Group Title: Arthritis Research & Therapy
Title: Major biological obstacles for persistent cell-based regeneration of articular cartilage
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Title: Major biological obstacles for persistent cell-based regeneration of articular cartilage
Physical Description: Archival
Creator: Steinert, Andre
Ghivizzani, Steven
Rethwilm, Axel
Tuan, Rocky
Evans, Christopher
Nöth, Ulrich
Publisher: Arthritis Research and Therapy
Publication Date: 2007
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Abstract: Hyaline articular cartilage, the load-bearing tissue of the joint, has very limited repair and regeneration capacities. The lack of efficient treatment modalities for large chondral defects has motivated attempts to engineer cartilage constructs in vitro by combining cells, scaffold materials and environmental factors, including growth factors, signaling molecules, and physical influences. Despite promising experimental approaches, however, none of the current cartilage repair strategies has generated long lasting hyaline cartilage replacement tissue that meets the functional demands placed upon this tissue in vivo. The reasons for this are diverse and can ultimately result in matrix degradation, differentiation or integration insufficiencies, or loss of the transplanted cells and tissues. This article aims to systematically review the different causes that lead to these impairments, including the lack of appropriate differentiation factors, hypertrophy, senescence, apoptosis, necrosis, inflammation, and mechanical stress. The current conceptual basis of the major biological obstacles for persistent cell-based regeneration of articular cartilage is discussed, as well as future trends to overcome these limitations.
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General Note: M3: 10.1186/ar2195
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Review

Major biological obstacles for persistent cell-based regeneration

of articular cartilage
Andre F Steinert1, Steven C Ghivizzani2, Axel Rethwilm3, Rocky S Tuan4, Christopher H Evans5
and Ulrich NSth1


1 Orthopaedic Center for Musculoskeletal Research, Konig-Ludwig-Haus, Julius-Maximilians-University, Warzburg, Germany
2Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL, USA
31nstitut for Virologie und Immunbiologie, Julius-Maximilians-University, Wurzburg, Germany
4Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of Health,
Department of Health and Human Services, Bethesda, MD, USA
5Center for Molecular Orthopaedics, Harvard Medical School, Boston, MA, USA


Corresponding author: Andre F Steinert, a-steinert.klh@mail.uni-wuerzburg.de


Published: 5 June 2007
This article is online at http://arthritis-research.com/content/9/3/213
C 2007 BioMed Central Ltd


Abstract
Hyaline articular cartilage, the load-bearing tissue of the joint, has
very limited repair and regeneration capacities. The lack of efficient
treatment modalities for large chondral defects has motivated
attempts to engineer cartilage constructs in vitro by combining
cells, scaffold materials and environmental factors, including growth
factors, signaling molecules, and physical influences. Despite
promising experimental approaches, however, none of the current
cartilage repair strategies has generated long lasting hyaline
cartilage replacement tissue that meets the functional demands
placed upon this tissue in vivo. The reasons for this are diverse and
can ultimately result in matrix degradation, differentiation or
integration insufficiencies, or loss of the transplanted cells and
tissues. This article aims to systematically review the different
causes that lead to these impairments, including the lack of
appropriate differentiation factors, hypertrophy, senescence,
apoptosis, necrosis, inflammation, and mechanical stress. The
current conceptual basis of the major biological obstacles for
persistent cell-based regeneration of articular cartilage is
discussed, as well as future trends to overcome these limitations.


Introduction
Structure and function of articular cartilage
Articular cartilage is a highly specialized tissue that protects
the bones of diarthrodial joints from forces associated with
load bearing and impact, and allows nearly frictionless motion
between the articulating surfaces [1,2]. The extracellular
matrix (ECM) of articular cartilage is distinct from that of other
connective tissues, consisting of an intricate network
containing predominantly fibrillar collagens and proteo-
glycans. The collagens, types II, IX and XI, form a fibrous
framework that gives the tissue its shape, strength and tensile


Arthritis Research & Therapy 2007, 9:213 (doi:1 0.11 86/ar2195)


stiffness [3]. Collagen type VI is found pericellularly around
chondrocytes [4], and collagen type X is found in calcifying
cartilage [5]. Although collagen type I is the most prevalent
collagen throughout the body, the primary constituent of the
articular cartilage matrix is type II, comprising 80% to 90% of
the collagen content [3]. The proteoglycans in articular
cartilage in their most abundant form exist as large hydro-
philic aggregates, which contain the fluid component and
control its movement. The level of compaction of the
proteoglycans within the collagen lattice will determine their
level of hydration and, in turn, the stiffness of the articular
cartilage. The synthesis, incorporation and degradation of
ECM proteins are orchestrated by chondrocytes that
populate the matrix at low density [3]. Because articular
cartilage is avascular, nutrients for the chondrocytes are
supplied from the capillaries of the synovium and must diffuse
into the synovial fluid and then into the cartilage matrix. Co-
ordinated synthesis and proteolytic breakdown of certain
ECM components by chondrocytes enables certain
components of the cartilage matrix to undergo turnover and
maintenance [3]. Factors that impair chondrocyte function
can disrupt the equilibrium of synthesis and catabolism in
favor of cartilage degradation, which over time can lead to
osteoarthritis (OA) [6,7].

Capacity of articular cartilage for repair
As a result of injury or disease, articular cartilage frequently
incurs damage, but has very limited ability to regenerate. In
chondral defects, where a lesion is contained within the
articular cartilage, there is no involvement of the vasculature.


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ACT = autologous chondrocyte transplantation; BMP = bone morphogenetic protein; ECM = extracellular matrix; FGF = fibroblast growth factor;
IGF = insulin-like growth factor; IHH = indian hedgehog; IL = interleukin; IL-1 Ra = IL-1 receptor antagonist; MMP = matrix metalloproteinase; MSC =
mesenchymal stem cell; NO = nitric oxide; OA = osteoarthritis; PTHrP = parathyroid hormone related peptide; RA = rheumatoid arthritis; SOX =
SRY (sex determining region Y)-box; TGF = transforming growth factor; TNF = tumor necrosis factor.








Arthritis Research & Therapy Vol 9 No 3 Steinert et al.



