Review

Special Issue: Celebrating 30 years of biotechnology

Extracellular matrix scaffolds for

cartilage and bone regeneration
Kim E.M. Benders1, P. Rene van Weeren2, Stephen F. Badylak3, Daniel B.F. Saris1,4,
Wouter J.A. Dhert1,5, and Jos Malda1,2
1

Department of Orthopedics, University Medical Center Utrecht, P.O. Box 85500, 3508 GA, Utrecht, The Netherlands
Department of Equine Sciences, Utrecht University, P.O. Box 80153, 3508 TD, Utrecht, The Netherlands
3
McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA, USA
4
MIRA Institute, Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands
5
Faculty of Veterinary Sciences, Utrecht University, Utrecht, The Netherlands
2

Regenerative medicine approaches based on decellularized extracellular matrix (ECM) scaffolds and tissues are
rapidly expanding. The rationale for using ECM as a
natural biomaterial is the presence of bioactive molecules that drive tissue homeostasis and regeneration.
Moreover, appropriately prepared ECM is biodegradable
and does not elicit adverse immune responses. Successful clinical application of decellularized tissues has been
reported in cardiovascular, gastrointestinal, and breast
reconstructive surgery. At present, the use of ECM for
osteochondral tissue engineering is attracting interest.
Recent data underscore the great promise for future
application of decellularized ECM for osteochondral repair. This review describes the rationale for using ECMbased approaches for different regenerative purposes
and details the application of ECM for cartilage or osteochondral repair.
The need for improved repair of osteochondral defects
Joint injuries are common in the young and active population and often result in cartilage or osteochondral lesions.
If untreated, these defects lead to joint swelling, pain, and
serious restrictions in daily activities and can eventually
progress towards osteoarthritis (OA), of which the only
end-stage, salvaging therapy is artificial joint replacement.
Over 151 million people suffer from OA worldwide [1],
representing a huge clinical and socioeconomic burden.
Established OA is notoriously difficult to treat, but prevention through successful treatment of cartilage lesions
will significantly reduce this socioeconomic impact.
Natural wound healing in full-thickness cartilage
defects leads to the formation of so-called fibrocartilage,
which is functionally and biomechanically inferior to the
original hyaline cartilage. This makes the tissue more
prone to further deterioration, and thus initiates a vicious
cycle.
Currently, many different cartilage repair-enhancing
treatments are applied in patients with (osteo)chondral
defects. These techniques are either based on cell therapy,
such as autologous chondrocyte implantation (ACI) [2] and
matrix-induced chondrocyte implantation (MACI) [3], on
Corresponding author: Malda, J. (j.malda@umcutrecht.nl).

replacement of the damaged tissue within the joint, for

example, by mosaicplasty [4] and osteochondral allografting (see Glossary) [5,6], or on the recruitment of mesenchymal stromal cells (MSCs) through, for example,
microfracture [7]. All of these techniques provide fairly
acceptable clinical results, but none results in restoration
of fully functional hyaline cartilage, making long-term
prognosis uncertain.
In an attempt to optimize the functional restoration of
cartilage, tissue engineering has been suggested as a good
basis for new regenerative therapies. The key to successful
engineering of cartilage with optimal restoration of function lies in finding the optimal combination of biomaterials,
biofactors, and cells [8]. Biomaterials currently used in the
field of cartilage tissue engineering can be grossly divided
into two groups: (i) natural biomaterials such as collagen
[9], gelatin [10], and fibrin [11], and (ii) synthetic biomaterials such as polycaprolactone (PCL) [12], and polylactic
acid (PLA) [13]. The synthetic materials often have good
biomechanical strength and their specific properties can be
tailored by changing the polymer composition. However,
the major challenge for these materials, which are foreign
to the body, is to achieve satisfactory tissue integration and
differentiation. Natural biomaterials may overcome this
challenge because they are biocompatible and biodegradable.
Despite the great advances that have been made in the
field of material sciences in mimicking the natural tissue

