Soluble Mediators in Cartilage Regeneration
Jan Willem van Rijswijk
Department of Orthopaedics
University Medical Center Utrecht
December 2012
Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Utrecht
University
by
Jan Willem van Rijswijk, BSc
3157954
Supervisor:
Anika Tsuchida, MD
Department of Orthopaedics, University Medical Center Utrecht
Examiner:
Laura Creemers, PhD
Department of Orthopaedics, University Medical Center Utrecht
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Contents
Acronyms ................................................................................................................................................................ 3
Introduction ............................................................................................................................................................ 4
Articular cartilage biology ....................................................................................................................................... 4
Natural history and Epidemiology ........................................................................................................................... 6
Treatment ............................................................................................................................................................... 7
Joint homeostasis .................................................................................................................................................... 8
Soluble mediators in cartilage degeneration .......................................................................................................... 9
The role of soluble mediators in cartilage regeneration ....................................................................................... 12
Summary and Discussion ...................................................................................................................................... 17
References ............................................................................................................................................................ 22
Acronyms
ACI
ADAM-TS
BMP
CPC
DAMPS
ECM
FGF
FGFR
ICRS
IGF-1
Ihh
IL-1
MAC
MACI
miRNA
MMP
MSC
NFκB
OA
OPG
PTHrP
RANK
RANKL
Sox9
TGFβ
TIMPs
TLR
TNFα
Autologous chondrocyte implantation
A disintegrin and metalloproteinase with thrombospondin motifs
Bone morphogenetic protein
Chondrocyte progenitor cell
Damage-associated molecular patterns
Extracellular matrix
Fibroblast growth factor
FGF receptor
Cartilage Repair Society
Insulin-like growth factor-1
Indian hedgehog
Interleukin-1
Membrane attack complex
Matrix-induced ACI
microRNA
Matrix metalloproteinase
Mesenchymal stem cell
Nuclear factor kappa B
Osteoarthritis
Osteoprotegerin
Parathyroid hormone-related protein
Receptor activator of NFκB
RANK ligand
SRY (sex determining region Y)-box 9
Transforming growth factor-beta
Tissue inhibitors of MMP
Toll-like receptor
Tumor necrosis factor-alpha
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Introduction
Articulating joints rely on a complex design to provide smooth and pain free articulation. The sliding surface of
bones is covered by a highly organized avascular tissue called articular cartilage. This hyaline cartilage consists
mainly of a matrix of type II collagen and proteoglycans and is maintained by sparsely distributed chondrocytes.
The chondrocyte is very sensitive and responsive to stressful stimuli. Joint trauma leads to a disturbance in the
highly regulated joint homeostasis, activation of chondrocytes, and a phenotypic shift causing cartilage
destruction. Cartilage has a limited intrinsic capacity for repair and untreated, initially focal, lesions may
eventually lead to development of osteoarthritis (OA) in which the whole joint, encompassing the cartilage,
synovium, and underlying bone, is either involved or affected. Severe pain, loss of movement, and a limited
intrinsic regenerative potential of cartilage after degradation currently make OA one of the most important
challenges in orthopedic surgery. Existing repair strategies result in cartilage of inferior quality as compared to
native cartilage. Understanding cartilage regeneration is essential for improved repair of articular cartilage
defects. The present thesis will summarize the changes in intra-articular homeostasis after focal defects of
cartilage and osteoarthritis, and focus on the role of soluble mediators in cartilage regeneration.
Articular cartilage biology
In order to fully appreciate the challenges in understanding cartilage disease progress and improving its
regeneration, this first section will cover the normal function and biology of articular cartilage.
Healthy articular cartilage is required for the joint’s basic function: articulation. Pain-free, smooth articulation is
facilitated by a thin layer of smooth, near frictionless, hyaline cartilage at the articulating ends of bones [1].
Moreover, friction is even further decreased in combination with the lubricating properties of synovial fluid [2].
Adult articular cartilage is an avascular and aneural tissue that consists of an extracellular matrix (ECM)
maintained by a relatively low number of isolated chondrocytes. The ECM mainly consists of two components:
collagen type II and glycosaminoglycans, which account for 60 and 30% of the dry weight of adult human
cartilage, respectively. Water however, comprises up to 80% of the wet weight [1]. When cut longitudinally,
four histologically distinct layers can be recognized in cartilage, from the articulating surface towards the
subchondral bone [3]. The top layer is called the superficial zone and consists of several layers of cells with a
flattened disc-like appearance, including chondrocytes and progenitor cells [4]. The ECM in the superficial zone
contains randomly organized horizontal bundles of thick collagen fibers, parallel to the plane of the articular
surface, and little proteoglycans. This layer is thought to deal with horizontal shear forces [5]. In the middle
zone, few increasingly rounded chondrocytes are found within a matrix consisting of proteoglycans and
collagen. The collagen fibers form a transitional network between the horizontal superficial layer and the
vertical deep layer collagen fiber orientation. The increase in proteoglycans attracts high amounts of water
which together with the collagen act as a mechanical cushion to absorb impact energy. The stiffer deep zone is
characterized by high proteoglycan content, vertically aligned collagen fibers, and clusters of chondrocytes
(chondrons) orientated along the vertical axis [3]. The bottom calcified layer of adult articular cartilage is
separated from the upper layers by the tidemark and provides a biomechanical junction into the underlying
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subchondral bone, which on a microscopic level shows direct coupling through collagen fibers [5]. These matrix
molecules combined with a highly organized architecture are essential for the mechanical properties of
articular cartilage, which requires a smooth, low friction surface, and stiffness against compression, forming a
cushion to protect the underlying bone from high mechanical peak stress [1].
The cartilage matrix is maintained by chondrocytes and possibly a progenitor cell population which take up
only about 5% of the cartilage volume [4, 5]. Chondrocytes are very sensitive to changes in the ECM, presence
of soluble mediators in the matrix, but also changes in mechanical loading. In response to these changes,
matrix components can be either synthesized or degraded. Matrix turnover is a slow process regulated by
catabolic and anabolic cytokines including interleukin-1 (IL-1) and insulin-like growth factor-1 (IGF-1),
respectively [1]. These cytokines in turn can mediate ECM remodeling by inducing expression of collagen and
proteoglycans, or matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and the aggrecanases -4 and
-5 [5]. In healthy cartilage, synthesis and degradation are in a delicate balance and the slightest disturbance can
cause serious damage [1, 5].
Because articular cartilage lacks vascularization and innervation it depends on oxygenation and nutrition by
diffusion of synovial fluid and by the underlying subchondral bone.
Synovium
The synovial tissue is the blood vessel-rich inner membrane that lines the joint cavity. The intimal layer facing
the articular cartilage consists of one to three cell layers of type A and type B synovial intimal cells. The type A
synoviocytes are tissue macrophages with antigen presenting capabilities, actively clearing cell debris and
matrix degradation products in the joint cavity [6]. Type B cells are fibroblast-like and involved in production of
hyaluronic acid, collagen and fibronectin for use in both the loose connective tissue and synovial fluid [7]. This
layer is thought to play an important role in joint homeostasis by providing nutrition to articular chondrocytes,
clearance of intra-articular debris, production of lubricating synovial fluid, and regulation of immune function
[6-8].
