympathetic neurotransmitters at
high concentrations have anti-inflammatory properties via Gαs-coupled cate-
cholamine receptors (Straub 2015). NE and its co-transmitter adenosine exhibit
anti-inflammatory effects mediated by β2-AR and adenosine A2A-/B-adenosine
receptors, respectively (Sanders and Straub 2002; Sitkovsky 2003). In addition,
the conversion of ATP to anti-inflammatory acting adenosine is elevated in RA,
because the ectonucleotidases (CD39 and CD73), which convert purine precur-
sor neurotransmitters such as ATP to adenosine, are increased in inflammation
(Straub 2015).
The early repulsion of sympathetic nerve fibers from inflamed tissue has been
positively selected to overcome acute wounding, because an anti-inflammatory
influence does not make sense before bacteria are cleared. Thus, after repulsion,
lower concentrations of NE are present locally and receptor signaling changes from
β2-AR to a predominantly α-AR signaling pathway, which is Gi driven and exhibits
mainly pro-inflammatory effects (Pongratz and Straub 2013).
Concomitant to TH-positive nerve fiber repulsion, TH-positive single cells

appear in the human and murine arthritic synovial tissue (Capellino et al. 2010,
2012) (Fig. 9.3). These cells synthesize and release catecholamines, possibly in
order to compensate the local loss of sympathetic nerve fibers (Jenei-Lanzl et al.
2015a; Miller et al. 2000, 2002). Interestingly, these recently described anti-
inflammatory TH-positive cells also appear in lymphoid organs at the same time
(Capellino et al. 2012). This appearance and function of TH-positive cells would
explain the time-dependent dual effect of sympathectomy observed in a murine
model of collagen-induced arthritis (CIA). In this model, the sympathetic nervous
system is pro-inflammatory in the early phase and anti-inflammatory in the late
chronic phase, where only TH-positive cells but no sympathetic nerves are present
in synovium (Capellino et al. 2012; Harle et al. 2005; Jenei-Lanzl et al. 2015a).
Cells of the inflamed synovium try to compensate sympathetic nerve fiber loss also
by upregulation of receptors for D1/5 dopamine receptors known for Gαs-mediated
signaling via cAMP (Capellino et al. 2014). However, even together with all these
neurotransmitters locally produced by cells, neurotransmitter levels are probably
still too low to exert anti-inflammatory effects.
Apart from NE and its co-transmitters, sympathetic nerve fibers after cholinergic
transition can secrete VIP, which is normally released by cholinergic parasympa-
thetic neurons (Steele et al. 1996). VIP-containing nerve fibers are present in human
and murine synovial tissue (Bjurholm et al. 1990). Similar to NE, VIP acts via Gαs-
coupled seven-transmembrane receptors, VIP receptor 1 and 2, by activating adeny-
lyl cyclase (Straub 2015). Thus, it is not surprising that VIP treatment of CIA mice
significantly reduced the incidence and severity of the disease and also destruction
of bone and cartilage (Delgado et al. 2001).
In addition, anti-inflammatory effects of VIP have been reported in patients with
RA (Gonzalez-Rey et al. 2006) and in animal experiments after CIA induction
(Muschter et al. 2015a, b). Here, it was shown that VIP reduced proliferation of
macrophages and has mostly protective effects on bone matrix by inhibition of
cathepsin K activity and decrease of osteoclast numbers, an observation probably
attributed to increased cAMP signaling. Therefore, the loss of sympathetic nerve
fibers results in the loss of anti-inflammatory neurotransmitters.
In general, locally produced catecholamines, VIP, or opioids are not powerful
enough to overcome the pro-inflammatory domination in chronic arthritis. Moreover,
in a most recent study, an unexpected pro-inflammatory switch from Gαs to Gαi
signaling depending on PDE4/β-arrestin interaction has been described in human
arthritic synovial tissue. This phenomenon is most probably responsible for reduced
efficacy of PDE4 inhibitors and Gαs agonistic neurotransmitters under these cir-
cumstances (Jenei-Lanzl et al. 2015b).
Multiple evidences confirmed that NPY influences the pathogenesis of RA
(Bedoui et al. 2004; Harle et al. 2006). Interestingly, NPY concentration is elevated
in RA joints, although the loss of NPY-containing nerve fibers from the superficial
synovium was reported assuming that these nerve fibers become restricted to deeper
zones of the synovium (Mapp et al. 1990; Larsson et al. 1991). One can speculate
that, similar to TH-positive single cells, also NPY-producing cells appear in the
synovium as a compensatory mechanism to nerve fiber loss (Capellino et al. 2012;

Jenei-Lanzl et al. 2015a; Miller et al. 2000). NPY potentiates inflammation by
manipulating immune cells via Y1/Y2 receptor signaling (Dimitrijevic et al. 2008)
or by boosting the effects of NE (Bedoui et al. 2004).
In addition, NPY has angiogenic activities in several tissues, however, only at the
healthy border of inflamed tissue because in the inner highly inflamed zone, the
sympathetic innervation, including NPY, is lost. However, sensory nerves are still
present, and in vivo experiments showed that SP induces angiogenesis in arthritic
synovium enabling the further circulation of immune cells leading to maintenance
of a pro-inflammatory condition (Ribatti et al. 2007).
This dominant and long-lasting pro-inflammatory situation in the synovium
under strong neuronal influence most probably affects the neighboring cartilage tis-
sue. Proliferating synovial pannus penetrates articular cartilage (Otero and Goldring
2007), and different cells of the pannus, e.g., fibroblasts, macrophages, and leuko-
cytes, release a broad spectrum of factors causing cartilage-matrix destruction and
bone erosion (Meyer et al. 2006; Knedla et al. 2007). Similarly, the action of inva-
sive synoviocytes and the contact of cartilage with the synovial fluid results in
destructive processes (Dodge and Poole 1989). In contrast to OA, where proteogly-
can and collagen loss occurs mainly in the cartilage superficial zone, in RA, also
middle and deeper zones are affected suggesting that the chondrocyte itself criti-
cally participates in destroying its own matrix by releasing ECM degrading enzymes
(Dodge et al. 1991; Mitchell and Shepard 1978; Kane et al. 2004).
Apart from synovial tissue and chondrocytes, also adipose tissue surrounding
the inflamed synovium secretes both pro-inflammatory and anti-inflammatory fac-
tors including leptin, IL-1RA, and IL-10 playing a role in cartilage physiology or
pathophysiology (Dayer and Burger 2004; Loeser 2003; Otero et al. 2006). Leptin,
one link between the neuroendocrine and immune system, is increased during
inflammation and has been shown to exhibit mainly pro-inflammatory properties
(Popa et al. 2005).
During skeletal development, mesenchymal progenitor cells in the growth
plate differentiate to chondrocytes providing the templates for the developing
bones (Goldring et al. 2006). A chondrogenesis-like process might also be impor-
tant for cartilage repair in adults since mesenchymal progenitors are present in
articular cartilage (Dowthwaite et al. 2004; Williams et al. 2010; O’Sullivan et al.
2011) and their number is increased in OA and RA (Pretzel et al. 2011; Fickert
et al. 2004; Grogan et al. 2009). As reviewed by Kondo et al., most pro-inflamma-
tory cytokines inhibit chondrogenic differentiation (Kondo et al. 2014). Thus, an
inflammatory situation, where sympathetic nerve fibers are lost and Gαi-mediated
effects are predominant or Gαs-mediated effects are not powerful enough, will
have negative influence on progenitors and consequently on cartilage defect
repair. In addition, catecholamines, released by sympathetic nerve fibers or by
immune cells, have been shown to enhance mesenchymal stem cell recruitment
from the bone marrow (Mignini et al. 2003; Stanojevic et al. 2013), given that
these cells express a variety of adrenergic receptors (Silva et al. 2003; Jenei-Lanzl
et al. 2014) (Fig. 9.1).
9.3.3 Intervertebral Disk Degeneration
The etiology of intervertebral disk degeneration (IVDD) is still unclear as is the

cause of low back pain; however, a correlation of both has been documented, and
the current consensus is that it is multifactorial (for review see (Vergroesen et al.
2015 and Chap. 10). Many changes in disk morphology and physiology have been
described but, still, no widely accepted disease model has been developed. However,
consensus exists that upon the degeneration process, IVDs become densely inner-
vated even in regions that normally lack innervation. This increased innervation
which is considered as part of a repair process concomitant with neovascularization
has been associated with discogenic pain (for review see Garcia-Cosamalon et al.
2010; Benneker et al. 2014).
Members of the neurotrophin family are likely involved in IVD hyperinnervation
(for details see Sect. 9.2.2). IVDs with symptoms of degeneration and those from
patients suffering from discogenic pain express increased levels of neurotrophins
and their receptors implying a positive correlation between levels of neurotrophins
and density of IVD innervation (Purmessur et al. 2008).
In human and animal models of IVDD, nociceptive fibers grow into usually
aneural parts of the AF and into the NP. These neurons are NGF sensitive and posi-
tive for SP and CGRP. Notably, the profile of these sensory neurons innervating
pathological IVDs remains identical to normal conditions; however, the nerve fiber
quantity is markedly increased constituting the phenomenon of sensory nerve fiber
sprouting (Takahashi et al. 2009). Freemont et al. report that levels of NGF are
higher in painful disks than in asymptomatic disks indicating that NGF may play a
role in sensitizing the painful disk (Freemont et al. 2002). Of note, there are contro-
versial reports that NGF expressing endothelial cells enter the IVD first followed by
sensory nerve fibers tracking alongside.
A recent study by Binch et al. observed nerve fiber ingrowth into the IVD also in
the absence of blood vessels challenging the hypothesis that neoinnervation requires
neoangiogenesis first (Binch et al. 2015). In addition to sensory nerve fibers, there
is growing evidence that afferents following sympathetic nerve fibers are also
increased in degenerating IVDs and that they play a significant role in lower back
pain (Takebayashi et al. 2006).
Binch et al. demonstrated that NP cells endogenously produce SP and CGRP
which become upregulated upon cytokine stimulation (Binch et al. 2014). In painful
IVDs, the levels of pro-inflammatory mediators such as TNFα, IL-1ß, and IL-8 are
higher as in asymptomatic IVDs which in turn upregulates NGF production and
secretion in cultured human IVD cells (Ahn et al. 2002; Lee et al. 2009). Notably,
cells isolated from human NP increase production of SP after stimulation with
TNFα, and levels of NGF together with SP were increased in human painful degen-
erated IVDs compared to non-degenerated NP cells (Purmessur et al. 2008;
Richardson et al. 2009). An association of TNFα with SP expression and IVD
degeneration is supported by the work of Chubinskaya and colleagues, who have
demonstrated downregulation of both genes in an anti-catabolic disk model
(Chubinskaya et al. 2007). Possibly, SP-containing nerve fibers contribute to
nociceptive processes within the degenerate IVD since expression of SP and its
receptor NK1R have been identified in both disk cells and nerve fibers growing into
the diseased IVD (Ohtori et al. 2006).
To this end, Kepler et al. reported that AF and NP cells expressed SP at low lev-
els, and expression did not change significantly with SP treatment but was signifi-
cantly upregulated after treatment with IL-1β/TNF-α (Kepler et al. 2013). The same
group detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin
receptor 3 (NK3R) in AF and NP cells (Kepler et al. 2015). Treatment of disk cells
with a specific NK1R antagonist was able to suppress expression of IL-1β, IL-6, and
IL-8 in a dose-dependent manner. Their study suggests that NK1R is responsible for
the pro-inflammatory effect of SP on IVD cells, and this effect can be blocked by
preventing binding of SP to NK1R. It became clear that SP mediates signaling in
disk cells through NK1R activating the pro-inflammatory p38-MAPK and ERK1/2
pathways.
9.4 ensory and Sympathetic Neurotransmitters and Their