Consequently, progenitor cells in blood and marrow cannot
enter the damaged region to influence or contribute to the
reparative process. Resident articular chondrocytes do not
migrate to the lesion, and no production of a reparative matrix
occurs. Thus, the defect is not filled or repaired and
essentially remains permanently [1,2].

Osteochondral cartilage defects have suffered damage to
both the chondral layer and bone plate, causing the rupture of
blood vessels and ingress into the bone marrow. In this case,
a repair response is initiated and generally begins with a
hematoma that forms when blood escapes the damaged
vasculature or marrow and enters the lesion [1,2]. The fibrin
network within the hematoma traps platelets that, in turn,
release various bioactive factors, including platelet derived
growth factor (PDGF) and transforming growth factor (TGF)-P,
that stimulate vascular invasion and migration of
undifferentiated mesenchymal progenitor cells into the fibrin
clot [1,8]. Within several days of injury, the progenitor cells
proliferate and may or may not differentiate into chondrocytic
cells that synthesize cartilaginous matrix. The resulting repair
tissue, however, is poorly organized and contains significant
amounts of collagen type I. This fibrocartilage tissue is
mechanically inferior and breaks down with time and loading,
becoming fragmented or disintegrating altogether [1,2,8].

Present status of cartilage repair
The specialized architecture and limited repair capacity of
articular cartilage, coupled with the high physical demands
placed upon this tissue, make it exceedingly difficult to treat
cartilage injury medically. Currently, there exists no pharmaco-
logical agent that promotes the healing of articular cartilage
lesions, whether chondral or osteochondral. Thus, physicians
have attempted various surgical methods to restore articular
surfaces, which fall into three broad classifications.

The first type of approach entails the use of mechanical
penetration of the subchondral bone by abrasion arthroplasty,
Pridie drilling, or microfracture to disrupt the vasculature and
marrow. This results in a large clot that fills the defect and
enables the natural repair response to form fibrocartilage
repair tissue, which is subordinate to normal cartilage in
terms of mechanical properties [1,2,8,9]. However, these
procedures are cost effective and clinically useful, as patients
often have reduced pain and improved joint function, and are,
therefore, generally used as first-line treatment for focal
cartilage defects [9-12].

The second approach involves attempts to regenerate hyaline
cartilage repair tissue through transplantation of tissues such
as periosteum, perichondrium or osteochondral grafts
[2,8,13,14]. Although short term results have been positive
for a number of patients, the long term clinical results are
uncertain, with tissue availability for transplant, especially in
large cartilage defects, being a major limitation [1,2,8].
Therefore, the autologous chondrocyte transplantation (ACT)


procedure has been developed and used clinically since
1987, in combination with a periosteal cover, to treat
chondral or osteochondral defects of the knee with good
clinical results [12,15-17]. However, for the first generation of
ACT, various adverse events have been reported, including
graft failure, delamination, tissue hypertrophy and chondro-
malacia [18,19]. In order to overcome some of these impair-
ments, modern modifications of this procedure involve
embedding chondrocytes in a three-dimensional matrix before
transplantation into cartilage defects [19-21]. To date, only a
few clinical studies have been conducted comparing ACT
with other repair procedures for large defects, and these have
shown no major differences in the respective outcomes
[22-26]. Despite these advances, with most surgical inter-
ventions resulting in improvement of clinical symptoms such
as pain relief, none of the current treatment options have
regenerated long-lasting hyaline cartilage tissue to replace
damaged cartilage [1,2,12,16].

Tissue engineering approaches that deliver a matrix seeded
with chondrogenic cells (chondrocytes or progenitor cells)
and chondrogenic factors have also been evaluated
experimentally, with basically the same long term overall
results in vivo thus far [27,28]. Despite good in vitro data
with different approaches, the reasons for the failure of cell-
based cartilage repair approaches to form hyaline repair
tissue in vivo remain largely unclear to date [2,27-29].
Although proof of principle has been provided that implanted
chondrocytes can contribute to structural cartilage repair, the
origin of cells persisting at the repair site, whether these are
the originally transplanted cells or migrated ones from the
subchondral marrow or the adjacent synovium, has not been
entirely clarified yet [2,30,31]. Emerging data are elucidating
not only the fate but also the function of the transplanted cells
at the repair site, for example, suggesting that transplanted
mesenchymal cells not only function as repair cells but also
as trophic mediators that stimulate the surrounding tissues
toward repair [32].

Biological obstacles to hyaline neocartilage formation
comprise differentiation insufficiencies, loss of transplanted
cells or tissues, matrix destruction and integration failures
(Figure 1), which all can occur due to various reasons.
Insights into these mostly interrelated mechanisms might help
us to systematically direct therapeutic approaches toward the
formation of a more hyaline cartilage repair tissue.

Differentiation cues
Cartilage development and chondrogenesis
During development, the process of chondrogenesis gives
rise to the formation of cartilage and bony skeletal tissues.
Chondrogenesis is typically initiated at sites of skeletal
element generation by localized proliferation of mesenchymal
cells guided by multiple growth factors and morphogens
such as Wnts, TGF-ps and fibroblast growth factors (FGFs)
[33,34]. Subsequent aggregation and condensation of these


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Figure 1


Inflammation


Mechanical stress Oxidative stress



*.-.x I..~
S... Apoptosis Necrosis -




,., "
h '.. cl $ .
., Chondrogenic cell .
-I-


Senescence
S -" Hypertrophy Senescence




Un- or dedifferentiation
E3ti


Osteoblast


Blood cell


o 0
Integration


W\ VW


* -I


Biological obstacles associated with cell-based approaches to
cartilage tissue engineering. Formation of hyaline neocartilage can be
hindered due to loss of transplanted chondrogenic cells by cellular
efflux, apoptosis or necrosis, differentiation insufficiencies, including
fibroblastic, hypertrophic or osteogenic differentiation (red arrows),
matrix destruction by mechanical, oxidative and/or inflammatory
stressors (red flashes), as well as integration failures within the
cartilage and/or bone compartment (green arrows) of the defect.


cells is mediated by both cell-cell (neural cadherin and neural
cell adhesion molecule) and cell-matrix (hyaluronan and
CD44, fibronectin, proteoglycans and collagens) adhesion,
as well as numerous intracellular signaling pathways trans-
duced by integrins, mitogen activated protein kinases, protein
kinase C, lipid metabolites and cyclic AMP and so on
[35-40]. At this stage, the undifferentiated mesenchymal cells
produce an ECM enriched with collagen type I, hyaluronan,
tenascin and fibronectin. Mesenchymal cells in the
precartilage aggregate and begin to synthesize and express
molecules associated with chondrogenic differentiation,
followed by their differentiation into chondroblasts and
synthesis of extensive ECM containing proteoglycan and
cartilage-specific collagen types II, IX, and XI [33,34].