Subchondral bone
Subchondral bone consists of the bone plate underlying the cartilage and is supported by a network of
trabecular bone. Subchondral bone and cartilage are biomechanically linked and the mechanical properties of
cartilage are probably dependent on the quality of the subchondral bone and the trabecular network, and vice
versa. Healthy cartilage requires a gradual stiffness gradient for optimal distribution and damping of
mechanical loading [9, 10]. In addition to mechanical load transfer, this region is also involved in cartilage
nutrition through structures called chondro-osseous junctions and/or micropores. These junctions are
prolongations of deep layer cartilage that extend through the calcified cartilage layer and contact the
underlying bone or marrow spaces. This contact implies a potential direct molecular communication pathway
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between bone cells, chondrocytes and synoviocytes. Joint homeostasis may thus be directly influenced by
cytokines and growth factors released from subchondral bone [11, 12]
Natural history and Epidemiology
The beginning of the natural history of OA is largely unknown but several factors may predispose joints to the
disease. It is most likely a combination of both endogenous risk factors like age, sex, genetics, and systemic
factors on one side, and environmental factors with injury, overload, and joint instability as most common
pathways on the other side. All articular joints including, fingers, knees, hips, and the spine can be affected,
especially the weight-bearing joints. OA has potentially devastating effects on these joints and on the overall
quality of life because of the involvement of pain, restricted mobility, and limitations of daily living activities
[13]. OA also represents a large economic burden mainly because of the previously described restrictions, but
also coexisting disorders, and the high cost of treatment, and is expected to increase in the future because of
the ageing population [14].
Knees, hands, and hips are commonly involved in OA of which the knee is most frequently affected in the
general adult population. The Framingham OA study (1983-1985) found radiographic knee OA in 34% of
women and 31% of men between the ages of 63 and 93 living in the United States. Radiographic knee OA is
defined as the radiological observation of joint space narrowing, multiple osteophyte formation, and
subchondral sclerosis. In women the prevalence of OA increased significantly with age, they showed 25%
radiographic OA at ages 63-69 and 52,6% at age ≥80, while at the same time in men this increase was far less
pronounced, with 30,4% displaying radiographic OA at ages 63-69 and 32,6% at age ≥80. Symptomatic knee OA
prevalence, associated with disability, was also more common in women than in men between age 63 and 93
years (11 vs. 7%) [15]. These numbers highlight the high prevalence of OA, and the significant effect of gender.
The onset of OA can often be traced back to joint trauma involving mechanical overload. Previous knee injury
significantly increases the relative risk of severe radiographic and symptomatic knee OA in both men and
women (relative risk for men, 3,46 vs. women, 2,18) [15]. Sport accidents (especially skiing and football) are the
main cause of acute traumatic damage in over halve of the cases. Accidents during daily living (work and at
home) account for another major fraction of cases [16, 17]. In 10-39% of the patients with cartilage damage
however the cause is unknown [16-18]. Daily wear and tear of the joints is also likely to contribute; hence age
as risk factor. But also obesity and diabetes are strong risk factors for developing OA. In the Framingham OA
study, pre-existing obesity strongly predicted the risk of later severe radiographic (RR men vs. women: 1,86 vs.
3,16) and symptomatic OA in men and women [15]. In addition to the obvious mechanical effects of obesity on
articular joints, it may promote a low grade inflammation in joints and production of adipokines, predisposing
to OA development [19]. Disorders to ligaments supporting the joint can also cause instability and altered
mechanical loads.
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In most cases, OA is initiated as a focal area of damage located at a load-bearing area of the cartilage and is
frequently not isolated to a single joint. The damage at this point is likely limited to disruptions in cartilage
matrix in the superficial layers and mostly asymptomatic with some minor temporary swelling, local pain,
locking, and catching [20]. The lesion size is dependent of the type and the severity of the trauma, and
according to the Cartilage Repair Society (ICRS) there are four anatomical gradations of the depth of cartilage
injury [21]. ICRS grade I describes near normal cartilage with only superficial defects, whereas in grade II lesions
the defect extends down to up to 50% of the cartilage depth. Grade III is a lesion with full thickness cartilage
damage up to but not including the subchondral bone, while the subchondral bone is also penetrated in grade
IV lesions. Smaller defects may eventually completely regenerate, whereas defects larger than 3 mm in
diameter do not repair spontaneously [20]. In these larger lesions the failing repair processes lead to a cycle of
increasing loss of homeostasis and cartilage matrix which may last for years or decades. Over time, the loss of
cartilage results in narrowing of the joint space which is visible on radiographs. At this stage, surrounding
innervated tissues are involved in causing pain, while loss of smooth cartilage causes the known clinical
problems including joint pain, inactivity stiffness, restricted movement, and crepitus [22].
Many patients that have become symptomatic will consult a physician. OA however is not always easy to
diagnose, e.g. there is only a weak correlation between radiographic changes in the joint, the gold standard,
and symptomatic OA progression, so it’s possible that some patients show no radiographic changes but display
all the symptoms, but it is also possible that radiographic OA is diagnosed while the patient is free of symptoms
[22]. For a more accurate diagnosis arthroscopy is required. During the procedure the source of symptoms and
a prognosis can often be determined [22]. Accurate diagnosis is very important because early detection
improves the chances of effective treatment [23].
Treatment
When the patient is presented at the clinic and is diagnosed with osteoarthritis, the first treatment is aimed at
reduction of pain and stiffness to maintain or restore mobility and improve the quality of life. The other goal of
treatment is to prevent the progression of cartilage degradation and to regenerate damaged tissue, when
possible. Therapy can be pharmacological for symptom relieve, non-pharmacological, or surgical, but more
commonly is a combination, depending on the patients’ state [24].
The first step always consists of informing the patient on OA and on the importance of changes in lifestyle like
weight loss in obese and therapeutic exercise to reduce pain and symptoms [24]. Patients may also use simple
painkillers and topical agents. More severe cases are also treated pharmacologically which involves the use of
anti-inflammatory drugs and other supervised therapies. Although these procedures (temporarily) alleviate
joint pain, they do not induce cartilage repair and without further treatment this can result in progressive
cartilage destruction. The development of pharmacological therapy targeted at disease causing factors is
challenged by the fact that OA is a multifactorial disease, induced by a combination of mechanical and complex
biochemical interplay [24]. However, many new targeted pharmacological approaches are in development and
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have passed pre-clinical stages. The drugs in phase I and II clinical trials include aggrecanase inhibitors, iNOS
inhibitors, and recombinant growth factors like FGFs and BMPs [25].