S
Receptors in Chondrocytes
9.4.1 Sensory Neurotransmitters
Besides their classical function in nociception, SP and CGRP appear to have extra
functions in the musculoskeletal system. Lately, it has been reported that newborn
murine costal and adult human articular chondrocytes endogenously produce SP
and its receptor NK1R (Opolka et al. 2012; Millward-Sadler et al. 2003). Prior, SP
was immunolocalized to articular cartilage of dog shoulder joints (Karahan et al.
2002). Expression and localization were increased in chondrocytes and within the
cartilaginous ECM after low-impact regimented exercise indicating a role in signal-
ing pathways through which chondrocytes respond to mechanical stimulation. This
was demonstrated by Millward-Sadler et al. who suggested that SP is involved in
mechanotransduction via the NK1R (Millward-Sadler et al. 2000). In these experi-
ments, SP was necessary for a hyperpolarization response of the cell membrane, and
concomitant changes in gene expression as response to mechanical stimulation indi-
cate a role of SP in maintenance of articular cartilage-matrix integrity and function
in the presence of mechanical stress. The same group demonstrated that normal and
OA chondrocytes reacted differently to mechanical stimulation in that OA chondro-
cytes upregulated gene expression of the SP encoding gene, Tachykinin 1, whereas
non-OA chondrocytes did not show this phenomenon (Howard et al. 2008).
In addition, we recently demonstrated that proliferation was increased in costal
chondrocytes from newborn mice when stimulated with SP, which also stimulated
cell–matrix adherence by inducing formation of focal adhesion contacts. These
effects are mediated specifically through the NK1R (Opolka et al. 2012) which is
expressed mainly in middle and deep zones in articular cartilage chondrocytes
obtained from OA patients (Fig. 9.5). Our observation implies that SP might modu-
late the proliferation rate of growth plate chondrocytes and consequently terminal
CGRP-Receptor in human OA cartilage NK-1 Receptor in human OA cartilage

articular surface superficial zone articular surface superficial zone
middle zone middle zone
deep zone deep zone
tidemark tidemark
Fig. 9.5 Immunofluorescence staining of cryo-sections from human OA cartilage for NK1R and
CRLR. In OA cartilage, chondrocytes in the middle and deep zones are immunoreactive for a
NK1R antibody, whereas chondrocytes of the superficial zones remain mainly negative. In OA
cartilage, chondrocytes in the middle and deep zones were immune-positive for a CRLR antibody,
whereas chondrocytes of the superficial zone remain mainly negative. Bars (overview pic-
tures) = 200 μm, bars (enlarged pictures) = 50 μm
Substance P NK1R combined
a Epiphyseal bone
Growth plate
Metaphyseal bone
b
Resting zone
Proliferation zone
Hypertrophic zone
Fig. 9.6 SP and NK1R immunofluorescence staining on epiphyseal sections from 14-week-old
wild-type mice. Proliferating and hypertrophic chondrocytes of the growth plate are positive for
both NK1R (red fluorescence) and SP (green fluorescence). Additional, there are NK1R- and
SP-positive regions in the epiphyseal and metaphyseal bone. (a) Bars = 200 μm, (b) Bars = 50 μm
differentiation during endochondral ossification. Unpublished data from our group

demonstrate expression of SP and NK1R in growth plate chondrocytes (Fig. 9.6). It
is thus conceivable that in chondrocyte physiology and in chondrogenic differentia-
tion during skeletal growth, endogenous SP acts as a trophic, anabolic factor and
does not function as a classical neuropeptide.
However, in adults, the detection of higher levels of SP in synovial fluid from

patients with RA and OA, and increased expression of NK1R, indicates possibly
catabolic effects of SP on articular cartilage (Inoue et al. 2001). In addition, trans-
forming growth factor (TGF)-β and basic fibroblast growth factor (bFGF) play an
important role as inductor or promoter for production of SP in synovial fibroblasts.
These data are supported by Im et al. who elegantly demonstrated that SP induces
IL-1ß release (Im et al. 2008). The authors propose a mechanism by which bFGF
together with SP reduces proteoglycan deposition and stimulates production and
release of matrix metalloprotease (MMP)-13 in human articular chondrocytes and
thus accelerates catabolic processes in cartilage.
Altogether, these observations suggest that SP has autocrine functions and dif-
ferentially modulates chondrocyte metabolism and cartilage homeostasis during
skeletal growth and in pathophysiology. Like in synovial cells, where SP is described
as potent mediator of inflammation by promoting secretion of PGE2, several MMPs
(Lotz et al. 1987), reactive oxygen species (Tanabe et al. 1996), IL-1β and TNFα
(Lotz et al. 1988), and SP might also act in a catabolic way on chondrocytes and to
promote cartilage degradation.
Up to date, there are no reports listed in PubMed with respect to production of
CGRP and its receptors in cartilage. However, our unpublished data demonstrate
expression of CRLR/RAMP-1 in articular cartilage chondrocytes obtained from OA
patients. Expression was detected mainly in middle and deep zones similar to the
NK1R (Fig. 9.5). Since in bone metabolism CGRP is described as an anabolic factor
by stimulating osteoblast activity and, thus, bone formation (Elefteriou 2005; Schinke
et al. 2004), one might hypothesize that CGRP has similar anabolic effects in cartilage
physiology. However, under pathophysiological conditions as in osteoarthritis, inhibi-
tion of CGPR effects by blocking its receptor with an antagonist, subchondral bone
sclerosis was attenuated in a murine surgical OA model (Destabilization of the Medial
Meniscus = DMM) (Nakasa et al. 2015). Consequently, articular cartilage erosion and
degeneration were delayed in the early stage in this OA model. Of note, these effects
were abrogated in a later stage of the disease (8 weeks after OA induction) indicating
that other factors affect bone turnover compensating for CGRP effects.
9.4.2 Sympathetic Neurotransmitters
Given the fact that sympathetic nerve fibers and/or neurotransmitter-producing cells
are present in joint tissue, except in articular cartilage and the avascular zone of the
meniscus (Jenei-Lanzl et al. 2014; Miller et al. 2000), neurotransmitters released
into the synovial fluid can influence cartilage tissue provided that specific neu-
rotransmitter receptors are present on chondrocytes (Fig. 9.1).
Takarada and colleagues detected β2-AR and α2a-AR gene expression at differ-
ent developmental stages in mice. In neonatal mouse tibial sections, β2-AR and
α2a-AR mRNA expression was found in chondrocytes by in situ hybridization
(Takarada et al. 2009). All other adrenergic receptors were not present. Similarly,
Opolka et al. described that β2-AR and α2a-AR mRNA but also α1b-AR and
α1d-AR were expressed in newborn murine costal chondrocyte cultures (Opolka

et al. 2012). A study performed by Lai et al. confirmed only the expression of β2-AR
on growth plate chondrocytes from ribs of embryonic E18.5 mice (Lai and Mitchell
2008) (Fig. 9.2).
Studies on cartilage tissue explants derived from OA patients after knee replace-
ment surgery and on chondrogenic progenitor cells isolated from human OA carti-
lage showed that also under degenerative conditions β2-AR and α2-AR are present
(Jenei-Lanzl et al. 2014; Lorenz et al. 2016). Regarding alterations in biomechani-
cal pathways in OA pathogenesis, it is important to consider that β2-AR might
contribute to disease development related to overloading of cartilage, because
β2-adrenergic drugs have been shown to influence mechanical events in bone tissue
(Elefteriou et al. 2005, 2014).
In addition, high expression of the A2a and A2b adenosine receptors has been
detected in OA chondrocytes (Koolpe et al. 1999). Both of these A2 adenosine
receptor subtypes play a role in attenuation of inflammatory processes elicited by
other cell types. It is known that NPY receptors play an important role in bone
homeostasis (Lundberg et al. 2007; Baldock et al. 2002). However, no data exists
about chondrocytes expressing NPY receptors, and only one study confirmed the
existence of the Y1 receptor on bone marrow stromal cells being able to contribute
to cartilage repair processes (Lundberg et al. 2007). Similarly, no studies described
the presence of VIP receptors on chondrocytes until now.
So far, no studies exist comparing different sympathetic receptor expression lev-
els between healthy and pathological conditions. In fact, sympathetic neurotrans-
mitters influence both chondrogenic progenitor cell differentiation and mature
chondrocyte function. Catecholamines released by sympathetic neurons or by
immune cells influence mesenchymal progenitor cell migration from the bone mar-
row. There is growing evidence that G-CSF plays a major role in MSC recruitment
driven by nervous stimuli (Dygai et al. 2012; Katayama et al. 2006; Saba et al.
2013). Interestingly, Maestroni and colleagues demonstrated that besides sympa-
thetic nerves in bone marrow, also bone marrow cells can synthesize substantial
amounts of catecholamines (Maestroni 2000; Maestroni et al. 1998). Moreover, NE
and dopamine release is subject to a circadian rhythm with early morning peaks.
Thus, increased sympathetic influences during stress or in an inflammatory situation
might critically influence progenitor function in bone marrow.
Furthermore, Takarada and colleagues treated murine pre-chondrogenic cells
with epinephrine and observed β2-AR-dependent inhibition of Sox9 and conse-
quently of chondrogenic gene expression via classical Gαs-cAMP signaling
(Takarada et al. 2009). Similar inhibitory effects of NE on BMSC and on OA
cartilage-derived progenitor cell chondrogenesis were reported (Jenei-Lanzl et al.
2014). NE in high concentrations inhibited collagen II and glycosaminoglycan
deposition via β2-AR signaling, and, in addition, it accelerated the expression of
hypertrophic markers like collagen X and MMP-13. NE in low concentrations, act-
ing preferentially via α-AR, had no effects.
In contrast to findings in progenitor cell chondrogenesis studies, Lai et al.
reported that the specific β2-AR agonist isoproterenol inhibited Indian hedgehog
(Ihh) and collagen X expression via cAMP and ERK1/2 activation in the murine
growth plate (Lai and Mitchell 2008) (Fig. 9.2). Furthermore isoproterenol stimu-
lated the proliferation of chondrocytes. In a later work, these authors elaborated on
the involvement of the transcription factor Jun-B, activated by β2-AR in chondro-
genesis, inhibiting the expression of Sox-6 and collagen II (Mitchell et al. 2011)
similar as in MSCs (Jenei-Lanzl et al. 2014). In costal chondrocytes isolated from
newborn mice, apoptosis decreased after NE stimulation; however, ECM formation
was not influenced by NE (Opolka et al. 2012).
Jikko and colleagues confirmed the cAMP-driven suppression of chondrocyte
hypertrophy by measuring alkaline phosphatase (ALP) activity and collagen X syn-
thesis after dibutyryl-cAMP treatment in rabbit growth plate chondrocyte cultures
(Jikko et al. 1996). Interestingly, Mauro et al. described completely contrary effects
in the chicken growth plate. NE did not affect collagen II expression, but enhanced
collagen X mRNA levels (Mauro et al. 2010) (Fig. 9.2). A further deviation from the
studies mentioned above is the involvement of α-adrenergic receptors in their data.
There is no explanation at present for these contrary findings regarding sympathetic
influences on growth plate chondrocytes other than possible switch of G-protein-
dependent signaling. High agonist concentrations, followed by permanently high
intracellular cAMP concentrations, might lead to switching of stimulatory
G-protein-mediated signaling to inhibitory G-protein-dependent signaling (Sun
et al. 2007; Jenei-Lanzl et al. 2015b; Baillie et al. 2003).
NE influences the function of human OA chondrocytes as well. In OA, espe-
cially in late stages, inflammation is a hallmark. However, not much is known
about the effect of inflammation on chondrocyte function in OA. For instance,
Lorenz and colleagues investigated the effects of NE together with inflammatory
stimuli like IL-1ß, simulating a late OA microenvironment (Lorenz et al. 2016).
NE in high concentrations decreased chondrocyte proliferation via β2-AR signal-
ing. In contrast, NE in low concentration increased the proliferation of OA chon-
drocytes via α1-AR signaling suggesting that NE might exhibit dual effects on
chondrocyte proliferation in OA depending on sympathetic activity. In addition,
NE reversed IL-1ß-mediated suppression of collagen II and glycosaminoglycan
synthesis at high concentrations. Furthermore, Lorenz et al. showed the first time
that TH+ chondrocytes are present OA cartilage. This indicates that the presence of
sympathetic nerve fibers (in contrast to RA) and a high sympathetic activity would
be beneficial in OA. However, no in vivo evidence exists at present confirming this
hypothesis (Fig. 9.1).
It is important to think about further sympathetic neurotransmitters that signal
via Gαs-coupled receptors and cAMP, e.g., adenosine. There is no evidence at pres-
ent that adenosine influences progenitor cell chondrogenesis or mature chondrocyte
function. However, one can speculate about similar effects compared to NE and
epinephrine, given the fact that different adenosine receptors signaling via Gαs pro-
tein are detected on MSCs and on chondrocytes (Carroll and Ravid 2013).
Taken together, all those reports discussed above are providing different data
which do not allow a clear prognosis in which way sympathetic neurotransmitters
contribute to chondrogenesis of progenitor cells and in mature chondrocytes to
articular cartilage repair in both a non-inflammatory and an inflammatory
microenvironment.
9.5 erve-Repellent Factors in Articular Cartilage