Glossary
Allograft: graft obtained from a donor of the same species.
bFGF: basic fibroblast growth factor; involved in angiogenesis, wound healing,
and embryonic development.
EGF: epidermal growth factor; stimulates proliferation and differentiation.
IGF: insulin-like growth factor; regulates cellular proliferation and apoptosis.
Mosaicplasty: surgical procedure during which a defect is filled with
osteochondral plugs taken from a non-load-bearing region of the joint.
Osseous phase: bone compartment.
Proteoglycans: glycoproteins of high molecular weight present in connective
tissue and cartilage.
TGFb: transforming growth factor beta; regulates many cellular functions
including proliferation, differentiation, and apoptosis.
VEGF: vascular endothelial growth factor; stimulates vasculogenesis and
angiogenesis.

0167-7799/$ see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2012.12.004 Trends in Biotechnology, March 2013, Vol. 31, No. 3

169

Review
environment to drive cell proliferation and differentiation,
oversimplified biomaterials for (cartilage) tissue regeneration are still being used. In fact, all tissues in the body are
composed of a complex mixture of different biomaterials
and this situation is not different for cartilage, notwithstanding its seemingly homogeneous and straightforward
appearance. In reality, the extracellular matrix (ECM) of
cartilage is a structurally complex 3D environment composed of various types of collagens and proteoglycans in
which multiple bioactive factors, such as growth factors,
integrins, and functional peptides, are incorporated. Even
highly sophisticated, newly developed biomaterials will
probably never reach this complexity.
The abovementioned circumstances and considerations
have driven the tissue engineering field towards increased
use of biomaterials or scaffolds based on (processed) natural ECMs, an approach that might be a very valid option for
cartilage repair as well.
ECM-based regenerative medicine
All tissues are composed of cells surrounded by ECM that
consists of a unique and tissue-specific 3D environment of
structural and functional molecules secreted by the resident cells [14]. There is reciprocal interaction between cells
and ECM; cellular products, including proteinases, modify
the ECM, and ECM-incorporated growth factors and cytokines act as functional cues, steering the metabolic and
secretory activity of cells. This situation becomes even
more complex because the intricate interplay of cells and

Box 1. The immune response to decellularized matrix

Several decellularized products for different regenerative purposes
are available for clinical use. However, the amount of cellular
material that remains after decellularization is variable [39]. There
are no clear-cut guidelines for the degree of decellularization
required, because cell remnants in devitalized tissue do not always
hinder tissue regeneration [44,69].
The immune response that may occur in response to implantation
of foreign cellular material is partly macrophage-mediated [39]. A
macrophage response to implantation of a scaffold is a necessary
event, because macrophages are involved in scaffold degradation.
However, macrophages release several soluble factors on activation
that can be both beneficial and detrimental to neotissue formation,
depending on macrophage phenotype. Activation of M1 macrophages leads to adverse remodeling through the release of catabolic
cytokines, whereas activation of M2 macrophages leads to constructive remodeling through anabolic cytokines [39]. For example,
M1 macrophages release IL-1b and IL-6, which are upregulated in
patients with damaged knee cartilage. The balance between M1 and
M2 macrophages after implantation tends to shift to M2 macrophages if decellularization is more successful [39].
The avascular nature of cartilage is one of the major challenges in
initiating intrinsic repair but may also be advantageous, because the
tissue is immunopriviliged to a large extent, which opens up many
more options in choosing the ECM source, including allogeneic and
xenogeneic sources, without rejection issues [70]. In addition, the
dense nature of cartilage ECM may further contribute to the weakly
immunogenic, or even non-immunogenic, status, because it
physically protects chondrocytes from T and NK cells that are
released in graft rejection [70]. The application of xenogeneic
products for cartilage repair is still in its infancy but should be
explored further, because it overcomes the limited availability of
human tissue or cells. The question remains, however, which tissue
components may lead to an inappropriate immune response, the
cells or the ECM.
170