The most commonly used surgical method for treatment of severe symptomatic chondral defects is the
microfracture technique. This technique relies, together with the drilling method, on penetration of the
subchondral bone plate to access underlying blood vessels and bone marrow. The resulting fibrin clot at the
surface of the defect is then repopulated by migrating bone marrow stem cells. However, the fibrocartilage
that is formed by these cells is of inferior quality and prone to deterioration [20]. Other surgical treatments
include autologous cartilage grafts (mosaicplasty) and chondrocyte implantation (ACI) and have resulted in
varied long-term outcomes, in part due to the unpredictable nature of repair. During mosaicplasty, cartilage
plugs are excised from ‘non-load bearing’ regions of the articular cartilage and transplanted to the site of the
lesion. ACI is an early tissue engineering technique in which chondrocytes are also isolated from a non-load
bearing area, but then expanded in vitro over the course of 2 to 3 weeks, and subsequently injected into the
defect, kept in place by a sutured autologous periosteal patch [26]. The ACI procedure yields good long-term
results in a large percentage of patients, and rapidly evolved into new, more efficient techniques like matrixinduced autologous chondrocyte implantation (MACI). Like ACI, MACI requires initial surgery to isolate
autologous chondrocytes; however the cells are now cultured on a three-dimensional bio-scaffold which better
preserves the chondrocyte phenotype. After several weeks the scaffold is glued onto the calcified cartilage
layer with fibrin gel. MACI has resulted in promising short term results, but long-term outcome is still unknown
and further investigation is required [27, 28].
Mosaicplasty, ACI, and MACI use autologous cells that are isolated during arthroscopic examination and this
requires the sacrifice of cartilage at a non-load bearing area. It is suggested however that the commonly
recommended sites for osteochondral harvest are in fact active sites of contact pressure and thus involved in
load bearing [29], and that this may lead to donor site morbidity. Sacrifice of perfectly fine cartilage should thus
preferably be avoided and with the increasing number of papers on stem cells in cartilage regeneration and the
effects of different anabolic factors this may soon become possible. Stem cells have been found in cartilage and
surrounding tissues and display chondrogenic capacity and regenerative potential when stimulated with the
appropriate growth factors [4, 30]. Furthermore, it is unclear in what way the cells seeded in the graft
contribute to repair or regeneration. A third generation of techniques, using biodegradable scaffolds for in situ
tissue engineering might represent the next step. These scaffolds can be seeded with MSCs and/or biological
cues to ‘guide’ derived and migrating cells towards chondrogenesis [31].
Joint homeostasis
One of the possible reasons why current treatments for cartilage defects fail to deliver is the fact that most
therapies focus exclusively on the cartilage even though the entire joint is involved. Cartilage is only one of the
players and restoration of this structure alone will not result in restoration of the whole joint function. In
healthy joints the interactions between cartilage, the synovium, synovial fluid, subchondral bone, but also the
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menisci and ligaments, create a highly organized dynamic environment with a balanced metabolism [32].
Cartilage, synovium and subchondral bone are known to directly communicate through the synovial fluid and
chondro-osseous junctions. Anabolic and catabolic factors, anti-inflammatory and pro-inflammatory
components are all exchanged between these structures and kept in a highly balanced state to ensure that
together they provide pain-free and smooth articulation. The concept of joint homeostasis is used to describe
this delicate equilibrium, in which joint tissues are capable of responding adequately to changes that fall within
physiological limits.
Loss of homeostasis is known to precede the onset of OA and is likely to be influenced by extrinsic mechanical
and biological factors such as loading, weight, age, and several predisposing disorders. Changes in e.g.
mechanical load distribution that lie beyond the normal physiological range will disrupt the local matrix and
disturb the metabolism of affected cells [1]. When a damaging factor is sufficiently forceful or repeated for a
sufficient duration of time, the metabolic equilibrium will shift towards an inflammatory catabolic state and
eventually leads to the alterations in joint homeostasis with involvement of the entire joint. The loss of
homeostasis directs the joint into a destructive cycle wherein matrix components are being degraded much
faster than they are synthesized, and where cartilage degradation is maintained by synovial inflammation and
subchondral bone remodeling [1, 23].
Soluble mediators in cartilage degeneration
Focal cartilage defects are often found after impact trauma and predispose to OA development. Mechanical
forces disrupt the ECM and cause cell deformation. Pathological loads and stresses that result in matrix
disruption, but also loads that produce no visible damage, may be translated to the chondrocyte by integrinmatrix interactions, which are essential for differentiation and viability, demonstrating the importance of the
cell environment [33, 34]. Indeed, a decrease in chondrocyte survival is often observed in vitro and in vivo in
the early phase after cartilage damage [35-38]. Traumatic impact causes death by necrosis primarily in the
superficial cartilage zone and some apoptosis in deeper layers as the lesion progresses [35, 37, 39, 40]. In
chondrocytes outside the necrotic and apoptotic zone, the mechanical load is mechano-transduced and may
activate or increase the expression of a numerous genes involved in inflammation and cartilage degradation
[41]. Within hours of initiation, the expression of cytokines including tumor necrosis factor (TNF)α, interleukin
(IL)-1β, IL-6, IL-1 receptor antagonist (IL-1Ra) [42], and proteases including matrix-metalloproteinases (MMP-1,
MMP-2, MMP-3, MMP-9, MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs
(ADAM-TS) dominates the chondrocyte expression pattern [39, 41, 43]. The result is a disturbance in the joint
homeostasis in which a shift to catabolic processes is observed. Cartilage ECM synthesis is inhibited while
matrix degradation is increased by the production of proteases.
Localized loss of proteoglycans is first observed and is followed by collagen type II degradation, leading to
cartilage softening and further matrix instability [42]. ADAMTS-4 and -5 appear to be responsible for the
majority of proteoglycan degrading [44], whereas collagen type II is mainly degraded by MMP-1 and MMP-13
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[45]. The increased release of matrix fragments from the cartilage surface into the synovial fluid can stimulate
the production of inflammatory cytokines and MMPs in the synovial membrane [46] which may last for
decades [43]. Synovial membrane inflammation is found in 50-90% of patients with early or late stage OA [8,
47] and is characterized by inflammation with production of pro-inflammatory cytokines and mediators,
infiltration of leukocytes, intimal thickening, and fibrosis. Synovitis is now recognized as a key factor in OA
pathology wherein cartilage alterations fuel synovitis, which in turn amplifies cartilage destruction. The exact
mechanism by which the synovium contributes to cartilage degradation is unknown, but it is thought that the
matrix fragments released during cartilage breakdown may act as damage-associated molecular patterns
(DAMPS) on Toll-like receptors (TLR) [48]. Synovial fibroblasts, macrophages, but also chondrocytes are known
to express these innate immune receptors and upon activation induce the TLR-activated transcription factor
NFκB and subsequent production of inflammatory cytokines and MMPs. In the synovium this leads to increased
mononuclear cell infiltration and neovascularization. Thus, involvement of the synovial membrane generally
intensifies cartilage degradation by increasing the inflammatory response [48].