N
Pathophysiology
Why is it that healthy and also diseased articular cartilage is mostly not innervated?
This is still not sufficiently clear. Possibly, the lack of blood vessels prevents inner-
vation or vice versa. Putative underlying molecular mechanisms for this unique
phenomenon might be found in specific axon guidance cues or nerve-repellent fac-
tors that are expressed in cartilage. Under physiological conditions, repellent factors
regulate cell–matrix interactions and cell–cell interactions of migrating cells during
embryonic development. This group consists of semaphorins and their receptors;
netrins and their receptors, deleted in colon carcinoma (DCC) and UNCs; slits and
roundabout receptors (Robo); and ephrins and the eph receptors (Hinck 2004; Miller
et al. 2004; Serafini et al. 1994). Notably, most of the members of these families are
expressed in cartilage already during embryonic limp development (Behar et al.
1996; Holmes and Niswander 2001; Wada et al. 2003; Kitsukawa et al. 1995).
9.5.1 Sensory Nerve-Repellent Factors
A selective repellent factor of SP- and CGRP-positive sensory nerve fibers is sema-
phorin (Sema) 3A which is present in neuronal tissue but also in developing carti-
lage and bone (Gomez et al. 2005), in intervertebral disks (Tolofari et al. 2010), and
in adult human articular cartilage (Okubo et al. 2011). Gomez et al. showed con-
vincingly that the semaphorin signaling system consisting of Sema3A and its recep-
tors plexin (Plx) A3/neuropilin (NPN)-1 is expressed in resting, pre-hypertrophic,
and hypertrophic chondrocytes in growth cartilage before onset of neurovascular
invasion during endochondral ossification (Gomez et al. 2005). They suggest that
the Sema3A/Plx-A3/NPN-1 pathway would inhibit neuro-vascularization of the
cartilage anlage early in skeletal development. Another group reported that gene
and protein expression of Sema3A, and its receptor Plx-A3/NPN-1, was signifi-
cantly elevated in chondrocytes from OA cartilage as compared to chondrocytes
from normal cartilage (Okubo et al. 2011). Sema3A expression is closely correlated
with chondrocyte cloning, which is a characteristic feature of OA cartilage. The
authors imply the possibility that Sema3A plays a role in the pathogenesis of chon-
drocyte cloning through antagonizing and inhibition of vascular endothelial growth
factor (VEGF)-mediated cell migration.
Our group described a role of the receptor DCC in chondrocyte migration
(Schubert et al. 2009b). We discovered strong induction of DCC in OA chondro-
cytes possibly induced by Sox9 and AP-1 suggesting that DCC mediates directed
chondrocyte migration in vitro in response to netrin-1, its ligand. To this end, these
findings may highlight an altered migratory phenotype of chondrocytes or misdi-
rected migration during OA, which has important consequences for cartilage
remodeling.
The induction of MMP1 and MMP3 via DCC in chondrocytes is an additional
indication of a possible functional relevance of DCC in the pathophysiology of
OA. Notably, treating the synovial fibroblasts (SF) with recombinant netrin-1
resulted in inhibition of migration of RA-SFs and OA-SFs, whereas control cells
were not affected (Schubert et al. 2009a). The stronger expression of UNC5B and
UNC5C receptors might contribute to the disordered phenotype of RA-SFs and
OA-SFs which in turn affect progression of cartilage degradation and severity dur-
ing pathophysiology of these diseases.
9.5.2 erve Growth Factors and Sympathetic Nerve-Repellent

N
Factors
Sympathetic nerve fiber density is regulated on one hand by NGF, which is an unspe-
cific growth factor for different nerve fiber types. On the other hand, sympathetic
nerve fiber density is regulated by specific nerve-repellent factors. In an inflamma-
tory situation, i.e., in late OA and in RA, activated synovial fibroblasts and recruited
macrophages produce NGF stimulating both sympathetic and sensory nerve fiber
sprouting (Aloe et al. 1992, 1993). These cell types also release semaphorin 3C and
semaphorin 3F resulting in the repulsion of sympathetic nerve fibers exclusively
(Fassold et al. 2009; Miller et al. 2004). This phenomenon might be responsible for
the preponderance of SP over sympathetic neurotransmitters in RA (Miller et al.
2000). Apart from NGF, other peripherally acting neurotrophins, e.g., brain-derived
neurotrophic factor (BDNF), have been discovered. The positive effect on nerve fiber
sprouting is similarly unspecific, because BDNF receptor expression has been con-
firmed on both sympathetic and sensory nerve fibers (Weidler et al. 2005).
Simao and colleagues did not find any correlation between plasma and synovial
fluid BDNF concentrations and knee OA severity, although mean plasma BDNF
levels of knee OA patients were significantly higher than in plasma of healthy con-
trols (Simao et al. 2014). Instead, BDNF seems to play a role in pain sensitivity in
knee OA. Given the fact that BDNF concentration is higher in RA synovium as well
(Weidler et al. 2005), an additional function besides stimulation of nerve fiber
sprouting would be possible. TH-positive single cells appear in both OA and RA
synovial tissue when inflammation starts. BDNF is known for its ability to induce
TH as shown, e.g., in dopaminergic differentiating stem cells (Trzaska et al. 2009).
Thus, BDNF might contribute to the appearance of TH-positive single cells in
inflamed synovium.
9.6 Perspectives
Sensory and sympathetic nerve fibers and their neurotransmitters are important neu-
ronal effectors regulating cartilage physiology and playing decisive roles in muscu-
loskeletal pathophysiologies (as OA and RA). Notably, many resident cells of the
osteoarticular system have receptors for sympathetic and sensory neurotransmitters,
and, thus, they can respond to these stimuli. Both types of neurotransmitters also
modulate embryonic limb growth and postnatal long bone growth by targeting
chondrocytes in the growth plate during endochondral ossification. More and more
evidence accumulates that neuronal signaling critically influences tissue regenera-
tion not only in joint disorders but also after bone and skin traumata.
In the meantime, many details with respect to sympathetic neuronal effects on
synovial tissue inflammatory responses in RA pathogenesis are known. However,
how changes in sensory and sympathetic joint innervation and their respective neu-
rotransmitters contribute to abnormal subchondral bone remodeling, cartilage deg-
radation, and osteophyte formation during the pathogenesis of OA is largely
unknown yet.
One can speculate that nerve-repellent factors, next to their known axon guid-
ance role, contribute to joint pathophysiology because several studies assign novel
functions to semaphorins and other repellent factors in chondrogenic differentiation
during embryonic development, cartilage degeneration, and synovial inflammation
in adults.
Taken together, it becomes more and more evident that sensory and sympathetic
nerve fibers and their neurotransmitters critically influence cartilage, synovial, and
other joint tissue function and homeostasis. Doubtless the peripheral nervous sys-
tem is crucially involved in the pathogenesis of musculoskeletal disorders such as
OA, RA, and IVD degeneration.
Acknowledgments The authors are supported by the DFG grants to SG (GR 1301-19) and to ZJL
(JE 642/4-1), both subprojects of FOR 2407-1.
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Intervertebral Disc Degeneration
10
Akansha M. Shah, Sarah Yoon Ji Kwon, Wilson C.W. Chan,
and Danny Chan
Abstract
The human spine consists of 23 intervertebral discs adjoining the vertebral
bodies. These discs provide mechanical support and spinal motion and func-
tion to distribute loads from daily activities. Tissues of intervertebral discs
share similarities to those of diarthrodial joints, such as a thin layer of carti-
lage that lines the interface between the joint and the bony elements and a
central space rich in extracellular matrix molecules that promotes lubrication
and maintains osmotic pressure. Like the pathophysiology of other cartilagi-
nous joints, intervertebral discs undergo biomechanical and structural changes
as a result of aging and mechanical insults. Due to higher mechanical loading,
lumbar discs are more susceptible to degeneration, which can lead to symp-
tomatic outcomes such as low back pain, sciatica, and other physical disabili-
ties. These affect quality of life as we age and present a significant burden to
the healthcare system globally. The etiology of intervertebral disc degenera-
tion is not fully understood, but is a consequence of the changing structure
and environment of its three interconnecting components, the nucleus pulpo-
sus, the annulus fibrosus, and the cartilaginous endplate, which function coop-
eratively to transmit load and regulate the cellular, biochemical, and nutritional
properties of the disc. Abnormal changes in one or more of these disc com-
partments will compromise disc integrity and culminate in a degenerated disc
state. Mechanistic insights into disc pathology can be gained through an
understanding of the development and homeostasis of the intervertebral disc
tissues. This chapter will provide an overview of the intervertebral disc in
health and disease.
A.M. Shah, PhD • S.Y.J. Kwon, PhD • W.C.W. Chan, PhD • D. Chan, PhD (*)
School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong
e-mail: aks.shah93@gmail.com; isyoonjis@gmail.com; cwilson@hku.hk; chand@hku.hk

230 A.M. Shah et al.
10.1 volution of the Vertebral Column

E
and the Intervertebral Disc
Formation of the vertebral column, a metameric structure of vertebral bones and

intervening intervertebral discs, marks a critical event in evolution. This was pre-
ceded by the presence of a rodlike midline structure (the notochord) in animals that
lack a backbone. The lifelong notochord alone offers rigidity to the organism, pro-
tects the overlaying spinal cord, and enables the formation of an extended body. In
higher organisms, the notochord exists only transiently in development as a primi-
tive axial skeleton until the formation of the spinal elements (Stemple 2005). In the
process, it functions as a signaling center, providing molecular signals that position
and specify fates of surrounding tissues (Pourquie et al. 1993). This progression in
evolution provides biological advantages that include the vertebral bones as attach-
ment sites to other axial and appendicular bones and muscles and the intervertebral
discs that allow flexibility and dissipate hydrodynamic loads along the spine.
10.2 Making of the Intervertebral Disc Tissues
While the nucleus pulposus is derived from the notochord, the annulus fibrosus and
cartilaginous vertebrae arise from the sclerotome (Christ et al. 2000). Genetic stud-
ies of murine development and advances in time-lapse live imaging demonstrate
that the three regions of the notochord along the anterior-posterior axis develop
from the organizer tissue differently. In advancing notochord development, the ante-
rior head process forms via a node-independent mechanism that involves the con-
densation of dispersed notochord progenitor cells located anterior to the forming
node, the trunk notochord originates from the node and elongates by convergent
extension, and the tail notochord arises from node-derived progenitors that migrate
toward the posterior end (Yamanaka et al. 2007). These different cellular origins
along the longitudinal body axis can introduce variations in the maintenance and
function of cells in the future nucleus pulposus along different regions of the spine.
In early vertebral development, signals from the notochord pattern the surround-
ing paraxial mesoderm to form an unsegmented, presomitic mesoderm (PSM). The
initial high levels of fibroblast growth factor (FGF) signaling maintain PSM cells in
their unsegmented state (Dubrulle et al. 2001), but its reduction leads to segmenta-
tion of the PSM in the anterior-posterior direction sequentially, giving rise to bilat-
eral pairs of somites on either side of the neural tube (Roughley 2004) (Fig. 10.1a).
Somites are transient structures that consist of outer epithelial cells surrounding an
inner mesenchymal core (the somitocoele) (Ferrer-Vaquer et al. 2010). Somitogenesis
and establishment of boundaries between developing somites are tightly regulated
by the periodic expression of cycling genes (segmentation clock) of the Notch and
Wnt signaling pathways (Kageyama et al. 2007; Maroto and Pourquie 2001;
Pourquie 2003; Saga and Takeda 2001). These two pathways cycle synchronously
with somite formation but slightly out of phase with each other, with cyclic Wnt
signaling acting upstream of cyclic Notch signaling. Genes that regulate Notch
10 Intervertebral Disc Degeneration 231
Development of mouse intervertebral disc