Available online http://arthritis-research.com/content/9/3/213



Synthesis of collagen type I is reduced, as is expression of
the cellular adhesion molecules. During development of the
limbs and other bony tissues, chondrogenesis is gradually
replaced by endochondral ossification. The chondrocytes
within the cartilaginous tissue mature, hypertrophy and
express collagen type X with reduced production of type II.
The cartilage becomes vascularized and is infiltrated by
osteoprogenitor cells. The chondrocytes undergo apoptosis,
while the osteoprogenitor cells differentiate into osteoblasts
and replace the cartilage with mineralized bone tissue
[33,34]. During the developmental process a portion of the
chondrocytes remains nonhypertrophic and expands to form
the permanent cartilaginous tissues [33,34]. In the adult
cartilage tissue, however, many of these developmental
mechanisms are lost or only partially restored once the tissue
is injured and repaired. Although adult cells, such as
chondrocytes and mesenchymal stem cells, show
fundamental biological differences compared to embryonic
stem cells, including their limited potential to self-renew, they
have been shown to be capable of greater plasticity than
previously expected and to retain their potential to undergo
chondrogenesis following considerable expansion in vitro
[27,41-44]. A further understanding of the differentiation
capacity of these cells, especially under aged and arthritic
conditions, might be key to the successful application of adult
chondrogenic cells in cartilage repair without evoking the
ethical and legal issues associated with the use of embryonic
stem cells [28,41,45-47].

Un- or dedifferentiated chondrogenic cells
Still a matter of debate is whether to transplant a fully in vitro
differentiated construct or a graft containing a homogeneous
population of rather undifferentiated cells and signaling
molecules, to ensure that the desired differentiation process
takes place in vivo under physiological conditions of mecha-
nical loading [2]. Each approach has its advantages and
disadvantages. The former allows chondrogenic differen-
tiation under controlled in vitro conditions, but is often
associated with biocompatibility and integration problems
[48,49]. The latter promotes integration well, but presents the
risk of uncontrolled and undesired differentiation processes
to occur [48,50]. Various cell types have been used to repair
cartilage lesions, including chondrocytes, perichondrial or
periosteal cells, and mesenchymal progenitor cells from bone
marrow and other sources.

Chondrocytes
As mentioned above, only autologous chondrocytes are used
in clinical practice. Human articular chondrocytes lose their
chondrogenic phenotype upon monolayer expansion and
change their morphology to a fibroblastic appearance [51].
However, dedifferentiated chondrocytes can re-differentiate
toward the chondrogenic lineage in three-dimensional
culture, particularly after conditioning by growth factors such
as FGF-2, epidermal growth factor (EGF), TGF-P3, or PDGF-
BB during monolayer expansion [52-54]. The same is


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Arthritis Research & Therapy Vol 9 No 3 Steinert et al.



observed for constructs generated from OA chondrocytes,
except with reduced rates of collagen production compared
to constructs made from non-OA chondrocytes [55]. Next to
age and underlying disease, a limited number of cell
doublings is also important to sustain proper commitment in
chondrocytes [42,45], while extensive in vitro expansion of
articular chondrocytes resulted in loss of in vivo cartilage
formation [56].

Chondrocytes have been employed in conjunction with
different matrix materials and growth factors in vitro and
cartilage repair approaches in vivo (for reviews, see
[2,29,57,58]). In each instance, improved repair was reported
for the respective treatments, with the lesion being filled to a
greater volume of tissue compared with untreated controls;
however, the repair tissue generated was mainly fibro-
cartilaginous in nature. The transplanted chondrocytes or
chondroblasts were concluded to have a beneficial effect on
the spontaneous repair response, over and above that
elicited by the matrix itself [2,27-29].

Perichondrial/periosteal cells
Cells derived from periosteum and perichondrium have been
shown to have considerable chondrogenic potential [59].
These cells reside within the proliferative stratum of the
cambial layer of the perichondrium and periosteum,
respectively, and have been isolated for tissue engineering
purposes [59]. The chondrogenic potential of perichondrial
and periosteal cells for their use in cartilage repair has been
tested extensively in vitro and in vivo following seeding into
various matrices [2]. However, the need for two surgical
interventions, the highly variable results achieved in
experimental studies thus far, as well as the long-term
instability of the repair tissue formed, may have prevented this
approach from entering clinical practice to date.

Mesenchymal stem cells
Mesenchymal stem cells (MSCs) are multilineage progenitor
cells responsible for the turnover and repair of mesenchymal
tissues, such as bone, cartilage, ligament, muscle, and fat
[41,44,60,61]. Although no clear definitive phenotype of
MSCs has been described, through the use of the proper
culture conditions, expanded MSCs can be stimulated to
differentiate along specific pathways, such as chondro-
genesis, adipogenesis, and osteogenesis [41,43,60,61].
With regard to cell-based cartilage repair approaches, MSCs
provide an attractive alternative to chondrocytes. Unlike
mature chondrocytes, which must be surgically harvested
from a very limited supply of non-weight-bearing articular
cartilage, MSCs are relatively easy to obtain from bone
marrow, and will maintain their multilineage potential with
passage, enabling considerable expansion in culture
[8,29,32,44,46,62,63]. For the purposes of cartilage repair,
extensive analyses of the appropriate microenvironment to
stimulate MSCs toward chondrogenesis in vitro have been
performed. MSCs are commonly isolated by adherence to


cell culture plastic or density-gradient fractionation, and,
therefore, represent a heterogeneous population of cells
[41,44,46]. Positive selection for chondroprogenitor cells has
been achieved by conditioning media with growth factors
such as FGF-2 or TGF-P during monolayer expansion [64].
Although no definitive MSC marker has been identified,
selection protocols for the same immunophenotype within the
MSC population might also be employed to improve
chondrogenic phenotype stability, with STRO-1, CD73 and
CD106 among the most promising candidates for positive
selection and CD11b, CD45, CD34, CD31 and CD117
among those for negative selection [44,46].