Trends in Biotechnology March 2013, Vol. 31, No. 3

ECM in a given microenvironment is not static but is

rather a dynamic event that responds to external influences, such as biomechanical triggers and hormonal
actions [15]. It is the eventual outcome of these dynamic
processes that determines tissue homeostasis and possible
aberrations thereof. Given the high complexity of these
processes and the multiple roles of the ECM, constructs
based on natural ECM sources are likely better prepared to
produce a tissue with optimal functionality than those
built from artificial compounds.
ECM-based tissue engineering strategies are already
successfully being used clinically for the regeneration of a
range of different tissues, including heart valves [16],
trachea [17], muscle [18], tendon [19], and abdominal walls
[20], with matrices derived from bladder and small intestinal submucosa [21] the most widely used implants. The
main advantage of ECM as a scaffolding material is that it
allows for so-called constructive remodeling [22], that is, it
supports and encourages specific tissue formation at the
implantation site rather than forming inferior and less
functional scar tissue. However, the functional outcome of
ECM-derived scaffolds depends on several factors, including retention of growth factors within the ECM, its surface
topology, modulation of the immune response (Box 1), and
the microenvironmental cues exerted on the cells, such as
biomechanical loading (Box 2) [23].
The underlying mechanisms are still not fully understood, but several potential explanations are possible for
the positive outcomes obtained with ECM-derived scaffolds. First, the process of dynamic reciprocity explained
above [24], which is vital for proper functioning of tissues,
is more likely to be effective in a natural tissue that
contains bioactive cues, such as growth factors, polysaccharides, and functional peptides, than in an artificial

Box 2. Biomechanical properties of decellularized matrix

The biomechanical characteristics of articular cartilage in terms of
resilience and stiffness are crucial to proper functioning of the tissue
in a strictly mechanical sense, but also with respect to tissue
homeostasis, because biomechanical cues steer chondrocyte behavior to a large extent via mechanotransduction pathways [71]. In this
context, biomechanical properties influence the growth factor
reservoir within the ECM and matrix stiffness may, for instance,
mediate TGFb-driven processes through which this reservoir is
continuously replenished and depleted [27].
The processes of harvesting, decellularization, and sterilization of
ECM scaffolds affect the hydration status and 3D configuration and
hence strongly influence biomechanical behavior. Washing steps
using SDS or other processes that lead to removal of GAGs entail
loss of water and produce a more loosely packed collagen network
and hence loss of viscoelastic properties [22,72]. Freezethaw cycles
may result in disruption of the collagen network through crystal
formation.
The biomechanical behavior of ECM scaffolds in vivo will depend
on the way the scaffold was processed, on the properties and
geometry of the surrounding tissue, the pattern and magnitude of
forces exerted on the scaffold, its degradation rate, and the extent to
which new ECM is formed [73]. The biomechanical properties of any
ECM-based scaffold will almost invariably be inferior to those of the
original tissue. The extent and rate at which neotissue is formed and
takes on more physiologic biomechanical characteristics depend
mainly on the capacity of the scaffold (and/or the cells seeded
therein, if any) to properly respond in an anabolic way to the cues
elicited by joint loading and motion.