~E10.5 ~E12.5 ~E13.5 Newborn
a b c d cEP
NP
AF
Somite
Sclerotome
Notochord
Cellularity changes in homeostasis and degeneration

Newborn Adult Degenerative
e f g
NCL NPC CLC PC FBC

Notochordal-like cell Nucleus Pulposus cell Chondrocyte-like cell Progenitor cells Fibrotic cell
Fig. 10.1 Development and cellular changes in intervertebral discs. A diagrammatic representa-
tion of the embryonic development of intervertebral discs in mice (a–d), illustrating the role of the
notochord in directing tissue formation and organization into the vertebral bodies and the interver-
tebral discs, and the formation of the nucleus pulposus from the notochord (e–g). The cellular
changes in the nucleus pulposus with age and degeneration: notochordal-like cells are replaced by
or progressively differentiate into nucleus pulposus and chondrocyte-like cells, and fibrotic cells
are present at later stages of degeneration. The presence of potential progenitor cells is also
depicted. Abbreviations: cEP cartilaginous endplate, NP nucleus pulposus, AF annulus fibrosus
signaling (Mesp2, delta1, Hes1, Hes7, and lunatic fringe) turn on and off in a wave-
like pattern in PSM cells, and each wave leads to the formation of a new somite
(Bessho et al. 2003; Chen et al. 2005; Jiang et al. 2000; Morimoto et al. 2005;
Pourquie 2011).
Once formed, cells in the somites differentiate with respect to their position start-
ing with the anterior-most somite. The dorsal epithelium of the somite forms the
dermomyotome, establishing the skeletal muscle and the dermis. Under the influ-
ence of bone morphogenetic protein (BMP) and Wnt signaling from the ectoderm
and sonic hedgehog (Shh) signaling from the notochord and floor plate, cells in the
somitocoele and ventral epithelium of the somite undergo epithelial to mesenchy-
mal transition (EMT) to form mesenchymal sclerotomal cells that surround the
notochord (Fig. 10.1a) (Borycki et al. 1998; Fan and Tessier-Lavigne 1994;
Monsoro-Burq 2005). Subsequently, the sclerotome derived from one somite segre-
gates into rostral and caudal halves, and re-segmentation occurs whereby the rostral
half of one segment joins with the caudal half of a neighboring segment (Bagnall
et al. 1988) (Fig. 10.1b). The sclerotome consists of multiple progenitor cells that
differentiate into all connective tissues cell types (Brand-Saberi et al. 1996; Brand-
Saberi and Christ 2000). Sclerotome cells specify into cartilage and annulus fibro-
sus lineages depending on the location of cells within the sclerotome and a complex
network of growth factor signaling.
The rostral and caudal halves of adjacent sclerotome give rise to the cartilaginous
vertebrae (Christ and Wilting 1992). Bone morphogenetic protein (BMP) activity
concentrated in these regions act alongside Shh and Pax1/9-induced Nkx3.2 tran-
scriptional repressors to activate Sox9 expression and subsequent chondrocyte dif-
ferentiation (Rodrigo et al. 2003; Tribioli and Lufkin 1999; Zeng et al. 2002). The
annulus fibrosus, conversely, is derived from cells adjacent to the von Ebner’s fis-
sure, the border between the two sclerotome halves of a single somite. However, it
is unclear whether the annulus fibrosus arises from the rostral or caudal side of this
border (Bagnall and Sanders 1989; Goldstein and Kalcheim 1992; Huang et al.
1994), with a most recent dye-labeling study in avian discs (Bruggeman et al. 2012)
suggesting that the annulus fibrosus originates from the rostral sclerotome.
Nevertheless, TGF-β signaling plays an important role in annulus fibrosus fate
determination. Targeted deletion of the TGF-β type II receptor (Tgfbr2) gene in
Col2a1-expressing sclerotome cells (Col2a1-cre) in mice results in a reduced or
absent intervertebral disc by E17.5, where the inner annulus fibrosus does not form
and more closely resembles a wild-type vertebrae (Baffi et al. 2004, 2006). This
suggests that TGF-β controls embryonic disc development by either precluding dif-
ferentiation of vertebral cartilage in the disc space or by directly stimulating annulus
fibrosus differentiation (Sohn et al. 2010).
At E13.5 in mouse embryonic development, the notochord also segments as it
contracts in the periphery of the emerging vertebral bodies and expands in the
developing intervertebral disc anlagen, giving rise to the nucleus pulposus
(Fig. 10.1c) (Aszodi et al. 1998). As no cell death in regression regions or cell pro-
liferation in expansion regions is observed during notochord segmentation, a pos-
sible mechanism of the pinching off process is that the developing vertebrae apply
a biomechanical force on the notochord, squeezing the notochordal cells toward the
less-compressed disc tissues while displacing them away from the vertebrae
(Grotmol et al. 2003).
Evidence that the nucleus pulposus is derived from the notochord is provided
through studies that utilize notochord-specific Cre mice to trace the fate of noto-
chordal cells. Noteworthy are studies that used Shh-Cre/Shh-CreERT2 (Choi et al.
2008), a molecule highly expressed in the notochord and later in the nucleus pulpo-
sus, and Noto-Cre (McCann et al. 2012), a homeobox gene that is restricted to the
node and posterior notochord during gastrulation (E7.5–E12.5) and is not detected
subsequently. These studies demonstrate that many cells in the adult nucleus pulpo-
sus can be traced back to their developmental origin, up to a period of 9 months for
the Noto-Cre (McCann et al. 2012) and 19 months for the Shh-CreERT2 (Choi et al.
2008). Whether other sources of cells contribute to the postnatal nucleus pulposus
is not clear.
While the intervertebral disc structure is established embryonically, in mice, signifi-
cant changes in cell and tissue morphology, such as the establishment of distinct annulus
fibrosus lamellae, demarcation of the annulus-endplate boundary, endplate chondrocyte

hypertrophy, and matrix gene expression, occur at early postnatal time points.
10.3 The Normal Disc: Working Together as a Functional Unit
Intervertebral discs are complex structures that connect adjacent vertebral segments
along the spine. Each disc consists of a central, gelatinous nucleus pulpous, con-
strained by a circumferential ring of fibrocartilage, the annulus fibrosus, and is situ-
ated between two cartilaginous endplates that are contiguous with adjacent vertebral
bodies (Fig. 10.1d). These components of the intervertebral disc are distinct in com-
position and structure, conferring mechanical properties that permit the interverte-
bral disc to distribute load while maintaining flexibility of the vertebral column.
Thus, different structural compartments of the disc act synergistically to produce a
functional unit.
10.3.1 Nucleus Pulposus
The nucleus pulposus is at the center of the intervertebral disc. Cells of the nucleus
pulposus produce the necessary proteoglycans, mainly aggrecan, to maintain a high
osmotic pressure in the disc environment (Roughley et al. 2006a, Roughley and
Mort 2014). The abundant water content gives the nucleus pulposus its gelatinous
appearance and enables distribution of compressive loads and maintenance of a
functional disc height (Boxberger et al. 2008). In early stages of life, the nucleus
pulposus is highly cellular, but with age, cell density decreases (Urban et al. 2000)
and changes in cell morphology are observed (Cappello et al. 2006)
(Fig. 10.1e–g).
Although cells in the nucleus pulposus are heterogeneous, early in life, it majorly
consists of notochordal-like cells (Fig. 10.1e). The notochordal-like (NCL) cells
appear in clusters, they exhibit a well-defined Golgi and extensive endoplasmic
reticulum, have very few mitochondria, and possess actin filament-bounded vacu-
oles that contain an osmotically active material (possibly unsecreted glycosamino-
glycans) (Adams et al. 1990; Smith et al. 2011; Trout et al. 1982a, b). Given that
cells in the nucleus pulposus originate from the developing notochord, they express
BrachyuryT, a known marker of the notochord and notochord tumors, and sonic
hedgehog (Shh). In addition, co-expression of cytokeratin 8 and vimentin are
observed in bovine notochordal-like cells (Gilson et al. 2010).
However, with growth and aging, the notochordal-like cells are replaced by or
progressively differentiate into mature nucleus pulposus cells (NPCs) that produce
the bulk of the extracellular matrix for the functionality of the nucleus pulposus
(Fig. 10.1f–g). This coincides with the appearance of chondrocyte-like cells (CLCs)
that exhibit morphology similar to chondrocytes, with a thin cortex actin surround-
ing the nucleus pulposus and a small cytoplasmic volume (Fig. 10.1b). Markers
common to chondrocyte-like cells include CD24, cytokeratin 18, cytokeratin 19,
and cadherin 2, and they can be distinguished from articular chondrocyte using
markers such as PAX1, FOXF1, CA12, and OVOS2 (Choi et al. 2015; Minogue
et al. 2010; Power et al. 2011). Since chondrocyte-like cells are reportedly less
metabolically active and more resistant to nutritional stress, a transition to chondro-
cyte-like cells with age may be induced by factors such as reduced nutrient supply
to the avascular nucleus pulposus upon calcification of the cartilaginous endplates
(Guehring et al. 2009). Finally, at a later stage in adult life, fibrotic cells populate
the disc nucleus pulposus (Arkesteijn et al. 2015; Ishii et al. 1991).
With different species and aging, the relative proportions of cells in the nucleus
pulposus vary. In humans and chondrodystrophic dogs, the notochordal cells
decrease rapidly with establishment of the mature spinal structure and aging
(Cappello et al. 2006; Hunter et al. 2003, 2004; Smolders et al. 2013). On the other
hand, in rodents and rabbits, pools of notochordal cells are maintained through
adulthood (Miyazaki et al. 2009; Poiraudeau et al. 1999). A disc that retains greater
number of notochordal-like cells through adulthood (i.e., non-chondrodystrophic
dogs) appears to sustain a healthy nucleus pulposus with a well-hydrated structure
(Hunter et al. 2004; Smolders et al. 2013). Conversely, disappearance of notochordal-
like cells causes a more fibrous nucleus pulposus and early degenerative changes
(Melrose et al. 1996).
A recent study of the human and mouse nucleus pulposus identified tyrosine-
protein kinase receptor (Tie2) and disialoganglioside 2 (GD2) as potential cell sur-
face markers for cells in the nucleus pulposus (Sakai et al. 2012). A hierarchal
expression pattern of progenitor cell markers was proposed, where Tie2+/GD2−/
CD24− cells behave as dormant stem cells. Upon activation they become Tie2+/
GD2+/CD24- with self-renewal potential and are potential progenitor cells. Upon
differentiation, Tie2 expression is lost, and the Tie2−/GD2+/CD24− cells are charac-
terized as cells of the early nucleus pulposus. Subsequently, cells lose expression of
both Tie2 and GD2 but maintain CD24 expression (Tie2−/GD2−/CD24+) that defines
a mature nucleus pulposus cell phenotype of chondrocyte-like cells in the disc
nucleus pulposus. This study suggests a chronological differentiation of cells in the
nucleus pulposus and supports the presence of progenitors that are able to commit
to the nucleus pulposus cell lineage. However, a decline in progenitor potential
(reduction of Tie2+ cells) is observed with aging, correlating with the onset of disc
degeneration in humans (Sakai et al. 2012).
10.3.2 Annulus Fibrosus
At E13.5–E14.5, the lamellar structure of the annulus fibrosus is not well defined in
mouse development, but resident cells in the region are oriented concentrically
around the newly formed nucleus pulposus (Peacock 1951). Organization of this
template sheet structure during development may be a result of pressures in the
nucleus pulposus that trigger the formation of actin stress fibers in annular fibro-
blasts, connecting the cadherin junctions of adjacent cells (Hayes et al. 1999). This
initial cellular organization may be necessary for the formation of tough
circumferential angle-ply layers and highly concerted matrix deposition that are
progressively established during postnatal growth. However, the molecular signals
that direct annulus fibrosus cells to assemble into a required lamellar orientation
remain unclear.
This microstructural composition and arrangement enable the annulus fibrosus to
contain the radial bulge of the nucleus pulposus and uniformly distribute torsional
and compressive loadings in the disc space. Furthermore, it facilitates joint flexibil-
ity by distention and rotation. In humans, the number of fiber bundles in the annulus
fibrosus can range from 20 to 62 (Marchand and Ahmed 1990), with the lamellar
thickness increasing with age, both circumferentially and radially. The inner and
outer layers of a fully developed annulus fibrosus differ substantially in their bio-
chemical and cellular composition and thus have potentially distinct biomechanical
roles. Cells in the outer annulus fibrosus are elongated, fibroblast-like, and extend in
the long axis of the fibrils, whereas those in the inner annulus fibrosus have a spheri-
cal morphology similar to chondrocytes (Bruehlmann et al. 2002). Being a tension-
bearing tissue, the annulus fibrosus has a high collagen content, with collagen fibers
in adjacent lamellae oriented in opposite directions. The progression from a pre-
dominantly collagen I matrix in the outer annulus fibrosus to a collagen II matrix in
the inner annulus reflects changes in the loading environment from more tension in
the outer regions to greater compression in the inner compartment.
The cellular origin of the annulus fibrosus is less well defined. Lineage tracing in
mice with Tbx18-Cre established that a pool of labeled cells in the anterior part of
the annulus fibrosus are derived from the sclerotome (Bruggeman et al. 2012).
Furthermore, using fluorescent lipophilic dyes, DiI and DiA, to differentially label
the rostral (upper) and caudal (lower) regions of the sclerotome in chick develop-
ment, it was shown that cells from the rostral half of the sclerotome contribute to
caudal half of vertebral body as well as the intervertebral space, while the caudal
half of sclerotome contributes to rostral half of adjacent vertebrae (Bruggeman et al.
2012). However, these studies cannot account for all the cells in the annulus fibro-
sus, and alternative sources of progenitor cells may contribute to the formation,
maintenance, and renewal of cells in the annulus fibrosus in postnatal life.
10.3.3 Cartilaginous Endplate
The cartilaginous endplate is the least studied intervertebral disc compartment,