Key steps in using MSCs for articular cartilage repair include
the development of effective methods to stimulate MSCs
toward chondrogenesis, maintenance of an articular cartilage
phenotype without ossification or fibrinogenesis, and a
delivery system to localize the cells within a lesion without
inhibiting chondrogenic differentiation or the integrity of the
repair tissue [2,8,27-29,44]. Building from these results,
transplantation of chondrogenic grafts or chondrogenesis in
osteochondral defects in vivo have been attempted with
varying levels of success [2,8,27-29,44,65,66]. Such
systems have been crucial to the elucidation of cell signaling
pathways during chondrogenesis and the contributions of
specific factors to this process, which include growth and
adhesion factors, as well as mechanical stimuli [2,27-29,44].
However, although short term success in generating hyaline-
like cartilage repair tissue after weeks in vivo has been
demonstrated, the long-term stability of the tissue formed has
been questioned, which is not surprising given the inferior
fibrocartilaginous nature of the retrieved tissue after longer
time points [2,27-29,44]. For the treatment of bone defects,
bone-marrow stromal cells within three-dimensional constructs
have already been applied to humans [67]. The first clinical
results of the transplantation of autologous bone marrow
stromal cells for the repair of full-thickness articular cartilage
defects have also been reported [68,69]. Three patients were
treated with collagen gel MSC-constructs, which were
covered with a periosteal flap, and fibrocartilaginous defect
filling was found after one year, as well as a significantly
improved patient outcome in the respective follow-ups after
one, four and five years [68,69].

Chondrocyte hypertrophy and osteogenesis
During development of the limbs and other bony tissues,
chondrogenesis is gradually replaced by endochondral
ossification as described above, with indian hedgehog (IHH),
parathyroid hormone related peptide (PTHrP) and Wnt/3-
catenin pathways among the main regulators of these
processes [33,34,70]. These regulatory mechanisms can be
partially recapitulated in vitro by using chondrocytes or
chondroprogenitor cells under the appropriate high-density
three-dimensional culture conditions [27,28,42,44]. Although
it was shown that in vitro chondrogenesis without significant
levels of end stage hypertrophy is possible by using normal


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articular chondrocytes [71,72], data from in vitro chondro-
genesis using MSCs [72-81] or OA chondrocytes [47,82,83]
together with members of the TGF-P superfamily reveal a
significant level of chondrocyte hypertrophy, indicated by high
levels of collagen type X expression. Although the use of
collagen type X as a marker of chondrocyte hypertrophy in
MSC-based systems has been questioned [84], it correlates
well thus far with the existing in vivo data. For example, MSCs
genetically modified to express bone morphogenetic protein
(BMP)-2 show a significant level of tissue hypertrophy and
osteophyte formation when transplanted orthotopically to
osteochondral defects [85] or ectopically [79,86] in small
animal models. Correspondingly, TGF-31 has been shown to
induce fibrosis in joints of nude rats when it was directly
delivered by first generation adenovirus [87]. Furthermore,
implantation of chondrocytes genetically modified to express
BMP-7 has been shown to generate good hyaline cartilage
repair tissue after 6 weeks in vivo, but only bad results have
been reported after one year, with loss of 72% to 100% of
the transplanted cells [88]. We argue that this might be
attributed to mechanisms of hypertrophic differentiation and
subsequent apoptosis, although experimental proof is not yet
available.

Possible strategies to avoid hypertrophic differentiation of
neocartilage constructs might be based on the use of cells
that are not subjected to high doses of TGF-P superfamily
members, but are to their inhibitors, such as noggin, chordin
[89,90] or that are modified using small interfering RNA-
mediated knock down approaches, while chondrogenesis
may be achieved via different pathways (for instance SOX9).
Other potent molecules that might be considered for the
facilitation of cartilage repair include IHH, a member of the
hedgehog family of cell surface-associated ligands. IHH is
expressed in prehypertrophic chondrocytes of the growth
plate and functions to inhibit chondrocyte hypertrophy by
maintaining expression of PTHrP through a negative
feedback loop [91,92]. Altering the expression of such
proteins during chondrogenesis may serve to delay the onset
of hypertrophy and formation of bone, while increasing the
pool of proliferating chondrocytes. Indeed, addition of PTHrP
has been shown to inhibit chondrocyte hypertrophy during in
vitro chondrogenesis of primary, adult MSCs [81,91,93-100].
Hypertrophic differentiation of chondrocytes, as well as bone
formation, is also regulated by the Wnt/p-catenin pathway
[101,102]. Specifically, recent developmental biology studies
indicate that the transition from a nonhypertrophic to a
hypertrophic chondrocyte appears to be under the control of
the canonical Wnt signaling pathway (Wnt-5a)
[34,70,102-104]. These molecules have also been shown to
be involved in in vitro chondrogenesis of adult MSCs, making
these molecules attractive targets for a possible therapeutic
use [101,102,105].

With regard to osteogenic infiltration from the underlying
subchondral bone, adequate sealing structures in replace-


Available online http://arthritis-research.com/content/9/3/213



ment of the tidemark (for example tissue sealants or semi-
permeable polymers) could be employed when treating
osteochondral defects in order to avoid the entry of
osteoblasts and red blood cells within the cartilage part of
the lesion (Figure 1; see also 'Integration failures' below)
[2,20].