Review
tissue that does not. Along the same line, incorporation of a
certain cell type in a scaffold made from the target ECM
will more easily drive the cell towards the appropriate
terminal differentiation [2527]. Second, naturally occurring ECM is the product of the resident cells and has a 3D
structure that may guide cell behavior, attachment, and
migration [28], but incorporated growth factors or other
functional proteins are often also associated with alignment of the collagen fibers that mostly make up the 3D
structure of a tissue and that give a tissue its biomechanical strength and resilience [29]. The biomechanical environment of the cell, which is largely dictated by the
biomaterial, can have a great influence on cell differentiation. For example, MSCs commit to the osteogenic lineage
in stiff biomaterials, but to the neuronal lineage in more
flexible biomaterials [30].
The mechanism behind the successful use of ECM-based
scaffolds seems to be generic to a certain extent and not
exclusively tissue-specific, because ECM scaffolds originating from tissues other than the target tissue have been
used with success. For example, small intestinal submucosa (SIS) ECM has been used as a scaffold for repair of the
musculotendinous junction between the gastrocnemicus
muscle and the Achilles tendon in dogs [31,32]. The scaffold
was recellularized by progenitor cells from its surroundings and was ultimately completely replaced by functional
contractile muscle and tendon, including one of the most
challenging types of tissue to regenerate, the neurovascular bed [31]. ECM-based scaffolds can even be of xenogeneic
origin [31,3335] after successful decellularization to remove cellular antigens.
Decellularization of tissues can be accomplished using
various methods or combinations thereof (Table 1). Physical treatments such as thermal shock, freezethaw cycles,
and mechanical crushing of the tissue will lead to cell lysis
and tissue breakdown, allowing for easier infiltration of the
chemical and enzymatic treatments that often follow [24].
Treatments with detergents or other chemicals, including
SDS and Triton X-100, are used to break down cellular and
nuclear membranes [36], which can then be removed in
subsequent washing steps. Enzymatic treatments depend
on the tissue type, but often trypsin and nuclease solutions
are used to break down peptides, DNA, and RNA [36].
Decellularization should ideally remove all cells and
cellular antigens while retaining the bioactive cues that
reside in the ECM. Decellularization of bladder submucosa
matrix using several washing steps with enzymatic agents
and detergents led to full decellularization but also ensured that important growth factors, such as VEGF,
TGFb1, bFGF, and EGF, typically remained present within the decellularized tissue [37]. In the case of cartilage,
preservation of proteoglycans, one of the main ECM components, may be important. Proteoglycans not only contribute to the mechanical characteristics of the tissue
through attraction of water by variations in fixed charge
density [38] but are also thought to be a reservoir of several
growth factors at times when these are not readily produced and released by the resident cells [37].
Both single tissues and whole organs can be decellularized, providing a biological scaffold of resident ECM with
the complex geometry of an organ and an intact vascular

Trends in Biotechnology March 2013, Vol. 31, No. 3

network that will enhance nutrient supply, benefitting

regeneration and recellularization [24]. In the case of organ
decellularization, it is imperative that the process does not
disrupt the natural integrity of the tissue; in the case of
tissue decellularization, the process can be more rigorous.
Certain criteria have been proposed for successful decellularization, or perhaps better denuclearization: (i) the
absence of nuclei on histological evaluation [hematoxylineosin or 40 ,6-diamidino-2-phenylindole, dihydrochloride (DAPI)], (ii) DNA quantification <50 ng/mg dry tissue,
and (iii) DNA fragments <200 bp [24]. However, these
criteria were based on the decellularization of loosely
organized tissues (SIS and urinary bladder matrix
(UBM)) and may not apply to more dense tissues such
as cartilage. Rigorous decellularization enhances loss of
structural integrity of the ECM and of certain ECM compounds. However, whether absolute decellularization is
necessary is still under discussion because ineffectively
decellularized ECM still induced similar host remodeling
to that induced by effectively decellularized material [39].
In addition to decellularization, artificial crosslinking of
ECM scaffolds is often applied to enhance the biomechanical
strength of the scaffold in the initial stages after implantation. However, this practice unequivocally affects ECM
properties. Artificial, and more specifically, chemical crosslinking will ultimately decrease the degradation rates and
thus the controlled release of bioactive factors [40]; chemical
crosslinking may also physically hamper tissue remodeling
because it elicits an adverse recipient immune response [41].
The successful application and encouraging results for
in vitro and in vivo work using ECM scaffolds in several
different fields hold great promise for this approach in
attempts to regenerate (osteo)chondral tissue.
Application of ECM-based scaffolds to treat
osteochondral defects
Treatments during which osteochondral plugs, taken either from a non-load-bearing region of the joint (mosaicplasty) or from a donor (allogeneic osteochondral grafting),
are used to fill the defect can theoretically be considered
ECM-based strategies because they imply the direct implantation of cartilage and bone matrix (Figure 1). However, the use of seeded or unseeded ECM-based scaffolds is a
new and emerging approach within the field of cartilage
tissue engineering, supported by a slowly increasing body
of evidence of success.
One of the major advantages of using the ECM as a
scaffolding material is its potential to retain the growth
factors that the tissue is naturally inclined to respond to.
For cartilage, some of the most important growth factors
are TGFb, FGF, and IGF [8]. The retention of bioactive
molecules will be especially beneficial in regenerating
cartilage, because this tissue naturally lacks a supply of
appropriate growth factors and nutrients owing to its
avascular nature.
Bioactive ECM for (osteo)chondral repair can be applied
in many different ways that fall in three general categories
(Figure 1). First, non-decellularized cartilage particles [42]
combined with a degradable biomaterial led to initial
clinical results that at least matched the outcomes for
microfracture. Even ECM particles from osteoarthritic
171