despite its important role in disc nutrition and mechanics. The endplate represents a
transition structure of hyaline cartilage that physically and biomechanically sepa-
rates the disc region from the vertebrae, barring the motion segment from applying
pressure onto the vertebral bone. In humans, its thickness varies from about 0.2 mm
in the center to 0.9 mm in the periphery and is thickest at birth and thins with age
(Moon et al. 2013). The endplate exhibits structural similarity with the articular
cartilage of synovial joints; thus, it consists of subpopulations of chondrocytes
embedded in an extracellular matrix of aggrecan and collagen II fibrils that are ori-
ented parallel to the longitudinal axis (Moore 2006; Sahlman et al. 2001). Collagen
X is expressed in the pericellular matrix of hypertrophic chondrocytes (Aigner et al.

1998). However, the origin of the collagen X-producing hypertrophic chondrocytes
remains unclear.
Calcification of the endplate initiates at around 6 human years of age and ossi-
fies by about 13 human years of age (forming a secondary ossification center)
(Bick and Copel 1951). In humans, the secondary ossification center adjacent to
the endplate fuses with the primary ossification center of the vertebral bone, ter-
minating skeletal growth and producing a “closed” vertebral growth plate at
puberty. In goats and rabbits, the primary and secondary ossification centers
remain distinct, and the osseous layer of the endplate is termed bony endplate
(Zhang et al. 2014). Although the vertebral growth plate remains “open” through
adult life in these animals, skeletal growth ceases. Unlike rabbits and goats that
contain only one to three layers of chondrocytes in their cartilaginous endplate,
mice contain three to seven layers of cells (Zhang et al. 2014). The cartilaginous
endplate of rodents manifests as a continuation of the growth plate with pockets
of secondary ossification centers in spines of elder mice (Zhang et al. 2014).
Nevertheless, the extent of calcification and growth plate – cartilaginous end-
plate continuous or discontinuous morphology – varies with age of the animal as
well as position of the disc along the spine.
Like tendon inserts into bone to form a contiguous tissue, the cartilaginous end-
plate and annulus fibrosus are tightly linked. The less organized inner annulus
fibrosus fiber bundles insert obliquely into the cartilaginous endplate, divide into
sub-bundles, and merge with the hyaline cartilage matrix. The junction at which
the two tissues intersect is defined as the inner annulus-endplate interface, which
contains a mixed cell population comprised of chondrocytes and cells with mor-
phology intermediate of elongated annulus fibrosus cells and round hypertrophic
chondrocytes. This histological feature suggests that cells in the two structures
may be closely related. Likewise, organized lamellar fibers of the outer annulus
fibrosus penetrate directly into the vertebrae with “Sharpey’s fibers” bridging the
two tissues (Hashizume 1980; Inoue 1981) or curve laterally at a 90° angle and
merge with the periosteum along the vertebrae (Nosikova et al. 2012). These inter-
faces, where soft tissue integrates into hard tissue, develop to withstand the wide
range of mechanical forces (shear, compression, tensile, and torsion) experienced
by the intervertebral disc.
10.4 Clinical Features of Intervertebral Disc Degeneration
Low back pain is a debilitating condition that is estimated to affect 84 % of the

population at some point in their life, with 10 % suffering from chronic disability
(Walker 2000). While there may be many causes of back pain, intervertebral disc
degeneration is a major contributing factor in 40 % of the cases (Cheung et al.
2009). Degenerated lumbar discs have been observed in patients as early as
11–16 years of age (Boos et al. 2002). In fact, 20 % of individuals in teen years
Normal High Disc Schmorl’s Modic

hydrated discs Dehydration intensity zone extrusion nodes changes
a b c d e f
Fig. 10.2 Common clinical feature of intervertebral changes by MRI. Selected human MRI of the
lumbar region showing common clinical features observed in the intervertebral disc with age and
degeneration. The colored arrows highlight the respective changes in each category. (a) An indi-
vidual with normal hydrated intervertebral discs. (b) An individual with a dehydrated nucleus
pulposus (yellow arrow). (c) An image depicting tears within the annulus fibrosus shown as a
brighter region (light green arrow). (d) Deformation of the annulus fibrosus and extrusion of the
nucleus pulposus impinging on the sciatic nerve (red arrow). (e) Abnormal structural changes the
cartilaginous endplates (light pink arrows). (f) Signal intensity changes in the bone marrow regions
of the vertebral bodies adjacent to a degenerative disc (light blue arrows)
procure mild signs of degeneration, which increases sharply with age such that
10 % of 50-year-old discs and 60 % of 70-year-old discs are severely degenerate
(Miller et al. 1988). Despite its wide prevalence, a unified definition of disc degen-
eration is lacking. In comprehending the scope of the degenerative condition, a
thorough evaluation of the structural and biological changes during degeneration
is essential.
Magnetic resonance imaging (MRI) is a useful technique to assess the quality of the
intervertebral disc by means of a T2-weighted sagittal scan (Fig. 10.2). The
Schneiderman (Schneiderman et al. 1987) or Pfirrmann (Pfirrmann et al. 2001) classi-
fications are the commonly used grading protocols for assessing degenerative changes
using MRI. An MRI scan of a healthy human lumbar disc is presented in Fig. 10.2a.
The bright nucleus pulposus represents a fully hydrated disc of normal height. The
most marked change in disc degeneration is a decrease in signal intensity (a dark disc),
indicating a loss of water content (Fig. 10.2b). Other features of degeneration include
tears in the annulus fibrosus shown as “high intensity zones” (Fig. 10.2c), extrusion of
the nucleus pulposus from the annulus fibrosus and its subsequent compression on the
sciatic nerve (sciatica) (Fig. 10.2d), and herniation of the nucleus pulposus to the carti-
laginous endplates which form structures called Schmorl’s nodes (Fig. 10.2e). In some
cases, Modic changes can be detected as signal variations in the marrow regions adja-
cent to the cartilaginous endplates (Fig. 10.2f) (Hu et al. 2009; Yu et al. 2012). The
relevance of Modic changes is not clear but many studies have shown a high
a b
Homeostasis Degenerative conditions
IVD cells IVD cells IVD cells IVD cells
Severity of degeneration
Fibrosis of tissue
Tissue hydration
Cellularity
Fig. 10.3 Extracellular matrix (ECM) changes influencing progression of intervertebral disc
(IVD) degeneration. Diagrammatic representation depicting the relationship between the extracel-
lular matrix and cells in a healthy disc (a) where a homeostatic environment can be maintained. (b)
Insults to cells or matrix initiate a cascade of events that lead to progressive changes in matrix
production or degradation and cellular differentiation to that of a degenerating intervertebral disc
association with symptomatic outcomes such as back pain (Albert et al. 2008). The
features described above can be presented in singleton or in combination.
10.5 The Etiology of Intervertebral Disc Degeneration
Cell tracing and fate mapping in mice suggest that cells in the nucleus pulposus are
long lived, but undergo biochemical and phenotypic changes upon natural aging and
degeneration. Degeneration has been described as an abnormal cell-mediated
response that leads to a gradual failure in tissue architecture (Fig. 10.3). Hallmarks
of disc degeneration include an overall breakdown of extracellular matrix, abnormal
matrix synthesis, and changes in cell number, cell morphology, and cell behavior.
These features alter the nutritional, biomechanical, and structural properties of the
nucleus pulposus, annulus fibrosus, and cartilaginous endplate, causing the disc to
function less efficiently in a vicious cycle that leads to disc degeneration (Fig. 10.3).
Degeneration is also marked by a reduced capacity for tissue maintenance and repair,
which possibly involves the mobilization of progenitor cell pools found within the
intervertebral disc tissues. The clinical features presented in MRIs suggest that
degeneration of the disc is complex, and it is likely that the anomalies described
previously are interconnected. However, a direct relationship between these changes
and their potential sequence of manifestation is not well understood. Furthermore, a
condition brought on by development, intervertebral “dysgeneration,” has been pro-
posed to describe cases where subtle differences exist in the disc from early on, caus-
ing an atypical cascade of events with age (Luk and Samartzis 2015).
Nucleus pulposus
Nutrition and O2 diffusion Nutrition and O2 diffusion ↓
Cartilaginous endplate Calcification of endplate
Subchondral plate
Blood vessel
Normal Degeneration
Fig. 10.4 Nutrient and oxygen supply in the intervertebral disc. Diagrammatic representation of
the blood supply to the intervertebral disc with blood vessels ending at the junction of the cartilagi-
nous endplate in normal and degenerate stages. The intervertebral disc proper is normally avascu-
lar and the cartilaginous endplate presents as selective barrier and the major route of nutrient and
oxygen supply. Conditions with impaired blood vessel formation and calcification of the cartilagi-
nous endplate will have an impact on nutrient supply and movement of solutes through the carti-
laginous endplate
10.6 iving in the Harsh Environment