Impact trauma may also lead to blood or serum leaking into the joint, changing the composition of the synovial
fluid and introducing innate immune factors like the complement system. Complement components have
indeed been observed in both healthy and diseased cartilage [49, 50]. Synovial fluids from patients with early
OA show increased levels of C3a and C5b-9 (MAC), indicating complement activation. The source of increased
complement activity may not necessarily be blood, as both cartilage and the synovium are able to produce
complement proteins and change their activity in cartilage disease [49, 51]. The trigger of increased activity is
thought to come from cartilage ECM degradation products. Collagen binding proteins have been found to be
potent activators of the classical and alternative complement pathways, suggesting they may contribute to the
disease progression [51, 52]. Complement may contribute by promoting pore-forming membrane attack
complex (MAC)-induced cell death or membrane instability in chondrocytes, the latter resulting in the
increased expression of degradative enzymes, inflammatory cytokines, and COX-2 [51]. Complement activation
also stimulates chemotaxis of leukocytes, which is reflected by synovitis. The importance of complement in the
pathogenesis after a cartilage defect is underlined by a study in which C5-deficient mice showed substantially
attenuated cartilage degradation and synovitis in an induced OA model as compared to C5-sufficient mice [51].
OA involves the entire joint, and starting from the moment of injury, the mechanical abilities of the cartilage to
transport loads to subchondral bone are altered. It is however also proposed that initiating events in the bone
lead to changes in cartilage load transmission and subsequent cartilage degradation. In either way, cartilage
remodeling and bone remodeling coincide and communication pathways between the tissues have been
demonstrated [11, 12]. It is suggested that pathological mechanic loads translate to osteoclasts, as it does to
chondrocytes, and induces an imbalance in bone formation and resorption. Initial mechanical stress however
does not appear to be a prerequisite to induce these changes [53]. Nonetheless, the shift in bone homeostasis
results in high bone resorption in early OA, likely to be mediated by phenotypically altered osteoblasts. These
changes in bone remodeling are in part due to increased activity of IGF-1 and TGF-β, which in turn are
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regulated by cytokines such as IL-1β, IL-6 and TNFα [9, 54]. It is hypothesized that these cytokines, produced by
osteoblasts, diffuse through channels in the calcified cartilage into the deep layer of the cartilage, and research
has already shown that factors secreted by OA bone cells are able to influence cartilage metabolism [55, 56],
and that OA chondrocytes can affect subchondral bone metabolism [57]. Whether one process precedes the
other or vice versa remains elusive but understanding this is essential for the development of treatments.
Besides IGF-1 and TGF-β, other proteins like receptor activator of NFκB (RANK), RANK ligand (RANKL), and
osteoprotegerin (OPG) similarly control subchondral bone remodeling and might also be involved in cartilage
metabolism [58, 59]. The ratio at which RANKL and OPG are produced in subchondral bone is impaired during
OA and leads to changes in bone remodeling and cartilage homeostasis [59]. Changes in subchondral bone are
also mediated by TGF-β/BMP signaling [9, 60]. In line with TGF-β/BMP signaling, a cross-talking pathway
represented by Wnt/β-catenin is involved in osteoblast differentiation and bone remodeling. Activation of the
Wnt pathway leads to increased levels of stable β-catenin in chondrocytes in vitro, inducing a catabolic shift
with increased expression of MMP-3, -13, and ADAMTS-4, and 5 [61]. Increased levels of nuclear β-catenin in
OA-like chondrocytes in vivo in a guinea pig model suggest the direct involvement of the Wnt/β-catenin
pathway in cartilage remodeling and degradation [61].
These persisting changes in both cartilage and bone homeostasis, in collaboration with the synovium, lead to a
vicious cycle of altered biomechanical properties, in which inflammation, cell death, and matrix degeneration
leads to the progression of cartilage damage to a ‘point of no return’. A recent study suggests that ‘about half
of the patients with traumatic joint injury develop OA’ [62]. The process of OA development after joint injury
appears to be a continuous process in which normal joint homeostasis is increasingly lost, catabolic activities
dominate over anabolic processes, and intrinsic repair fails. Most inflammatory mediators and effectors
involved in the joint response after injury remain present during OA progression [50]. Several temporospatial
differences in expression have been observed however.
Progression of OA by progressive degradation of cartilage collagen is mediated by MMP-13 expression, which is
found in increasing concentrations with increasing stages of OA [63]. Mild synovitis is present in all stages from
trauma to late-stage OA [48], but the evolution of synovitis with the progression of OA appears to be variable
and may depend on an unknown factor [64]. Several studies report an increase in synovial inflammation as
disease progresses, with the greatest intensity during late-stage OA [65], while others observe the opposite,
with increased numbers of infiltrating macrophages, lymphocytes, and neovessels in early OA [64, 66].
Nonetheless, increased levels of secreted pro-inflammatory cytokines (primarily IL-1β and TNFα) in the synovial
membrane activate other synovial cells and chondrocytes and induce expression of inflammatory mediators
including iNOS, COX-2, and the proteases MMP-3, -9, -13, and ADAMTS-4 [67]. Besides the proteolytic
properties of the MMPs, they are also involved in regulating the activity of other proteases and growth factors
[68]. More recently several microRNAs (miRNAs) have been identified that show differential expression in OA
cartilage and bone tissue when compared to healthy joints. Deregulation of miRNAs including miR-9, -98, -146
[69], and miR-455 [70] is thought to contribute to cartilage destruction by altering gene expression and thereby
11
regulating proteins as IL-1β, TNF-α, MMP-13, and TGF-β. It thus appears that miRNAs also play a role in
changing cartilage homeostasis and mediating cartilage destruction [69-71].
In summary, the process leading to cartilage degradation and OA is multifactorial, altering expression and
secretion of numerous genes, factors and soluble mediators in the whole joint. Mechanical overloading can
cause heterogeneous lesions in all layers including the bone plate. Chondrocytes are killed on impact and
disruption of the cartilage matrix leads to dysfunction of surrounding cells. Increased catabolic activity, matrix
degradation fragments and released pro-inflammatory cytokines spread through the joint. TNF-α and IL-1β
appear to be the main mediators of inflammation and cartilage destruction by inducing expression of NFκBregulated inflammatory genes in the cartilage, synovial membrane, and subchondral bone [45]. Increased
expression of ADAMTS-4 and -5, and several MMPs, most importantly MMP-13, leads to destruction of
cartilage matrix while homeostatic changes in the cartilage decrease matrix synthesis. The release of cytokines
and matrix fragments into the synovial fluid enhances or initiates synovitis by complement activation and
DAMPS-induced TLR activation, which in turn increase cell death and inflammation intensity by the release of
more cytokines and proteases. OA synovial intimal cells are an important driving factor in disease progress.
Parallel to, or even preceding the events in the cartilage and synovium is the aberrant remodeling of bone
tissue by imbalanced IGF-1, TGF-β/BMP, RANKL, and OPG expression. Remodeling of the bone alters its
loadbearing properties which are reflected in mechanical changes in the overlying cartilage. Furthermore, the
expressed factors in the subchondral bone are able to directly mediate changes in chondrocyte metabolism
through communicating channels. Because of the lack of effective endogenous cartilage repair, cartilage matrix
is increasingly degraded, fueling inflammation in the three joint tissues by release of ECM fragments and
changes in load transduction. Further understanding of the timing of events in the joint is needed to grasp the
complex pathways that are responsible for homeostatic changes and disease in OA.