Review

Trends in Biotechnology March 2013, Vol. 31, No. 3

Table 1 (Continued )

Table 1. Possible decellularization techniques for

(osteo)chondral repair
Decellularization method
Cartilage tissue
1. Rinsing in PBS
2. Lyophilization
3. Tissue grinding
4. Trypsin treatment
5. Rinsing in PBS
6. Nuclease treatment
7. Hypotonic TrisHCl treatment
8. Incubation in Triton X-100
9. Rinsing in PBS
10. Lyophilization
11. Crosslinking with UV
12. Sterilization by ethylene oxide
1. Rinsing in PBS
2. Shattering of the tissue in PBS
3. Differential centrifugation
4. Incubation in Triton X-100
5. Hypotonic TrisHCl treatment
6. Nuclease treatment
7. Rinsing in PBS
8. TrisHCl treatment
9. Rinsing in PBS
10. Lyophilization
11. Dehydrothermal treatment
12. Crosslinking with
carbodiimide
13. Rinsing in PBS
14. Sterilization by cobalt
g-irradiation
1. Rinsing in distilled water
2. NaOH treatment
3. Rinsing step
4. Defatting in ethanol
5. GndHCl and NaOAc treatment
6. Rinsing step
7. H2O2 treatment
8. Rinsing step (0.9% NaCl)
1. Rinsing in PBS
2. Freeze and thaw cycles
3. Hypotonic TrisHCl
treatment
4. SDSEDTA treatment
5. Rinsing in PBS
6. Nuclease treatment
7. Rinsing in PBS
8. Peracetic acid treatment
9. Rinsing in PBS
1. SDS treatment
2. Rinsing in water
3. Lyophilization
Bone Tissue
1. Rinsing in demiwater
2. NaN3 treatment
3. Chloroform and
methanol treatment
4. Incubation in Triton X-100
5. SDS treatment
6. Rinse in PBS
1. Defatting in acetone
2. Rinsing in saline
3. Trypsin treatment
4. Rinsing in saline
5. Rinsing in acetone
6. Crosslinking with
hexamethyl diisocyanate
7. Rinsing in acetone
8. Rinsing in saline
9. Sterilization by g-irradiation
172

Tissue type

Refs

Bovine cartilage

[49]

Human cartilage

[48]

Human nasal cartilage

Porcine nasal cartilage
Porcine meniscus

[35,47]

Porcine cartilage

[72]

Cartilage ECM
sheets of 10 mm

[52]

Human cancellous
bone

[57]

Porcine trabecular
bone

[56]

Decellularization method
Cultured cell matrices
1. Incubation in Triton
X-100 with NH4OH
2. Rinsing in PBS
3. Rinsing in double
distilled water
4. Freeze and thaw cycles
5. NH4OH treatment
6. Rinsing in double
distilled water
7. Na3PO4 treatment
8. Rinsing in double
distilled water
1. SDS with nuclease and
EDTA treatment
2. Rinsing in PBS
3. Culturing for 4 weeks
4. SDS with nuclease and
EDTA treatment
5. PBS rinsing
1. Freeze and thaw cycles
2. Rinsing in PBS
3. Rinsing in double distilled
water
4. Perfusion based washing in
bioreactor
1. Freeze and thaw cycles
2. Rinsing in distilled water
3. Lyophilization

Tissue type

Refs

Human MSC matrix

[27]

Human MSC matrix,

normal human articular
chondrocyte matrix,
and normal human
dermal fibroblast
matrix cultured on
PLGA meshes