L
of an Intervertebral Disc
Disc cells require a sufficient supply of nutrients and adequate removal of metabolic
waste for normal function and survival. An imbalance between nutrient availability
and consumption may lead to aberrant cellular behaviors that contribute to disc
degeneration (Grunhagen et al. 2011; Urban et al. 2004). This is a challenge for
cells in the intervertebral disc as the tissue is avascular, and cells at the center of the
adult human disc are approximately 8 mm away from the nearest blood supply that
terminates at the cartilaginous endplates (Fig. 10.4) (Urban et al. 2004). Nutrients
such as glucose and oxygen and substrates for matrix production such as amino
acids enter via bloody supply in the cartilaginous endplates and the margins of the
annulus fibrosus (Naresh-Babu et al. 2016). Cells in the outer annulus fibrosus
engage in metabolite exchange via blood vessels within the soft tissues at the disc
periphery, as well as small capillaries that penetrate 1–2 mm into the outermost
lamella in adult humans (Hassler 1969; Rudert and Tillmann 1993). The remaining
majority of disc cells rely on nutrient supply via capillaries that penetrate channels
in the subchondral plate and terminate at the border of the cartilaginous endplate
(Holm et al. 1981). Small solutes are transported from capillaries to cells across the
disc matrix by diffusion (Holm et al. 1981; Naresh-Babu et al. 2016), and cellular
wastes such as lactic acid are removed in the opposite direction (Magnier et al.
2009).
The dense extracellular matrix of collagens and polyanionic proteoglycans
restricts the size and charge of solutes transported to the disc (Magnier et al. 2009;
Roberts et al. 1996). The negative charge of aggrecan enhances the penetration of
small cations (sodium), whereas anions (sulfate and chloride) are partially excluded
(Urban and Maroudas 1979). Moreover, a high aggrecan concentration minimizes
the spaces between glycosaminoglycan chains and sterically excludes large solute
molecules (cytokines and growth factors), which make use of load-induced fluid
movement for their transport (Chandran and Horkay 2012; Ferguson et al. 2004).
The rate of solute transport depends on many parameters, from the blood supply
to compartmental permeability and cellular metabolic demand. A fine balance
between the rate nutrient supply and consumption is a key for tissue homeostasis
(Urban et al. 2004). A sustained imbalance will lead to cellular and biochemical
changes.
10.6.1 The Role of Cartilaginous Endplate in Nutritional Supply
The cartilaginous endplate acts as a selective barrier, and this property influences
the movement of solutes into and out of the intervertebral disc (Magnier et al. 2009;
Roberts et al. 1996). A major risk factor for intervertebral disc degeneration is cal-
cification of the endplate (Roberts et al. 1996) or occlusion of endplate channels
(Benneker et al. 2005), which have been demonstrated to disturb transport into the
disc region (Rajasekaran et al. 2004; Roberts et al. 1996) that could lead to cell
death (Galbusera et al. 2013). MRI studies of human intervertebral discs using con-
trast medium showed that diffusion into discs via the cartilaginous endplate was
lower in early degenerative discs (Nguyen-minh et al. 1998; Rajasekaran et al.
2004), suggesting impaired solute transport upon degeneration. Furthermore, a
gradual change of the endplate to fibrocartilage and focal necrosis upon aging may
also impede diffusion of solutes (Boos et al. 2002), although this has not been
experimentally demonstrated.
10.6.2 Impact of the Vasculature on Nutrient Supply
The impact of systemic blood circulation on disc cell health is evident in the correla-
tion between disc degeneration and disorders that obstruct blood supply to the ver-
tebral column or the capillary beds of the endplate. These include atherosclerotic
calcification of the abdominal aorta (Kurunlahti et al. 1999), Gaucher’s disease and
Caisson’s disease (Jones 1997), sickle cell anemia (Babhulkar 1997; Jones and
Engleman 1966), lipidemia, and hyperuricemia (Nerlich and Boos 2016).
Furthermore, post-contrast MRI studies of human intervertebral discs found that
signal intensity increased inside the disc after administration of vasoactive drugs,
suggesting that adjustment in the rate of blood flow via muscarinic receptors of
vascular channels in the subchondral plate affects the delivery of nutrients to the
disc (Rajasekaran et al. 2008). Reduction of vascularity in the endplate also occurs
with aging (Boos et al. 2002; Edelson and Nathan 1988) resulting in general reduc-
tion of blood supply to the disc, which may explain a higher predisposition of aged
discs to degeneration. Obliteration of blood vessels in the endplate is paralleled by
structural changes such as cartilage disorganization and an increase in cartilage
cracks and microfractures (Boos et al. 2002).
10.6.3 Factors Influencing Nutrient Demand
Factors such as endplate calcification or changes in blood flow patterns can reduce
nutrient supply leading to disc degeneration. However, increasing the rate of nutri-
ent utilization per cell (the cellular demand) can also add to the risk. Cellular
demand can be increased by growth factors or cytokines that enhance lactic acid
production and glucose utilization; these factors are shown to be elevated in herni-
ated human lumbar discs (Andrade et al. 2013; Gronblad et al. 1994; Weiler et al.
2005). Moderate levels of growth factors are essential for maintaining a healthy disc
by stimulating matrix production (Li et al. 2004). However, upon loss of proteogly-
cans and an increased permeability of the matrix in degeneration increased levels of
growth factors (and cytokines) infiltrate, affecting cellular metabolism and the bal-
ance between nutrient supply and demand (Grunhagen et al. 2011).
Addition of exogenous growth factors (GDF-5, TGF-β, FGF, or EGF) to bovine,
canine, or human cell cultures in vitro (Chujo et al. 2006; Kim et al. 2003; Gruber
et al. 1997; Thompson et al. 1991) or injection into mouse tail discs in vivo (Walsh
et al. 2004) can stimulate cell proliferation and cell activity. Increased cell activity
and cell numbers can boost glucose consumption rates, which may deplete nutrients
from the intervertebral disc niche (Mokhbi Soukane et al. 2009). Furthermore,
upregulated proinflammatory cytokines in the disc, such as tumor necrosis factor
alpha (TNFα) and interleukin-1 (IL-1) (Johnson et al. 2015), propagate local inflam-
matory signals via prostaglandin E2 (PGE2) (Bachmeier et al. 2007; Rannou et al.
2000; Shinmei et al. 1988; Takahashi et al. 1996), stimulate angiogenesis (Doita
et al. 1996), and the production of matrix degrading enzymes in disc cells (Le
Maitre et al. 2005). These changes will result in enhanced metabolic activity,
although this has not been addressed directly.
Mechanical stresses can also increase nutritional requirements of cells beyond
diffusional capacity of the disc. For instance, as the disc deforms under mechanical
loads, the hydrostatic pressure inside the disc increases and fluid moves out of the
disc. This change in fluid dynamics increases the interstitial fixed charge density,
attracts more ions into the tissue, and elevates osmotic pressure in the disc (Boos
et al. 1993; Chan et al. 2011; Ishihara et al. 1997; Urban et al. 1994). The hyperos-
motic extracellular environment leads to a decrease in cell volume (Pritchard et al.
2002; Verkman et al. 1996) and triggers recovery mechanisms such as activation of
ion channels to restore intracellular water and chemical composition (Erickson et al.
2001). Since a vital sodium gradient is maintained across plasma membranes by the
Na+/K+ pump, which utilizes 50–70 % of the cellular adenosine triphosphate (ATP),
changes in ionic distribution can increase Na+/K+-ATPase activity in a cell, thereby
increasing cellular demand of energy (Horner et al. 2001; Mobasheri et al. 1997).
Thus, alterations in the physical environment surrounding cells due to loading can
cause changes in cellular metabolism, resulting in a different biosynthetic activity
of the cell.
10.6.4 B
alancing Nutrient Supply and Demand for Tissue
Homeostasis
The concentration of nutrient available to disc cells (the steepness of its concentra-
tion gradient) is determined by the balance between rate of influx (e.g., rate of blood
flow) and the rate of consumption by cells (Urban et al. 2004). Thus, a high cell
density and rate of cell metabolism will increase the net nutrient consumption,
thereby the rate of nutrient diffusion (Magnier et al. 2009). However, cell number
and metabolism are also regulated in a negative feedback loop, where a decreased
nutrient supply will restrict cell mass and metabolic rates, thus maintaining homeo-
stasis among different regions of the disc. In the intervertebral disc, concentrations
of nutrients are highest at the disc periphery and lowest at the center (Magnier et al.
2009), and cell density in the disc is also highest at the outer edge of the annulus
fibrosus, closest to the nutrient supply, and falls rapidly toward the inner annulus
fibrosus and the nucleus pulposus (Maroudas et al. 1975; Urban et al. 2004). This
correlation between nutrient levels and cell density across the disc suggests that cell
numbers may be regulated by nutritional constraints.
Cells in the nucleus pulposus and inner annulus fibrosus are often exposed to
suboptimal microenvironments with low oxygen tension, limited glucose availabil-
ity, relatively low pH (due to lactic acid buildup), and high metabolic waste accu-
mulation (Magnier et al. 2009). In this avascular environment, disc cells
predominantly rely on glycolysis for energy production, which requires glucose and
produces lactic acid (Holm et al. 1981). Therefore, disc cells are particularly sensi-
tive to changes in glucose and lactic acid concentrations. A reduction in glucose
concentration to below 0.5 mmol/L or a decrease in pH to below 6.4 comprises cell
viability of bovine cells in vitro (Bibby et al. 2005; Horner and Urban 2001).
Moreover in a low pH environment, bovine disc cells continue to produce active
matrix metalloproteinase (MMPs) that break down matrix macromolecules, while
matrix production is inhibited (Razaq et al. 2003).
On the other hand, the effect of oxygen concentration and hypoxia on disc cell
function is currently under debate. Studies in canine and bovine discs have demon-
strated sulfate incorporation for matrix production is highest in the nucleus pulpo-
sus at 5 % O2 and at pH 7.0. In vitro studies suggest that disc cells have been adapted
to hypoxic environments (Agrawal et al. 2007; Feng et al. 2013; Mwale et al. 2011;
Risbud et al. 2005) in part by activating hypoxia-inducible factors (HIF), particu-
larly HIF-1. HIF-1 enhances the expression of matrix genes such as aggrecan in
nucleus pulposus cells in vitro (Agrawal et al. 2007) and also promotes cell survival
by modulating apoptosis and regulating glucose metabolism (Risbud et al. 2005)
However, an oxygen concentration below 5 % causes cellular inactivity (Horner and
Urban 2001) and reduced matrix synthesis (Ishihara and Urban 1999). Thus, the
effects of depleted nutrients and increased metabolites have a cumulative detrimen-
tal effect on metabolism and survival of cells in the intervertebral disc niche, as well
as on the integrity of the disc matrix.
10.7 he Extracellular Matrix Environment