Senescence
Another mechanism limiting the function of transplanted cells
for cartilage repair is aging or senescence. Articular
chondrocytes and MSCs, like all human cells, can only
undergo a finite number of replications in vitro, a
phenomenon known as Hayflick's limit or replicative
senescence [106-109]. Cellular senescence is associated
with many molecular mechanisms, including telomere erosion
and oxidative damage caused by 02, H202 and tert-butyl-
hydroperoxide, leading to activation of p53, retinoblastoma
gene and insulin-like growth factor (IGF)/Akt pathways [106-
109]. In articular cartilage tissue, the number of chondrocytes
decreases with increasing age, chondrocyte function
deteriorates, and the ability of the cells to maintain or restore
the tissue declines [110,111]. In parallel, senescent cells
accumulate with age, and produce degradative enzymes and
pro-inflammatory cytokines, which can disrupt the tissue
structure and consecutively decrease tissue function
[110,111]. The cells synthesize smaller aggrecans and less
functional link protein, leading to the formation of smaller,
more irregular proteoglycan aggregates [110,111].
Chondrocyte mitotic and synthetic activities decline with age,
as well as age related responses to anabolic cytokines
[110,112]. In parallel, chondrocyte and MSC telomere length
declines and senescence-associated P-galactosidase
expression increases with advancing age, suggesting that
senescence contributes to the age-related deterioration of
chondrocyte function, limiting their use for cartilage repair
approaches [45,109,112-114]. Mediators that also promote
premature stress-induced senescence of resident and
transplanted cells include ethanol, ionizing radiations,
mitomycin C, or excessive mechanical stress, and have been
associated with age-related pathologies such as OA, which
also involves pro-inflammatory cytokines such as IL-1 or
tumor necrosis factor (TNF)-a [110,112,115,116]. Thus,
senescent cells might contribute to aging and age-related
pathology by stimulating tissue remodeling and/or local
inflammation, which would compromise tissue structure and
function. Telomerized cells have been used for tissue
engineering applications [117], and other measures to inhibit
replicative senescence have also been proposed [118]. It
remains to be seen if such measures can be used to improve
the outcome of cartilage repair approaches, especially when
dealing with aged cells and patients [112].

Delivery of chondrogenic factors and gene therapy
With regard to cell-based therapies for articular cartilage
regeneration, it is widely thought that exposure of chondro-
genic cells (chondrocytes, periosteal/perichondrial cells,


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Arthritis Research & Therapy Vol 9 No 3 Steinert et al.



MSCs) to specific stimuli that promote chondrogenic differen-
tiation and maintenance of the chondrocyte phenotype could
significantly enhance the repair potential of this type of
procedure and improve the clinical outcome. Potentially
useful in this respect are members of the TGF-P superfamily,
including TGF-P3 1, 2, 3, and several BMPs, IGF-1, FGFs, and
EGF, among others (reviewed in [29,66]). Another class of
biologics that may be useful in cartilage differentiation repair
are transcription factors that promote chondrogenesis or the
maintenance of the chondrocyte phenotype. SRY (sex
determining region Y)-box 9 (SOX9) and related transcription
factors L-SOX5 and SOX6 have been identified as essential
for chondrocyte differentiation and cartilage formation
[99,100,119]. Signal transduction molecules, such as
SMADs, which are mediators of TGF-P and BMP signals, are
also known to be important regulators of chondrogenesis
[120,121]. However, the short half-lives of recombinant
proteins and a lack of effective delivery methods for
intracellular acting molecules hinder the clinical application of
these factors.

Gene transfer offers an alternative approach to protein
delivery that may satisfactorily overcome the limitations of
conventional methods [66,95,1 22,1 23]. By delivering cDNAs
that code for therapeutic proteins to specific target cells, the
genetically modified cell is converted to a biofactory for
protein production [66,122,123]. Through the localized
delivery of gene transfer vectors or genetically modified cells
to specific sites of cartilage damage, sustained protein
synthesis can be concentrated at the site of injury or disease
with minimal collateral exposure of non-target tissues [66].
Gene transfer approaches in vivo using various transgenes,
including IGF-1, BMP-2, BMP-7, FGF-2, and SOX9,
accelerated the healing of cartilage defects in various repair
models [66,85,88,119,122-133]. Although short-term
success in generating hyaline cartilage repair tissue after six
weeks in vivo has been demonstrated, long-term in vivo
analyses indicate only unsatisfactory results [66,88,123]. The
possible causes are reviewed in this article. For a successful
gene therapy approach for cartilage repair, the mode of
delivery, level and duration of transgene expression, as well
as the type and dosage of vectors used, have to be well
considered [66,123]. For example, ubiquitous intra-articular
expression of strong anabolic transgenes such as TGF-P1
can cause severe fibrosis in joints [87], and excessive
adenoviral loads are inhibitory to chondrogenesis of primary
MSCs [134].

Loss of transplanted cells or tissues
As adult human articular cartilage is avascular and thought to
be a post-mitotic tissue, with virtually no cell turnover and
resident cells being encased within the dense extracellular
matrix, there is no compensatory mechanism for loss of native
and transplanted chondrogenic cells. In cartilage defects,
cells can be lost due to leakage of cell suspension, apoptosis
and necrosis.


Delivery, surgical and biomaterial issues
Surgical procedures such as tissue harvest for the ACT
procedure or lesion-edge conditioning (performed to improve
the fitting of an implant) inevitably affect healthy cartilage
tissue surrounding the lesion site. These procedures are
associated with an extensive margin of apoptosis and/or
necrosis along the cut surfaces that is likely to create an
unfavorable biological environment for graft integration, with
scalpel margins being less affected than trephine wounds
[2,48]. When periosteal flaps are used in first generation ACT
procedures, significant problems were reported, including
delamination, loss of flaps and cell suspension [18,135].

Because of this and other reasons, chondrocytes have been
incorporated in various matrix materials before transplantation
in newer generations of ACT, including collagen and hyaluronic
acid [19,136-138]. Various other scaffold materials have also
been tested in vivo and in vitro for cartilage tissue engineering
purposes (extensively reviewed elsewhere [2,19,20,27,29,
139-142]). From a biological perspective, beneficial material
features would include not only a three-dimensional
environment for differentiation and biocompatibility (no signs of
cytotoxicity, apoptosis, senescence), but also would promote
the release of factors supporting one or more biological
aspects of repair, as outlined in other chapters of this review.
Such so called third generation biomaterials have been manu-
factured for tissue engineering purposes [143-145]. For
example, polylactic acid scaffolds have been engineered to
release recombinant BMP-2 in a sustained fashion, which was
sufficient to induce a favorable chondrogenic response in vitro
[146]. However, the ideal material properties for a successful
cartilage regeneration approach in vivo remain to be defined,
including the biomechanical stability of the matrix, kinetics of
resorption, selection of bioactive factor, cellular targets) and
mechanism of stimulation [142,146-148]. Given these impon-
derables, a guided biomaterial development focusing on cell
and cartilage tissue specific requirements seems desirable.