The role of soluble mediators in cartilage regeneration
OA causes irreversible loss of cartilage by increasing inflammation and failing attempts to repair. The low
intrinsic capacity for self-repair in adult cartilage make it one of the key obstacles in orthopedic treatment. In a
joint environment where homeostasis is disrupted and catabolic factors dominate, the only way to halt
progression and start regeneration appears to be to restore homeostasis before irreversible damage is
inflicted. Unfortunately, this means necessity for early treatment; a period in which most individuals are
asymptomatic and do not present themselves for treatment. Treatment during this period of relatively normal
joint homeostasis positively influences the outcome of cartilage formation. In contrast, late treatment of older
defects, with highly disturbed homeostasis, does not lead to repair. This indicates the presence of a factor, or a
change in ratio of factors in cartilage that affect the regenerative potential [23].
It is well known that while mature articular cartilage has low potential for repair, fetal and immature cartilage
have the ability to spontaneously regenerate [72]. In some species, like the eastern newt and the axolotl
salamander, adult animals maintain the capacity to completely regenerate damaged cartilage and even
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complete limb and tail structures [73, 74]. Mechanisms involved in cartilage regeneration in these animals are
remnants of embryonic cartilage development. It was found e.g. that the transcription factor SRY-box 9 (Sox9),
which is essential for early chondrocyte differentiation, was increased over time after injury and associated
with increased matrix synthesis, suggesting reinstatement of latent chondrogenic capacities [73]. In adult mice
cartilage, Sox9 appears to have taken on a central role in mediating the expression of numerous chondrocytespecific genes and maintaining a healthy cartilage homeostasis [75], and loss of Sox9 expression in OA cartilage
may promote loss of cartilage homeostasis by inducing hypertrophy, cell death and decreasing ECM content
[75, 76]. On the other hand, articular cartilage lesions lead to local proliferation and matrix expression in Sox9rich chondrocytes in an OA mouse model. These studies suggest that, like in salamanders, mammalian Sox9 is
involved in the induction of chondrogenesis in mature cartilage after injury [76]. The repair processes initiated
by these chondrocytes however are insufficient at overcoming the progressive degeneration of cartilage.
Interestingly, in an OA rabbit model, implantation of Sox9-transduced bone marrow stem cells enhanced
cartilage repair by taking on a stable chondrocyte phenotype [77]. Sox9 may also be involved in regulation of
vascularization and subchondral bone growth via suppression of several genes including VEGFA, RUNX2,
MMP13, and RANKL [78], suggesting that cartilage and bone homeostasis are also linked via Sox9. This is
underlined by an in vitro co-culture study in which OA subchondral osteoblasts decreased Sox9 and
subsequently COL2A1 gene expression in chondrocytes [56].
Sox9 activity in turn is highly regulated by soluble upstream mediators including bone morphogenetic proteins
(BMPs), fibroblast growth factors (FGFs), IGF-1, parathyroid hormone-related protein (PTHrP), and Indian
hedgehog (Ihh) [76]. BMPs are well known powerful bone and cartilage inducing cytokines of the transforming
growth factor-beta (TGF-β) superfamily and may regulate Sox9 via BMP-Smad signaling [79]. BMPs anabolic
properties in this way contribute to mature joint homeostasis [80].
More specifically, BMP-2 and -7 and its receptor BMPR-1A are detected in the synovial membrane, in normal
cartilage, and at lesion borders, and an important role for BMP-2 and -7 has been predicted in cartilage repair
[81]. Furthermore, BMP-2 mRNA was found up regulated after cartilage injury [82] and deletion of the BMPR1A
gene results in OA-like cartilage degeneration in mice [80]. BMP-2 addition in an OA mouse model leads to an
increase in ECM turnover, indicating a role in remodeling or repair [83]. BMP-7 (also known as OP-1) also plays
a key role in cartilage homeostasis and enhancing repair by improving chondrocyte survival and improving the
quantity and quality of matrix repair after injury [84, 85]. BMP-7 is able to regulate both anabolic and anticatabolic processes at the level of the ligand, receptor, and downstream targets [86], and stimulates
production of collagen II, proteoglycans, IGF-1 and the IGF-1 receptor. Conversely, inhibition of the BMP-7 gene
leads to depletion of matrix components from the cartilage ECM [86]. BMP-7 belongs to a subgroup of BMPs
that also includes BMP-6. This protein is known to promote chondrocyte differentiation in the growth plate and
possibly other early chondrocyte metabolic processes. The role of BMP-6 in mature cartilage is unclear but
expression at both mRNA and protein level was observed in healthy and OA cartilage, and in vitro stimulation
of OA and healthy chondrocytes with BMP-6 stimulated proteoglycan synthesis [87]. These findings suggest
13
that BMPs play a key role in cartilage maintenance, repair, and homeostasis, and appear to do so in a Sox9
dependent manner.
BMP-6 however may also influence cartilage repair in a different way. In a study on chondrocyte progenitor
cells (CPCs), isolated from late stage OA cartilage, it was demonstrated that these cells migrate to OA cartilage
ex vivo and that BMP-6 can serve as a chemoattractant [30]. In combination with TGFβ3, BMP-6 also positively
regulates matrix formation by CPCs by increasing collagen type II expression while at the same time reducing
collagen type I and MMP13 mRNA expression. Although Sox9 mRNA expression was unaltered after
TGFβ3/BMP-6 stimulation, Runx2 expression was reduced and this explains the changes that enhance the
chondrogenic potential of CPCs. This transcription factor is found increased in OA cartilage and promotes
disease by inducing MMP13 expression [30].
The previous study brings us to the role of progenitor cells in cartilage regeneration. Bone marrow stem cells
and adipose derived stem cells have demonstrated chondrogenic potential and have been studied extensively.
Furthermore, bone marrow cells are already indirectly clinically applied with moderate success in the microfracturing and drilling therapy, so called marrow stimulation [88]. The use of stem cells to induce tissue
regeneration is highly attractive, especially when the native tissue shows low regenerative capacities.
Increasing evidence suggests that cartilage itself also contains a large progenitor cell pool [4, 30, 89-91]. These
progenitor cells are thought to be remnants of bone growth during childhood in which cartilage is appositioned
from the epiphysial plate to increase bone length [89]. This is supported by the apparent distribution of
progenitor cells in cartilage, namely primarily in the superficial [4, 89, 91] and middle cartilage zones [91]. The
exact role of the progenitor population in mature cartilage is unclear. Several studies demonstrated an
increased frequency of stem cell markers in OA cartilage which suggests the involvement of progenitor cells in
the course of OA [89, 90]. One explanation for this may be an increase in migratory mesenchymal cells from the
bone marrow through micro-fractures that penetrate the subchondral bone, which have been observed in OA
joints [90]. In either way, the observed progenitor cells appear multipotent and show a MSC-like differentiation
pattern and have the potential to regenerate structural and biomechanical properties of diseased cartilage [30,
90]. These observations suggest that the intrinsic reserve capacity for regeneration of diseased cartilage is
larger than previously thought and may provide a basis for regenerative therapies [91].