T
of an Intervertebral Disc
The extracellular matrix is a dynamic network of insoluble fibers (collagens and

elastin), soluble polymers (glycosaminoglycans and proteoglycans), and adhesive
glycoproteins (e.g., fibronectin, COMP, matrillin, laminin) (Sivan et al. 2014). Each
disc compartment consists of a different combination of extracellular matrix pro-
teins for proper function, and cells maintain a balance between anabolic and cata-
bolic processes for tissue homeostasis. Cells regulate their behavior by sensing their
changing microenvironment via cell surface receptors such as integrins that physi-
cally link the matrix proteins with intracellular cytoskeletal elements (Gilchrist
et al. 2007).
10.7.1 E
xtracellular Matrix Organization in Tissues
of the Intervertebral Disc
The nucleus pulposus is composed of large amounts of proteoglycans, predomi-

nantly aggrecan (30–50 % of dry weight) (Adams and Muir 1976; Eyre 1979),
which generates a swelling pressure inside the disc to resist compressive loads.
Aggrecan is retained in the nucleus pulposus by interacting with hyaluronic acid
to form supramolecular aggregates (Morgelin et al. 1988), and the sulfate groups
on its glycosaminoglycan chains (chondroitin and keratin sulfate) attract water
molecules (70–85 % of total weight) by osmosis (Watanabe et al. 1998). Human
chondroitin sulfate may also serve to restrict neural ingrowth, according to recent
in vitro studies (Johnson et al. 2002; Purmessur et al. 2015). In humans, aggrecan
content increases in juvenile years, peaks at late adolescence, but subsequently
declines in adult life so that the nucleus pulposus becomes less hydrated with
aging (Roughley et al. 2006a). In the nucleus pulposus, proteoglycans are
entrapped in an unorganized network of fibril-forming collagens (collagen II and
small amounts of collagens XI) that are decorated with fibril-associated collagen
with interruptions in the triple helix (FACITs) (collagen IX) (Wu et al. 1992; van
der Rest and Mayne 1988). These collagen copolymers (20 % of dry weight)
form a structural framework that contains the swelling pressure of the nucleus
pulposus. Collagens IX and XI also exist to possibly link collagen molecules to
other matrix components.
In a healthy human annulus fibrosus, collagens and proteoglycans make up 70 %

and 10 % of the dry weight, respectively, and the water content is 50 % (Eyre 1979;
Eyre and Muir 1976). The collagen II fibers in the inner annulus fibrosus have wider
interfibrillar spaces that retain a greater volume of water than collagen I in the outer
annulus fibrosus (Han et al. 2013; Schollmeier et al. 2000). This confers a superior
ability of the inner annulus fibrosus region to undergo deformation and endure high
mechanical pressures. A co-localized network of elastin fibers concentrated between
lamellae may also contribute to restore collagen and lamellar organization upon
distortion (Yu et al. 2005, 2007a). Collagen I forms heterotypic fibrils with colla-
gens III and V, and the translamellar bridging network of the outer annulus fibrosus
connects fibers of adjacent lamellae to provide a structural alignment that limits
interlamellar movement (Melrose et al. 2008; Smith and Elliott 2011).
Like the nucleus pulposus, the cartilaginous endplate is also composed of a
network of collagens II, IX, and XI and aggrecan in the pericellular microenviron-
ment of chondrocytes, although their relative quantities differ. Total collagens
make up 60–80 % of the dry weight, whereas glycosaminoglycans constitute 17 %
of the dry weight (Setton et al. 1993). Alternative splicing of the COL2A1 gene
produces procollagen mRNA that include (IIA) or exclude (IIB) an exon encoding
a cysteine-rich N-terminal domain. In mice pro-IIA mRNA exist in non-chondro-
genic and pre-chondrogenic cells, and expression switches to collagen IIB upon
chondrocyte differentiation (Sandell et al. 1991). Moreover, collagen X, encoded
by the COL10A1 gene, is mainly expressed in the central region of the endplate in
hypertrophic chondrocytes and regulates mineralization along with collagen II, by
entrapping matrix vesicles and facilitating calcium influx (Lammi et al. 1998;
Shen 2005). The pericellular matrix is believed to influence physiochemical sig-
nals sensed by chondrocytes and augment their protective response to mechanical
compression.
10.7.2 The Role of Proteinases in Matrix Remodeling
The matrix is a dynamic structure that is routinely broken down by proteinases

such as matrix metalloproteinases (MMPs) and a disintegrin and metalloprotein-
ase with thrombospondin motifs (ADAMTS) produced by disc cells. MMPs are
capable of cleaving most matrix constituents, with MMPs 1, 8, and 13 degrading
intact collagens I and II and MMPs 2 and 9 (gelatinases) cleaving partially
degraded collagen molecules (Nagase and Woessner 1999). The ability of MMPs
to degrade aggrecan, however, is significantly lower than that of the ADAMTS
aggrecanases (ADAMTSs 1, 4, 5, 9, and 15) (Durigova et al. 2011; Nagase and
Kashiwagi 2003; Pockert et al. 2009). These molecules along with other proteo-
lytic enzymes are implicated in the homeostatic turnover of disc extracellular
matrices. Equilibrium between synthesis, breakdown, and accumulation of matrix
macromolecules determines matrix integrity and influences the mechanical behav-
ior of the intervertebral disc as well as the maintenance of an avascular/aneural
microenvironment of a healthy disc.
10.7.3 The Changing Matrix Environment in Disc Degeneration
Intracellular events affecting glycosaminoglycan synthesis or the extracellular deg-

radation of aggrecan can cause disc dehydration, a common feature of degeneration.
In response to excessive mechanical loading (Neidlinger-Wilke et al. 2006) or infil-
tration of inflammatory cytokines such as TNFα and IL-1 (Hoyland et al. 2008; Le
Maitre et al. 2005, 2007a), disc cells produce MMPs and ADAMTS proteinases
(Adams and Roughley 2006; Roberts et al. 2000). Their proteolytic cleavage of
aggregated and non-aggregated aggrecan causes an extensive decrease in fragment
size and untimely loss from the disc tissue. Changes in disc hydration may also
result from shortening of chondroitin sulfate chains. The increasing avascularity of
degenerating discs can further reduce chondroitin sulfate synthesis, which requires
the oxidation of hexose to uronic acid. These changes, in addition to the steady
decline of aggrecan with aging, cause dehydration of the nucleus pulposus and an
increasing inability to recover from compressive deformation (Lyons et al. 1981;
Urban and McMullin 1988). Moreover, as the hydrostatic pressure of the disc falls,
mechanical loading may apply inappropriate stress concentrations along the end-
plate or annulus fibrosus forming fissures and tears between collagen fibrils, dis-
rupting the lamellar structure (Adams et al. 1996). Prolonged stresses on the disc
can cause the nucleus pulposus to protrude through the weakened annulus fibrosus
(herniation) or though the endplate (Schmorl’s nodes) (Adams and Dolan 2012;
Urban and Roberts 2003).
High concentration and charge of aggrecan in the normal disc also prevents
movement of large uncharged molecules (growth factors and cytokines) into the
disc microenvironment (Maroudas et al. 1975). Upon loss of aggrecan, infiltration
of these molecules may advance degeneration by affecting cellular behavior and
matrix catabolism. For instance, IL-1 induces fibrotic changes that include an
upregulation of MMP3 and 13 and ADAMTS4 in the nucleus pulposus, a shift in
expression from collagen II to collagen I in nucleus pulposus and annulus fibrosus
cells (Le Maitre et al. 2005, 2007b), and further degradation of aggrecan. It is also
considered to induce apoptosis and senescence of disc cells. Breakdown of normal
disc extracellular matrix and formation of annular clefts and tears in turn stimulate
neovascularization and accelerate the degenerative processes (Carreon et al. 1997;
Karamouzian et al. 2010; Vernon-Roberts et al. 2007).
Finally, ectopic expression of collagen X has been identified in the nucleus
pulposus and annulus fibrosus at late stages of degeneration in humans (Boos
et al. 1997). Induction of collagen X signifies cell hypertrophy and can be accom-
panied by increased expression of alkaline phosphatase (ALP) and Runt-related
transcription factor 2 (Runx2), causing abnormal calcification (Boos et al. 1997;
Rutges et al 2010).
Although the exact sequence of changes in degenerative cascade has not been
clearly defined, changes in matrix composition in the disc, particularly the loss of
aggrecan, impedes its ability to maintain osmotic pressure and remain intact under
mechanical loading. Changes in the extracellular matrix also hinder cellular main-
tenance and activity, resulting in a downward spiral of destructive events.
10.8 echanical Loading and Intervertebral Disc

M
Degenerative Events
Association between the mechanical environment and mechanical properties of disc

cells with degeneration has been demonstrated through in vivo studies that use
external loading devices, in vitro organ culture studies, and application of hydro-
static pressure in in vitro cell cultures. A disc cell’s response to stresses depends on
the type, magnitude, frequency, and duration of the loading. While physiological
loading conditions (<1 MPa magnitude, <3 Hz frequency, and <24-h duration) are
anabolic, loads outside this range are catabolic. Outside the “safe window” of tissue
mechanics, initially overload and eventually immobilization affect tissue homeosta-
sis and cell mechanobiological response (Stokes and Iatridis 2004).
The “wear-and-tear” or overload hypothesis represents a single, excessive
loading cycle or a frequent number of moderate loading cycles that accumulate
microscopic damage. The damage heals slowly due slow tissue turnover rates
and progressively weakens the ability of the disc tissues to withstand mechani-
cal pressure, increasing their susceptibility to fatigue failure. For instance, the
laminated structure of the annulus fibrosus requires damage propagation
through multiple micro-failure (matrix cracking, delaminations, and fiber fail-
ure). Damage is propagated mainly by delamination in presence of high inter-
laminar shear stresses after initial tears in the annulus, while fiber failure
occurs under severe loading conditions. Structural changes such as the decrease
in the number of layers and an expanded layer from increased spacing of col-
lagen fibers may also alter disc cell mechano-responsiveness and advance the
degenerative process.
Various mechanical interventions on cadaver discs and animal motion seg-
ments have been demonstrated to cause structural and compositional changes in
the discs. Administration of high compressive loading with bending on normal
cadaver discs (aged 40–50) produced disc prolapse or herniation (Adams and
Hutton 1982; McNally et al. 1993), and application of cyclic compressive load-
ing caused endplate fractures, Schmorl’s nodes, and a decreased disc height
(Brinckmann et al. 1988). Endplate fractures and Schmorl’s nodes reduce pres-
sure in the nucleus pulposus and generate compressive stress peaks in the annu-
lus fibrosus (Adams et al. 2000). Repetitive loading on endplate-damaged discs
further intensifies stresses on the annulus fibrosus, causing inversion of the inner
annulus, extreme outward bulging of the outer annulus, radial fissures, and disc
prolapse.
The zonal variations in mechanical environment, cell population, and cell
mechanical properties between the annulus fibrosus and nucleus pulposus result in
a distinct cascade of load-induced behaviors. An Ilizarov-type device used to apply
dynamic loading on skeletally mature Wistar rat tail discs demonstrated that while
changes in gene expression in the annulus fibrosus is dependent on magnitude of
stress, the nucleus pulposus is more frequency dependent (Maclean et al. 2004). In
the nucleus pulposus, low frequencies increased aggrecan and collagen mRNA,
whereas higher frequencies stimulated aggrecanase and collagenase expression. In
contrast, the annulus fibrosus exhibited increased aggrecanase and collagenase

mRNA levels with higher magnitudes of compression regardless of the frequency of
the applied compression.
While the consequences of cyclic loading are determined by both the magnitude
and frequency of the applied load, mechanosensitivity of disc cells to static loading
is largely magnitude dependent. An in vivo study analyzed mouse tail discs after
1 week of continuous loading at different stress levels (Lotz et al. 1998). Increasing
loading induced disorganization of lamellar structures in the inner annulus fibrosus
and replacement of fibroblast-like cells with chondrocyte-like cells, whereas the
outer annulus fibrosus was able to maintain its organization. Collagen II expression
was reduced in all areas of the disc regardless of magnitude of compression, whereas
aggrecan mRNA decreased in areas of highest loading. In the nucleus pulposus,
large cell clusters were dissociated into smaller islands of cells.
Static bending of mouse tail discs for 1 week also presented lamellar inversion at
the concave (compressed) side (Court et al. 2001), and as in human scoliotic discs,
the concave annulus fibrosus contained a greater number of dying cells. When the
bending is released, chondrocyte-like cells are detected in the concave annulus, sug-
gesting three possible differentiations of fibroblasts into chondrocytes, selective
loss of fibroblast-like cells, or chondrocyte migration from the endplate. Comparable
observations can be made using a compression bending mouse model induced by
tail looping that can be used to study cellular response to stretch and compressive
forces (Sakai et al. 2015).
When the structural interrelationship of disc compartments fails at late stages of
degeneration, tissue stiffness and hypomobility result. In vivo animal models dem-
onstrate that the immobilization condition shows reduced protein synthesis due to
lack of mechanical stimulus or lack of nutritional supply. Yet further investigation
on the effects of hypomobility on the progression of disc disease is necessary.
10.9 Genetic Risk Factors of Disc Degeneration
Intervertebral disc degeneration is considered to be a complex, multifactorial dis-