Apoptosis and necrosis
As pointed out earlier, various stressors can affect neo-
cartilage formation and integration, including pro-inflammatory
cytokines such as IL-1 or TNF-a, nitric oxide (NO), free
radicals and oxidants, mechanical stress, glucocorticoids and
others [53,149]. Depending on the cell type, as well as dose
and type of stressor, the transplanted cells will react in
different ways. For instance, a high, cytotoxic dosage of a
stressor could cause a level of damage such that cellular
biochemical activities decrease, leading to cellular death by
necrosis. The level of damage sustained by cells determines
whether programmed cell death (apoptosis) can unfold or, if
the damage is lower, senescence can occur [150,151].
Since even after in vitro selection, transplanted cell popula-
tions are not homogeneous, the dose of the stressor will shift
the percentage of cells executing each of the possible
programs cellular proliferation or inflammation, senescence
(see above), apoptosis, and necrosis [152].


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As outlined above, apoptosis naturally occurs during limb
development following end stage terminal hypertrophic
differentiation of chondrocytes, before osteoprogenitor cells
infiltrate the cartilage anlagen and chondrogenesis is
gradually replaced by endochondral ossification. Furthermore,
it has been suggested that apoptosis of chondrocytes during
development may act to control chondrocyte number in
cartilage tissue, and current evidence indicates that chondro-
cyte apoptosis is involved in OA pathogenesis [153-155].
However, in cartilage repair, apoptotic cell death is obviously
an undesired phenomenon, either when it occurs within the
transplant or along with necrosis at the cut lesion edges of
cartilage [48,156]. Apoptosis is characterized by specific
hallmarks, such as loss of cellular membrane asymmetry, cell
shrinkage, release of cytochrome c, chromatin condensation
and degradation of nuclear DNA to 180 base-pair
oligomeres, with caspases 3, 7, 9 and others mediating the
apoptotic signaling program [109,153,157,158]. In vitro,
chondrogenic cells respond to the same apoptosis-inducing
signals as other cells, including FasL/CD95, TNF-a, TRAIL
(TNF-related apoptosis inducing ligand), IL-1, matrix
metalloproteinase (MMP)-9, calcium and phosphate ions,
NO, ceramide, retinoic acid, or serum deprivation and
mechanical forces [150,154,159]. For example, TNF receptor
mediated signaling simultaneously stimulates pathways for
apoptosis induction via caspases and pathways for
suppression of apoptosis via nuclear factor (NF)-KB
[109,150,154]. Thus, the balance of these two signaling
pathways determines the fate of cells after TNF-a stimulation.
Interestingly, BMPs, specifically BMP-2, -4 and -7, have been
identified as key regulators inducing apoptosis during digit
development, which was reversed by the BMP antagonist
noggin [160]. Next to the avoidance of stressors named
above, the use of apoptosis inhibitors that directly bind and
inhibit caspases, such as Bcl-2 or Bcl-XL, might be of
therapeutic value in order to expand the lifespan of
transplanted and resident cells [160-162]. Remarkably, it has
also been shown that the dedifferentiation of chondrocytes,
as well as the inhibition of apoptosis, is regulated by the
Wnt/p-catenin pathway [34,163]. Under arthritic conditions,
Wnt-7a induces dedifferentiation of articular chondrocytes by
stimulating transcriptional activity of P-catenin, whereas NO-
induced apoptosis is inhibited via the activation of cell
survival signaling, such as phosphatidylinositol 3-kinase and
Akt, which block the apoptotic signaling cascade. Whether
this mechanism can be harnessed therapeutically for the
maintainance of chondrocyte populations, capable of building
hyaline neocartilage, remains to be elucidated [163].

Besides induction of apoptosis, where cells can be
eliminated without inflammation, cell death can be mediated
by necrosis following a significant inflammatory response,
due to the leakage of cell contents. Whereas the term
'necrosis' in the strict sense refers to changes secondary to
cell death by any mechanism, including apoptosis, it is used
here to describe a caspase-independent mode of cell death,


Available online http://arthritis-research.com/content/9/3/213



as used previously by other groups [164-166]. Necrotic cell
death is characterized by swelling of the cells and organelles,
uncontrolled release of lysosomal enzymes and nuclear
condensation pycnosiss) [164-166]. Thus, necrosis for
cartilage repair is especially detrimental because of the
associated inflammatory response, which interferes with
chondrogenesis and the integrity of the cartilage matrix. The
avoidance of cytotoxic stimuli during cartilage repair
procedures, such as trephine wounds, needle stitches, and
others (see above), is of ample importance for a successful
repair, as well as anti-inflammatory measures where necrosis
cannot be completely avoided.

Given the lack of cell supply in cartilage, major cell loss in
cartilage and neo-cartilage will inevitably lead to tissue failure,
as chondrogenic cells are the only source of matrix synthesis
in theses tissues. Furthermore, recent studies are beginning
to indicate a central role of the collagen framework in the
maintenance of cartilage cells. However, whether cell loss is
primary or secondary to cartilage matrix destruction is still a
matter of debate [154,167].

Matrix degradation
Damage to the cartilage ECM can arise focally after acute
cartilage injury in an otherwise healthy joint, or from an
underlying disease process, such as in OA or rheumatoid
arthritis (RA).