The presence of a progenitor cell population in the cartilage (or in the underlying bone marrow) also indicates
that the use of exogenous cell transplantation may not be necessary for cartilage regeneration. This is
emphasized in the previously mentioned study with TGFβ3/BMP-6 as a chemoattractant for CPCs but also in
another recent study. Lee et al. demonstrated in a model that acellular bioscaffolds impregnated with TGFβ3
could induce full regeneration of articular cartilage defects [92]. TGFβ3, not specifically known for its homing
abilities, was able to recruit stem cells from surrounding joint tissues in a 3D cell migration system, including
from bone marrow and adjacent adipose tissue, and induced these cells to undergo chondrogenesis [93]. Thus
14
stimulation of the latent regenerative potential by augmenting progenitor cell homing and enhancing
chondrogenic potential seems feasible.
Another member of the TGFβ-family, TGFβ1, which is abundantly expressed in normal articular cartilage, is also
able to stimulate chondrogenesis in bone marrow progenitor cells in addition to synovial progenitors.
Furthermore, TGFβ1 stimulates ECM production and reduces catabolic processes, including MMP13 expression
in chondrocytes [94]. TGFβ1 delivery can enhance cartilage homeostasis and regeneration in rabbits [95], and
TGFβ1 deficiency leads to OA-like cartilage [94]. High synovial fluid TGFβ1 levels are observed in OA joints and
are associated with osteophyte formation and synovial hyperplasia and fibrosis [96]. TGFβ1 thus appears
crucial for cartilage homeostasis but at the same time its spatial expression and receptor sensitivity must be
highly regulated witnessing the adverse reactions at the osteochondral border and the synovial membrane.
Besides BMPs and TGFs several other growth factors are also able to modulate cartilage repair responses. A
number of studies demonstrated improved cartilage repair response after stimulation with FGF-2 (bFGF) in vivo
and in vitro [97-100]. FGF-2 is a growth factor known to effectively stimulate bone marrow mesenchymal cells
to grow and expand, and it is suggested that FGF-2 is involved in cartilaginous repair by stimulating expansion
and chondrogenic potential of CPCs in articulating joints [98-100]. It may do so by a dose dependent increase of
Sox9 expression in resident cartilage CPCs and their derived chondrocytes [99]. Interestingly, other research
has defined FGF-2 as a degenerative mediator in OA, with effects ranging from inhibition of proteoglycan
expression to upregulation of Runx2 and MMP13 [101]. It is thought that FGF-2 can mediate effects on CPCs
and chondrocytes via different receptors. Binding to the FGF receptor 1 (FGFR1) results in mainly catabolic
effects while activation of FGFR3 induces anabolic effects like chondrogenic differentiation and ECM formation.
FGF-18, another member in the family, possibly exerts the same effects by activating FGFR3 [102]. FGF2 is
secreted by chondrocytes and partly stored in the pericellular matrix. Possibly upon traumatic impact with
matrix disruption these factors are released in increased numbers and mediate changes in joint homeostasis
[103]. Increased levels of FGF-2 have been detected in OA synovial fluid which decreased when cartilage
regeneration was observed [104]. On the other hand, FGF2 deficient mice developed accelerated OA but could
be rescued by delivery of exogenous FGF2 [105]. There appears to be a dynamic balance between the levels of
FGF-2 and the availability of its receptors FGFR1 and FGFR3. A traumatic mechanical event (and possibly aging)
may cause disturbance in this homeostasis with resulting upregulation of catabolic factors [106].
Most of the growth factors and proteins discussed in this chapter mediate their anabolic effects through the
Sox9 transcription factor. This is no different in a protein that is often associated with its positive effects on
chondrocytes in vivo and in vitro: IGF-1. IGF-1 is secreted locally by chondrocytes, stimulates matrix production
in chondrocytes and bone marrow stem cells, and aids in cell survival in part by stimulating Sox9 expression
[107, 108]. IGF-1 is also capable of counteracting catabolic effects, e.g. IL-1 and TNFα induced matrix
degradation [109, 110]. However, it has been demonstrated that IGF-1 production decreases with age in
humans [111], IGF-1 responsiveness also showed an age-related decline in non-human primate cartilage, but
15
had no effect on cell viability [112]. IGF-1 deficiency causes OA-like lesions in cartilage of rats indicating that IGF
is required for normal articular cartilage integrity [113]. Furthermore a decrease in IGF-1 response was
detected with increasing OA in monkeys [112]. When added exogenously in an OA horse model, IGF-1
facilitated repair of cartilage defects with increased collagen type II production in the deep layers and
protection against matrix degradation [109]. A combined growth factor treatment with IGF-1 and BMP-7 in in
vitro 3D culture showed even more matrix production than was observed for each factor alone [85]. It is
suggested that growth factors like IGF-1, FGF-2, and TGFβ1 are able to modulate each other’s response [114].
Other proteins that target Sox9 and thereby may affect cartilage homeostasis are PTHrP and Ihh. These factors
are involved in chondrocyte maturation and endochondral ossificiation [115], and participate in the
maintenance of cartilage homeostasis in growing and adult mice in a load-induced fashion [116]. Ihh and PTHrP
form a feedback loop to control chondrocyte differentiation and it is suggested that dysregulation of this
system may contribute to OA in the form of disturbed hypertrophic chondrocytes [116].
In summary, mature cartilage may possess more intrinsic repair capacities than originally suspected. Many of
the growth factors and other proteins involved in chondrogenesis are still expressed in mature articular
cartilage and take on regulatory roles. BMP-2, -6, and -7, TGFβ1 and -β3, FGF-2 and -18, and IGF-1 have shown
great potential as cartilage anabolic factors involved in cartilage maintenance, repair, and homeostasis. These
growth factors not only act on chondrocytes via Sox9 by inducing matrix synthesis, improving matrix quality,
and counteracting effect of numerous catabolic mediators, but in addition have similar effects on stem cells.
Stimulation of bone marrow and adipocyte stem cells with BMPs, TFGβs, FGFs, or IGF-1 augments
chondrogenic potential and cell expansion; furthermore BMP6 and TGFβ3 have demonstrated chemoattractant
properties in articular cartilage. Recruitment of progenitor cells and their subsequent chondrogenic
differentiation may be the key in effective cartilage regeneration since homeostatic disturbance in disease
cause a significant loss in the number of functional chondrocytes. Progenitor cells are found in increased
numbers in OA and may include native and migrated cells from bone marrow and other joint tissues. However,
despite the effort these cells are not able to overcome the present inflammatory and degradative processes
found in OA joints.