ease involving many genetic risk factors that interact with one another and the envi-
ronment. This is supported by a study of an Arabic pedigree which established
familial inheritance as a risk factor for intervertebral disc degeneration and sug-
gested that lumbar disc degeneration is a polygenic disease with a complex mode of
inheritance (Livshits et al. 2001). Indeed, lumbar disc degeneration (LDD) is esti-
mated to have a heritability rate from 47 to 74 % in familial and twin studies (Battie
et al. 2008; Sambrook et al. 1999). To understand the cause of intervertebral disc
degeneration, genetic studies provide the most direct and informative route to eluci-
date the genes and cellular pathways involved, followed by functional testing using
established model systems.
Much effort has been made to identify the genetic risk factors associated with
intervertebral disc degeneration in the lumbar spine. The initial focus was based on
the candidate-gene approach with genetic association studies assessing specific
genes as potential risk factors. Many genes in the inflammatory pathways and com-
ponents of the extracellular matrix were tested based on our initial understanding of
the biology of disc degeneration. Positive associations were identified, such as col-
lagen IX genes (COL9A2 and COL9A3) (Annunen et al. 1999; Jim et al. 2005;
Paassilta et al. 2001); aggrecan (ACAN) (Kawaguchi et al. 1999; Roughley et al.
2006b); MMPs-1, -2, -3, and -9 (Dong et al. 2007; Song et al. 2008a; Sun et al.
2009; Takahashi et al. 2001); and IL-1 (Solovieva et al. 2004a, b, 2006).
However, many of the studies have not been replicated and only showed low
levels of association, likely due to small cohort size and variable phenotype defini-
tion. A recent review of 52 association analyses (Eskola et al. 2012) suggested mod-
erate levels of evidence for six candidate genes, ASPN (asporin) (Song et al. 2008b),
COL11A1 (collagen XI α1) (Mio et al. 2007), GDF5 (growth differentiation factor
5) (Williams et al. 2011), SKT (Sickle tail) (Karasugi et al. 2009), THBS2 (thrombo-
spondin 2) (Hirose et al. 2008), and MMP9 (matrix metalloproteinase 9) (Hirose
et al. 2008).
A popular alternative approach for the study of complex diseases is perform-
ing genome-wide association studies (GWAS) to identity candidate loci across
the genome in an unbiased assessment (Hirschhorn and Daly 2005). However,
this requires a very large cohort size to reach statistical significance, as thou-
sands of single nucleotide polymorphisms (SNPs) must undergo multiple testing
corrections. Due to the lack of large cohorts that provide GWAS data for inter-
vertebral disc degeneration, an alternative is to perform a meta-analysis of
smaller cohorts to increase the genetic power. In a meta-analysis study totaling
4,683 subjects, a strong association (p = 2.8 × 10−8) for rs926849 which is located
in an intron of the Parkinson protein 2, E3 ubiquitin protein ligase (PARK2) gene
on chromosome 6 was detected (Williams et al. 2013). The relevance of this vari-
ant in lumbar disc degeneration is not clear, and further confirmation and func-
tional assessment are needed. More recently, an approach combining linkage
analysis of families with early onset lumbar disc degeneration and GWAS studies
totaling 32,642 subjects across three ethnic populations identified chondroitin
6-O-sulfotransferase (CHST3) as a risk factor (Song et al. 2013). The associated
functional SNP is on a miRNA binding site and may possibly alter the level of
CHST3 in affected individuals. CHST3 is enzyme required for proper chondroi-
tin sulfate synthesis, which is essential for water retention and inhibition of neu-
ral ingrowth.
10.10 Concluding Remarks
In the last decade, we have gained significant insight into the pathobiology of inter-
vertebral disc degeneration. From detailed studies on humans and animal models,
we are beginning to decipher the nutritional, mechanical, cellular, and biochemical
contributions to the degenerative process. While there are differences in the devel-
opment, anatomy, cellular characteristics, and mechanical properties between ani-
mal species and humans, research is most effective when an optimal animal model
is used to address well-defined and specific research questions, and subsequent vali-
dations are carried out in human cells or tissues. It is also essential to look beyond
the dehydrating nucleus pulposus as the major cause of disc degeneration. Cellular
and biochemical changes in the nucleus pulposus could be the consequence of ear-
lier changes in the surrounding tissues and disc environment.
The topics discussed in this chapter highlight the complexity of disc degenera-
tion, and to comprehensively study its causes and consequences, we must take
advantage of the most recent advances in technology. For instance, single cell tran-
scriptomics (Tang et al. 2009) has proved a powerful approach for dissecting cellu-
lar heterogeneity. Thus, it can be readily applied to recognize the identity of cells in
the nucleus pulposus and surrounding disc tissues, to understand the cellular
changes in a degenerating disc, and also to identify potential local progenitor cells
that may be used to stimulate endogenous repair or exogenous cell therapy strate-
gies. Furthermore, a comparative deep proteomic analysis of degenerate and non-
degenerate human intervertebral discs can provide supplemental information to
better understand how cells in different disc compartments may respond to a chang-
ing environment.
Findings from genetic studies are vital for understanding the cause of human disc
degeneration. However, as of yet, very few genes have been identified and the effect
size is small. Since variations in phenotypic parameters that define the degenerative
status of the disc confounds genetic studies, an accurate assessment of the pheno-
type and development of a common classification system for disc degeneration is
necessary. This improvement, alongside the collection of larger cohorts and the
advent of next-generation sequencing, will enable meaningful meta-analysis studies
to increase the statistical power of detection and identify rare genes with bigger
effect size.
Combining human genetics with in vitro or in vivo model systems for functional
assessment studies provides an ideal scenario. Upon identifying individuals that
harbor specific risk factors or even protective factors of disc degeneration, induced
pluripotent stem (iPS) cells (Yu et al. 2007a, b) can be generated from blood or urine
samples (Loh et al. 2009; Zhou et al. 2012) and then differentiated into the desired
cell type(s) for further analysis of disease outcome. These are all feasible technolo-
gies and approaches to consider for future studies. Understanding the etiology of
intervertebral disc degeneration could offer potential therapeutic strategies that not
only relieve procuring symptoms but also hope to restore the form and function of
intervertebral discs.
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Susanne Grässel · Attila Aszódi Editors

ISBN 978-3-319-45801-4 ISBN 978-3-319-45803-8 (eBook)

Library of Congress Control Number: 2017931314

© Springer International Publishing Switzerland 2017

Printed on acid-free paper

This Springer imprint is published by Springer Nature

epiphyseal dysplasia (MED) define a disease spectrum typified by varying degrees

Regensburg, Germany Susanne Grässel

1 Pathogenesis of Osteoarthritis in General ������������������������������������������������ 1

Attila Aszódi, PhD Laboratory of Experimental Surgery and Regenerative

Stephanie J. Gauci, PhD University of Melbourne Department of Paediatrics and

Heather Stanton, PhD University of Melbourne Department of Paediatrics and

M.B. Goldring (*) • K.L. Culley • M. Otero

© Springer International Publishing Switzerland 2017 1

1.2 Disease Etiologies and Therapeutic Prospects

1.3 Articular Cartilage Physiology

Adult articular cartilage is composed of a specialized matrix of type II collagen and

Proteases Adipokines Proteases

Tidemark → Catabolic factors

chondrocyte in a low-turnover, survival state by protecting it from interacting with

1.4 Articular Cartilage Pathology in OA

Of clinical importance is the ability of the chondrocyte to react to mechanical stim-

responses. The impact of cytokines on chondrocyte function, particularly with

Proteoglycan degradation is mediated by aggrecanases of the a disintegrin and

1.5 I nfluence of Synovitis and Inflammation on OA

1.7  ross Talk Between Articular Cartilage

these channels involving increased osteoclast activity, infiltration of inflammatory

FGF signaling is important in skeletal development and cartilage and bone

There is, however, considerable controversy about whether OA pathology

MMP-13 expression in osteoarthritis in humans and mice. J Immunol 182(12):7937–7945.

Kloppenburg M (2014) Hand osteoarthritis-nonpharmacological and pharmacological treatments.

Loeuille D, Chary-Valckenaere I, Champigneulle J, Rat AC, Toussaint F, Pinzano-Watrin A,

The role of mechanical overload/injury in osteoarthritis development cannot be

H.M. Ismail • T.L. Vincent (*)

© Springer International Publishing Switzerland 2017 27

Table 2.1 How mechanical factors contribute to OA risk

tendon/meniscal injuries leading to joint destabilisation, etc.) (reviewed in Brandt

2.2  rticular Cartilage: Structure and Mechano-Sensing

The articular cartilage is a paucicellular type II collagen- and proteoglycan-rich tis-

2.3 Chondrocyte Injury Assays

A number of different methods for applying injurious mechanical stress to a chon-

Table 2.2 In vitro cartilage injury models

embedded within three-dimensional artificial matrices, e.g. by stretching or mechan-

2.4  ellular Response to Cartilage Injury: Activation

2.4.1 Focal Adhesions: Src, FAK and Integrin Signalling

Integrins have been defined as ‘mechanoreceptors’ in other cell types, triggered by

cluster in focal adhesions to create transmembrane protein complexes that form

2.4.2 MAPK and NFkB Signalling

The mitogen-activated protein (MAP) kinases, p38, extracellularly regulated kinase

viability especially in the superficial zone. Reduction in cell viability post-injury

2.4.3 Smad and Wnt Signalling

2.4.4 Ion Channel Signalling

Mechanically induced Ca2+ signalling has been studied extensively in chondrocytes.

of the chondrocyte’s response to injury. GsMTx4 significantly protected chondro-

2.5 Cellular Response to Cartilage Injury: Cell Death

2.6 Cellular Response to Cartilage Injury: Gene Regulation

Several groups have studied gene regulation in response to mechanical cartilage

2.7 In Vivo Models of Cartilage Injury

Increasingly, we have seen an attempt to validate injury pathways in vivo

(reviewed in Campbell 1969). For instance, experiments of rabbit articular carti-

Regensburg, Germany Susanne Grässel

1 Pathogenesis of Osteoarthritis in General �� 1

1.2 Disease Etiologies and Therapeutic Prospects

1.3 Articular Cartilage Physiology

1.4 Articular Cartilage Pathology in OA

1.5 I nfluence of Synovitis and Inflammation on OA

1.7 ross Talk Between Articular Cartilage

2.2 rticular Cartilage: Structure and Mechano-Sensing

2.3 Chondrocyte Injury Assays

2.4 ellular Response to Cartilage Injury: Activation

2.4.1 Focal Adhesions: Src, FAK and Integrin Signalling

2.4.2 MAPK and NFkB Signalling

2.4.3 Smad and Wnt Signalling

2.4.4 Ion Channel Signalling

2.5 Cellular Response to Cartilage Injury: Cell Death

2.6 Cellular Response to Cartilage Injury: Gene Regulation

2.7 In Vivo Models of Cartilage Injury

3.1 Cartilage Matrix: Composition and Function

3.1.1 Aggrecan Structure and Function

3.1.2 Type II Collagen Structure and Function

3.1.3 SLRP Structure and Function

3.2 Cartilage-Degrading Proteinases in OA

3.2.2 ADAMTS Enzymes

3.2.3 Regulation of MMP and ADAMTS Enzymes

3.2.4 Aggrecan Degradation

3.2.5 Aggrecan Neoepitopes as Markers of Cartilage Catabolism

3.2.6 Type II Collagen Degradation

3.2.7 he Relative Contributions of Aggrecanolysis

3.2.8 SLRP Degradation

3.3 artilage Degradation Products Promote Inflammatory

methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl) met hyl)-2H-tetrazol-

4.2 Large Proteoglycans

availability of α-dystroglycan (DAG1) and the low-density lipoprotein receptor-

4.3 Small Proteoglycans

4.4 Small Leucine-Rich Repeat Proteoglycans (SLRPs)

5.1 Cytokines in Osteoarthritis

5.2 rigin (Synovial Membrane, Cartilage, Fat Pad, Bone/

5.3 ytokine Profiling in Synovial Fluid of Osteoarthritic

5.4 Profiling in Trauma and Early OA

5.5 Synovial Fluid Cytokines as Therapeutic Targets in OA?

5.6 Cytokines as Biomarkers

6.2 Cartilage Development and Endochondral Ossification