Mechanical factors
As mentioned above, mechanical factors are integrally
involved in cartilage matrix integrity, as well as survival and
death of chondrogenic cells in native cartilage as well as
repair tissues [157,168,169]. Quantitative analyses revealed
that cartilage repair tissue after ACT has inferior mechanical
properties compared to healthy cartilage in a large animal
model [170,171]. In vitro studies using bovine cartilage
explants showed that compressive stress, as low as 4.5 MPa,
was able to induce apoptosis, while other parameters of matrix
degradation, such as disruption of the collagen fibril network,
tissue swelling, release of glycosaminoglycans, and increased
nitrite levels, were apparent only at higher stress levels [172].
Beyond a threshold of 6 MPa, cell viability is inversely
proportional to the applied stress [173]. In contrast, physical
parameters have also been identified that aid the cartilage
tissue quality in vitro [174-176] and in vivo after cell-based
cartilage repair, comprising intermittent active or continuous
active/passive motion and other techniques (reviewed in [2]).
Therefore, post-operative care and physical therapy also play
important roles in determining the healing outcome.

Inflammation and degeneration
Joint trauma, cartilage repair responses with or without
different matrix materials, and diseases such as OA or RA
can cause joint inflammation of varying intensities. The
cytokines involved, including IL-1 and TNF-a, have direct and
indirect pro-inflammatory effects to regulate the degradation


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Arthritis Research & Therapy Vol 9 No 3 Steinert et al.



of ECM components by stimulating secretion of proteolytic
enzymes and other mediators, such as MMPs, aggrecanases
and other members of the 'a disintegrin and metallo-
proteinase with thrombospondin motifs' (ADAMTS) gene
family, NO, and prostaglandin E, [149,177,178]. The
importance of IL-1 is reinforced by the fact that it has been
shown to inhibit chondrogenesis in chondrogenic cells in
vitro by downregulation of chondrocyte differentiation state
marker genes, such as those encoding SOX9, collagen type
II and others, while upregulating NO, prostaglandin E, and
MMP-3, and the finding that the effects could be inhibited by
the addition of the IL-1 receptor antagonist (IL-1Ra)
[179-181]. However, much of this work was derived from
research studying OA and RA [179,182-184]. Cell-based
cartilage repair in OA knees has been attempted, with equal
results compared to cartilage repair procedures in non-
arthritic knees [50]. Inhibition of pro-inflammatory cytokines
such as IL-1 may offer a useful supportive approach to the
management of cartilage injury by reducing gene expression
of genes involved in cartilage matrix degradation, and thus
favoring a beneficial repair environment for transplanted
chondrogenic cells [132,185-187]. The application of anti-
inflammatory cytokines is hindered by delivery problems. For
example IL-1Ra is available as the drug 'Kineret' for the
treatment of RA, and is self-administered at a daily dose of
100 mg by subcutaneous injection, because it is neither
feasible nor safe to deliver IL-1 Ra by repeated intra-articular
injection [179,184]. This treatment leads to a peak IL-1Ra
serum concentration of approximately 1 pg/ml, which is
transient; the concentrations of IL-1Ra achieved in synovial
fluid are unknown but they are likely to be low and also
transient, explaining the limited success thus far using this
drug in RA [179,184]. Gene delivery strategies to overcome
these delivery issues have been devised [188-193], with a
phase I clinical study having been completed for the
treatment of RA [194,195] and an ongoing study for the
treatment of OA [196]. Should these technologies prove to
be safe and effective in the future, their use could be
beneficially extended to the area of cartilage repair.

For treatment of local defects the use of growth factors, anti-
inflammatory cytokines or matrix molecules offers much
promise as an approach to promote hyaline cartilage repair
[29,66,122,197]. Since local defects are contained within
healthy cartilage, it is likely that expression of certain growth
factor and chondroprotective genes will be required for the
length of time necessary to promote cartilage healing. The
potential for gene therapy to promote cartilage repair in
chronic diseases like RA and OA is more limited, due to more
extensive cartilage loss, and altered metabolic responses of
OA and RA chondrocytes.

Integration failures
One of the main biological challenges associated with cell-
based cartilage repair procedures is integration of the graft
tissue with the host. Depending on the nature of the cartilage


injury, whether it is a chondral or osteochondral lesion,
different aspects of transplant integration with cartilage and
bone have to be appreciated.

With respect to integration of cartilage with cartilage, the
nature and status of the tissue that comprises the wound
lesion edge is central to tissue integration. As mentioned
above, controlling aspects of cell differentiation, apoptotic or
necrotic cell death together with matrix synthesis is the main
factor influencing successful integration. Despite some cell
migration potential from cartilage tissue in young experimental
animals [49,198], chondrocytes appear to have only a limited
ability to infiltrate existing cartilage matrices and even to
occupy empty chondrocyte lacunae [48]. However,
integrative repair and chondrogenesis is not only a function of
gross matrix synthesis, but is also affected by adjacent tissue
structure and composition [199].

Another aspect is that blunt trauma to cartilage induces a
greater proliferative response that extends to a greater
distance from the lesion edge compared to burst and sharp
trauma. However, in the case of sharp trauma, the basal cells
enter proliferation before surface zone chondrocytes, which is
not the case in blunt wounds [48]. Several measures have
been used to promote this process by establishing good
contact between cartilage transplants and native articular
cartilage, including the use of collagen cross-linkers, brief
enzymatic digestion and biological glues (tissue transgluta-
minase and other adhesives; reviewed in [2]).

With respect to the repair of osteochondral defects, two
distinct types of tissues, articular cartilage and subchondral
bone, are involved. In designing a multiphase implant, the
healing of the underlying subchondral region of the defect site
is critical as it supports the overlying neocartilage regeneration
[49,200-202]. In order to protect the cartilage graft from
stromal cell invasion from the underlying bone and
dedifferentiation in osteochondral lesions, there have been
attempts to mimic the tidemark with the use of biological
sealants [20]. However, there have not been any controlled
studies to date to validate this procedure. Initial studies have
been performed to generate biphasic transplants in order to
promote graft integration with the underlying bone in vitro, with
a tidemark being already integrated [49,200-202]. Also, initial in
vivo studies regenerating the subchondral layer by using MSC-
seeded poly-E-caprolactone scaffolds have been performed
with successful results [203]. Although the fabrication of
neocartilage with an appropriate zonal organization has been
attempted [204,205], exact reproduction of the different zones
of native articular cartilage, including the tidemark and
underlying bone, has not been achieved to date. A fruitful
interplay between cells, morphogens and scaffold technology
will be crucial for successful cartilage-like patterning of
transplants for successful integration. Optimization and
development of these approaches will determine whether
constructs are applicable for partial- or full-thickness defects.