The expression pattern of the anabolic growth factors but also their receptors is highly regulated but also highly
dynamic in healthy cartilage. Growth factor effects can be modulated by other growth factors and can be
mediated by different receptor subtypes, while at the same time spatial expression prevents inappropriate
exposure. The response of anabolic growth factors may also change with age, as demonstrated for IGF-1 with a
decrease in production and responsiveness with increasing age.
The results from the studies described in this chapter suggest the presence of an intrinsic regenerative capacity
of the articular joint, which upon stimulation with appropriate anabolic factors can be reinvigorated. Resident
stem cells or homing cells from adjacent tissues are able to repopulate damaged cartilage and regenerate
16
hyaline cartilage. On the other hand, the complex and dynamic pathways indicate that stimulation with growth
factors alone is not sufficient for effective clinical treatment. This is underlined by demonstration of the
importance of receptor subtypes, age, and posttranslational modification on growth factor responsiveness.
Thus, anabolic growth factors have great potential for OA therapy because of their ability to induce
chondrogenesis in diseased tissue, but unanswered questions on the timing of application, side effects, and the
involvement of many other proteins have prevented it of being successful. Such a therapy will likely have to
consist of a cocktail of growth factors and anti-inflammatory drugs which is designed for specific subgroups or
individual patients, dependent on the patient’s age, the stage of disease, and the expression profile for the
affected joint. In combination with gene therapy, to increase expression of beneficial receptors and other
anabolic and anti-catabolic factors, this may result in depression of inflammation and induction of
chondrogenesis.
Summary and Discussion
In the present thesis I have described the processes involved in the progression of focal cartilage defects to
advanced osteoarthritis, a disease that can linger unnoticed for decades before exposing itself. Furthermore I
have focused on the role of some of the most studied soluble mediators in cartilage regeneration, a process
which appears to be latent in diseased mature cartilage but may be revived by appropriate stimulation.
OA is a very common disorder of synovial joints, affecting people of all ages but is more common in the elderly
[15]. It is characterized by destruction of load-bearing articular cartilage, inflammation of the synovial
membrane and changes in the subchondral bone plate. OA has potentially devastating effects on the quality of
life in affected individuals because of associated joint pain, restricted mobility, and the corresponding
limitation of daily living activities. OA is an increasingly important public-health problem and it is expected that
the incidence will increase steeply with the increasing population and fraction of elderly [24].
Healthy articular cartilage is a highly specialized avascular tissue, secreted and remodeled exclusively by
chondrocytes and a population of chondrocyte progenitor cells. Cartilage homeostasis is maintained by its
chondrocytes, i.e. anabolic and catabolic factors, anti-inflammatory and pro-inflammatory components are
kept in a highly balanced state. Damage can be initiated via several mechanisms and factors. Traumatic injury
to cartilage or ligaments, changes in subchondral bone, age-relate wear and tear, age related changes in
protein functionality, obesity, diabetes, congenital joint disorders, and syndromes that cause joint instability
are all factors that predispose to onset of OA [45]. These different and often unknown etiologies and driving
factors make OA very hard to treat effectively.
Pathological changes in load distribution and alterations in biochemical pathways cause disruptions in joint
homeostasis which results in an equilibrium shift towards an inflammatory catabolic state. First changes after
traumatic impact are observed within hours. Increased expression of inflammatory cytokines like TNFα, IL-1β,
and IL-6 mediate upregulation of catabolic mediators and suppress Sox9-induced anabolic activity. Cartilage is
17
slowly degraded while at the same time matrix synthesis is decreased. At this early stage some chondrocytes
will also respond by increasing proliferation and increasing matrix synthesis to repair cartilage but in most
cases this attempt will fail. This will cause a slow destruction of articular cartilage. At the same time the release
of cytokines and matrix fragments causes synovitis and changes in subchondral bone remodeling. Synovitis and
bone remodeling are important driving factors in disease progression. OA has long been called a noninflammatory disease but this view is slowly changing as more evidence on involvement of inflammatory
processes is found. This is underlined by the role of synovitis in OA but also the early involvement of Toll-like
receptors and the complement system [45].
Up to this point, which may take years, the cartilage matrix has been progressively degraded but the collagen
network is still relatively intact. It is thought that if disease progression is halted at this point, the chondrocytes
are able to compensate for the matrix loss and fully regenerate. When the collagen network is damaged, many
researchers tend to speak of having reached the ‘point of no return’. Whether this infamous timepoint really
exists remains to be established. It is not unlikely however that at some point in disease progression the joint
homeostasis is so out of balance that the chance of intrinsic repair is negligible [23]. Nonetheless, there is
progressive loss of collagen, fuelled by a vicious cycle of degradation followed by increased release of catabolic
factors, which in turn leads to accelerated degradation. Over time, the loss of cartilage leads to problems with
joint movement due to increased friction. Disruptions in innervated tissues like the synovium and subchondral
bone may also cause joint pain [22]. Without proper treatment OA may in some cases lead to total loss of
cartilage which ultimately requires joint replacement. Interestingly, progression to severe OA requiring joint
replacement is observed in only a small number of OA patients, suggesting a yet unknown differentiating
mechanism [22].
Intrinsic repair capacity of articular cartilage has long been described as being insufficient. In part this is true,
witnessing the progressive cartilage destruction in OA. However, research has shown that pathways involved in
early chondrogenesis are still functional, albeit in a different role, in mature cartilage, and that by stimulation
with growth factors the chondrogenic potential could be reinstated or augmented. Especially the transcription
factor Sox9 appears to play an important role in inducing repair and maintaining a healthy cartilage
homeostasis [75]. Anabolic growth factors like TGFβ1, BMP-2, IGF-1, and FGF-2 all, at least in part, are able to
stimulate cartilage formation via Sox9 by increasing cell survival, matrix production, and suppressing catabolic
factors. These factors not only affect chondrocytes but are also able to induce chondrogenesis in resident
chondrocyte progenitors, bone marrow stem cells, and adipose derived stem cells [92, 93, 100]. The
chondrogenic capacity of progenitor cells opens new possibilities in regenerative therapies. These
undifferentiated cells have shown to be able to induce regeneration in full thickness defects in animal models
which is even increased when Sox9 is overexpressed [77].
Although recent research on cartilage regeneration has delivered promising results and numerous potential
pharmacological targets for therapy, the road to clinical application is still a long one. In part this is due to the
18
seemingly infinite number of experimental models which make it difficult to compare studies and even harder
to draw conclusions that reach outside of the scope of specific studies. First of all a wide variety of animal
models is used, ranging from transgenic mice and rats to rabbits, dogs, sheep, goats, and horses. Within these
animals there are numerous methods of inducing OA-like lesions. Surgical induction of cartilage damage is
done by drilling, biopsy punches, cutting, or scraping. Mechanical ways of inducing damage are also intra
articular fracturing, impact loading, and meniscectomy to create joint instability. Damage can also be induced
by intra articular injection of IL-1β or by a high-fat diet in transgenic mice. Each method causes different
wounds which of course reflect the multifactorial disease that is OA, but makes it hard to compare.