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Available online http://arthritis-research.com/content/9/3/213


Table 1

Therapeutic strategies to augment cell-based cartilage repair


Impairment

Differentiation
insufficiencies


Cell loss


Matrix degradation


Integration


Pathomechanism


Un- or dedifferentiated cells






Hypertrophic differentiation




Osteogenesis


Senescence
Age
Telomere erosion
Oxidative damage
Chemical stress
Mechanical stress

Inefficient cell delivery
Apoptosis
(NO induced, stress)


Necrosis
Age

Mechanical stress
Chemical stress
Oxidative stress

Matrix degradation
Inflammation


Mechanical stress (shear
stresses, compressive
forces)



Cartilage to cartilage
Cartilage to bone


Therapeutic strategy


References


Timed growth factor release systems/gene delivery
TGF-P superfamily members
FGFs
SOX/Smads
Cell selection
Growth factor selection
Immunophenotype selection
Inhibition of hypertrophy
BMP inhibitors: noggin, chordin, siRNAs
PTHrP/IHH
Wnt5a
No dexamethasone
Inhibition of osteogenesis
BMP inhibitors (noggin, chordin), siRNAs
Establishment of a barrier/tidemark to bone
Senescence protection
Low oxygen tension
Use of telomerized cells
Anti-oxidative selenoproteins, superoxide dismutase
Anti-inflammatory agents (IL-1 Ra, slL-1 R, sTNFR)
Mechanoprotection

Guided, homogeneous cell delivery
Anti-apoptotic measures
Bcl-2, Bcl-XL, anti-FasL
Anti-inflammatory agents (see also above)
Anti-oxidative agents
Necrosis
Surgical protection (no needle stitches, no
unnecessary harm to cartilage lesion borders)
Mechanoprotection
Anti-inflammatory agents
Anti-oxidative agents

Delivery of matrix components
Anti-inflammatory agents (IL-1 Ra, slL-1 R, ICE inhibitor,
sTNFR, anti-TNF-antibodies, TACE inhibitor, TIMP-1,
-2, MMP inhibitors, IL-4, -10, -11, -13, GFAT)
Mechanoprotection
No trauma
Avoidance of non-physiological loads
Establishment of correct knee axis and stability
Antioxidants

Cartilage matrix crosslinks
Tidemark formation
Stimulation of cell migration
Chondroblasts above tidemark
Osteoblasts below tidemark


[76,85,88,134]
[131]
[119-121,124-126]

[52-54,64]
[41,46]

[89,90,160]
[81,91-95]
[29,103,163]
[75]

[89,90,160]
[49,198]

[107,110]
[117]
[115,117,118,151,187]
[132,185-188,190,191,193-195]
[157,168,169]

[2,48]

[151,161,162,164]
[132,185-188,190,191,193-195]
[115,117,118,151,187]

[2,48]

[2,48;168,169]
[132,185-188,190,191,193-195]
[115,117,118,151,187]

[197]
[132,185-188,190,191,193-195]



[2,48;168,169]
[1,2,8,9,12,20]
[1,2,8,9,12,20]
[115,117,118,151,187]

[20,49,200-202]
[49,198]
[27,29,66]


BMP, bone morphogenetic protein; FasL, Fas-Ligand; FGF, fibroblast growth factor; GFAT, fructose-6-phosphatase amido transferase; ICE, IL-1
converting enzyme; IHH, indian hedgehog; IL, interleukin; IL-1 Ra, IL-1 receptor antagonist; MMP, matrix metalloproteinase; NO, nitric oxide; PTHrP,
parathyroid hormone related peptide; slL-1 R, soluble IL-1 receptor; siRNA, small interfering RNA; SOX, SRY (sex determining region Y)-box;
sTNFR, soluble TNF receptor; TACE, TNF-alpha converting enzyme; TGF, transforming growth factor; TIMP, tissue inhibitor of matrix
metalloproteinases; TNF, tumor necrosis factor.


Conclusions and future perspectives


quality and stability. Although current treatments have limited


Cell-based approaches to cartilage repair have only been effectiveness, an improved clinically useful repair approach
introduced to the clinic by means of the ACT procedure, with does not necessarily have to demonstrate complete
good clinical results, but are still far from generating a repair regeneration of normal tissue. The aim of this review is to
tissue that is comparable to native cartilage in terms of tissue critically analyze the current clinical and experimental


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Arthritis Research & Therapy Vol 9 No 3 Steinert et al.




cartilage repair approaches from a cell and molecular
biological perspective in order to encourage the development
of more rational approaches to repair. Considering the main
biological obstacles to cartilage repair as outlined in this
review (Figure 1), an ideal construct would contain hyaline
differentiated tissue, and become fully integrated with the
adjacent cartilage and bone, without the induction of an
inflammatory response, senescence, apoptosis or necrosis.
This aim may be overly optimistic to achieve in the near future,
but the enabling technologies exist to address these issues.
Efficient delivery of chondrogenic, anti-inflammatory and anti-
oxidative factors seem to be of key importance (Table 1). As
most of these factors are recombinant proteins, which have
short half-lives, a repeated local administration is likely to be
necessary to achieve the desired result, creating delivery
problems. Gene transfer techniques could be adopted that
might help us to overcome the limitations of the current
treatments for damaged articular cartilage. Nonetheless, what
remains to be determined are the exact factors needed for
hyaline repair, including their bioactive level and duration of
expression to meet the complexities of treating this tissue.

Competing interests
The authors declare that they have no competing interests.

Acknowledgements
This work is supported by grants from DFG (STE1051/2-1 to AFS and
UN), IZKF (D-23/1 to AFS and AR), and NIH NIAMS (Intramural
Research Program Z01 AR41131 to RST, and grants AR48566 and
AR50249 to SCG and CHE). We apologize to investigators whose
work could not be cited due to space limitations.

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