When cartilage regeneration is studied in vivo, the joint is damaged and, in many cases, attempts at
regeneration are started immediately and in the same procedure. This is highly unrealistic since in the clinic OA
is often diagnosed and treated years or decades after initial damage the joint. This is relevant because the
degree of change in the cartilage homeostasis over time is indicative for the repair potential. Furthermore,
animals involved in these studies are most likely young mature specimens while in humans OA is more likely to
occur in older individuals. This is important for treatment because it is known that the severity of OA increases
with age and that in e.g. rats, primates, and humans it has been demonstrated that anabolic growth factor
production and responsiveness decreases with age. This suggests that regenerative results obtained in younger
animal test subjects may not be relevant for the large group of elderly with OA. Another problem with the use
of animals in general is that subtle differences exist in protein expression and in protein function [106].
Besides in vivo studies, in vitro research has provided large amounts of basic knowledge on molecular pathways
involved in cartilage destruction and regeneration. The problem with in vitro research in general is that tissue
or cells are isolated from their native environment while we try to extrapolate these data to whole tissue native
conditions. Cartilage cannot be seen as an independent tissue inside the joint, but is rather the result of
complex interplay between various joint tissues but also systemic factors. This is underlined by the observation
that cartilage gene expression patterns may be changed upon explantation [106], much like any traumatic
injury would. Chondrocytes for in vitro study can be obtained from various sources, most studies use surgical
waste material, organ donors, post mortem samples, or abattoir waste. Surgical waste cartilage is often
obtained after total arthroplasty, which is a procedure mostly limited to the elderly population. Other sources
are post mortem cartilage which occasionally is isolated 48 hours after death. Chondrocytes isolated from
these tissues are most likely a mix of healthy and diseased cells and may display age related changes as
described previously.
After isolation, cells are classically plated and cultured on flat polystyrene surfaces, but as previously stated,
cellular behavior depends on its environment, its ‘context’. This important fact was first observed over 15 years
ago when the differences in cell behavior between 2D and 3D cultures were demonstrated. These results
showed the importance of tissue phenotype dominance over the cellular genotype, i.e. the ECM modulates
tissue homeostasis and may regulate cell growth, survival, and differentiation [117]. This has also been
19
observed in chondrocyte culture. Chondrocytes cultured in monolayers have an unstable phenotype which
leads to dedifferentiation into a fibroblast-like cell type. This is mainly due to down-regulation of Sox9, collagen
type 2 and aggrecan expression [118, 119]. Because of this unstable phenotype, several culture systems have
been developed to maintain chondrocytes in vitro, many of which involve 3D culture. These 3D culture systems
more closely mimic the native chondrocyte environment and may provide structural support and allow for the
important cell-matrix interactions. 3D culture methods like micromass- and pellet culture, but also the use of
alginate beads or other scaffolds considerably increase Sox9 expression and cartilage matrix formation [119].
It has become evident from in vivo and in vitro observations that the key in maintaining or restoring
chondrocyte phenotype is its environment. Whether it is in healthy or diseased cartilage, in 3D or 2D cultures,
the state of the matrix and the availability of growth factors dictate chondrocyte behavior. Maintenance of
Sox9 expression is especially important for the chondrocyte phenotype, and a successful regenerative therapy
may only be realized when appropriate expression of this transcription factor is secured. Several pathways
leading to its activation have been identified in vivo and in vitro and include the previously mentioned growth
factors TGFβ1, BMP-2, IGF-1, and FGF-2. (Pre-)Clinical application of these growth factors for cartilage
regeneration is limited to several phase I/II trials, and their effectiveness so far remains to be proven [25].
Although animal- and in vivo-studies show great potential for these anabolic factors, in my opinion it is unlikely
that treatment with a single growth factor will lead to an effective treatment in the majority of OA cases. Early
lesions in young individuals might benefit from such a therapy since the cartilage matrix and other joint tissues
are relatively unaffected, and by increasing the chondrogenic capacities of resident chondrocytes but also
CPCs, the homeostasis and matrix may be restored before progression into OA. However, treatment of
advanced OA may require other strategies. Extensive matrix destruction, total loss of homeostasis, and age
related changes in gene expression and protein functionality are some of the difficulties needed to overcome.
The overall complexity of OA will likely require a combination of anti-inflammatory drugs, a cocktail of anabolic
factors, gene therapy, and tissue engineering.
Tissue engineering has been proposed as an alternative to current MACI procedures which require the sacrifice
of ‘non-load bearing’ cartilage. In tissue engineering, autologous stem cells with chondrogenic capacities are
seeded onto biodegradable scaffolds and stimulated with anabolic factors to induce cartilage formation in
vitro. Acellular scaffolds may also be implanted directly and depend on recellularization and tissue formation in
vivo by resident chondrocytes [31]. When loaded with growth factors these scaffolds can induce or augment
intrinsic matrix deposition and reduce inflammation. Gene transfer may be a highly effective way to deal with
(age related) changes in gene expression and protein functionality. Bone marrow stem cells may be isolated
and altered by gene transfer before being re-implanted, or target cells are manipulated in vivo by the use of
viral vectors [120]. In a rabbit OA model, Sox9 transduced bone marrow stem cells were seeded onto a
degradable scaffold and implanted into a cartilage defect and showed improved short-term repair when
compared to MSCs without Sox9 transduction. Temporary overexpression of Sox9 by these cells leads to
induction of chondrogenesis [77]. Gene transfer as a therapy in general has great potential to overcome the
20
limitations of the current treatments in countless diseases. However, almost 70 years after its initial discovery
this has only led to a handful of successful clinical applications, in part because of safety concerns due to
potentially lethal side effects [120].
In conclusion, OA is an increasing problem in orthopedics. Many factors may contribute to its development,
including mechanically induced impact trauma, normal wear and tear, joint instability, but also complex age
related changes and obesity. Lesions often start as a focal defect, and due to changes in homeostasis and the
progressive degradation of cartilage matrix often progresses into OA. Cartilage damage causes changes in
chondrocyte expression pattern that lead to increased catabolic- and decreased anabolic-processes. A lot is
known about disease pathogenesis, but this has not yet resulted in an effective clinical therapy to halt OA
progression and induce regeneration. Over the last years, cartilage regeneration has gained increasing
attention and this has led to the identification of soluble mediators and CPCs with chondrogenic capacities in
native cartilage. The combination of anabolic growth factors and stem cells has demonstrated great
regenerative potential in vivo and in vitro. These observations might prove crucial in the development of new,
improved OA therapies. Several challenges however remain to be solved. The underlying cause of OA can be
one of many and disease progression is also dependent on many different factors including the patient’s age
and lifestyle. An effective therapy will have to take into account patient specific expression profiles (‘OA
phenotype’) of affected joints like e.g. relevant receptors and other anabolic and anti-catabolic factors. Based
on these results, a therapy can be designed for specific subgroups or individual patients. A regenerative
response to OA damage is closer than ever in this era of tissue engineering and rapid technological innovations.
However, further understanding of the complex pathways that initiate and lead to progression of OA,
separated by spatial and temporal expression, is necessary to improve clinically relevant OA models.
21
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