[51]

Immature bovine
chondrocyte matrix
cultured in agarose
wells

[46]

Human MSC bone

matrix cultured on
polyesterurethane

[58]

Human MSC bone

matrix cultured on
tissue plastic

[59]

patients can be used for this purpose [43]. A combination of

OA cartilage particles that had undergone freezethaw
cycles (devitalization) with MSCs in fibrin glue for implantation in subcutaneous pockets in mice led to better shape
fidelity; glycosaminoglycan (GAG) content and chondrogenic gene expression were also enhanced compared to
non-supplemented glue [43]. Cartilage tissue can also be
processed into cartilage microparticles [44] that may be
used as an additive to enhance current cell-centered techniques (ACI or MACI) by mixing it with the cell suspension
or biomaterial that fills the defect. The addition of microparticles to pellet cultures leads to upregulation of chondrogenic gene profiles and moderately decreases
hypertrophic gene expression [44].
Second, cartilage matrix can be harvested from allogeneic or even xenogeneic sources and then used in a scaffold
form. Preclinical results underscore the benefits of devitalized or decellularized tissue over implantation of living
cartilage, because the formation of neocartilage of the
latter tends to lag behind [45]. Decellularized cartilage
matrix can be obtained from different sources and through
different decellularization processes. Owing to the dense
nature of cartilage ECM in which the cells are embedded,
more vigorous protocols are required to decellularize cartilage than for many other tissues. This inevitably leads to
greater destruction of the ECM components; GAGs will be
especially affected [46]. Moreover, cartilage thickness
decreases and the tissue loses some of its biomechanical
resilience [46]. The effect of GAG loss on the final concentrations of bioactive cues such as growth factors still needs
to be evaluated for different decellularization protocols.
Decellularized cartilage ECM can also be rebuilt into a

Review

Trends in Biotechnology March 2013, Vol. 31, No. 3

Osteochondral
auto/allogra

Devitalized
osteochondral
allogra

Decellularized
osteochondral
gra
Cell-derived
decellularized ECM

Living carlage
parcles

Decellularized
carlage

Decellularized
carlage parcles

TRENDS in Biotechnology

Figure 1. Various possibilities for matrix-based approaches to (osteo)chondral repair: osteochondral defects can be filled with fresh, devitalized, or decellularized
osteochondral grafts, which can be from autologous or allogeneic origin. Defects can be treated with allogeneic living cartilage particles [42], a decellularized cartilage graft,
or decellularized cartilage particles [45]. In addition, use of in vitro produced cell-derived decellularized matrix is also being actively explored [27,28,50,51].

scaffold through lyophilization [4749]. In rabbits, this

type of scaffold resulted in the regeneration of hyaline
cartilage when combined with rabbit MSCs [49].
Finally, the ECM to produce a scaffold for cartilage repair
can be harvested from cultured cells to create so-called cellderived ECM scaffolds [27,28,50,51]. Cell-derived ECM
overcomes the issues of possible exogenous pathogen transfer and allows ECM produced by the patients own cells to be
used. Moreover, different cell types can be mixed to create
the appropriate ECM for more complex tissues, and use of
thin ECM sheets allows much easier decellularization and
recellularization [50]. ECM sheets seeded with MSCs or
chondrocytes show superior chondrogenesis compared to
pellet cultures [50,52]. The main challenge in using cellderived ECM is finding a way to upscale the process in such a
way that it can be clinically applied for human regenerative

therapies. One way to accomplish this is to stack several

different decellularized cartilage sheets to create a layered
construct [52,53].
The process of decellularization paves the way for the
use of xenogeneic material, the major advantages of which
are cost-effectiveness and the relatively limitless availability of ECM. With a xenogeneic matrix, the age of the source
animal should be taken into account. Young individuals
heal better than adults and the tissues may be morphologically different. SIS ECM, for example, is thinner in older
animals and has lost its elastic properties, as well as some
proteoglycans and growth factors [23]. Therefore, use of
tissue from younger donors may be advantageous [23]. For
a tissue such as cartilage that is metabolically stable in
mature individuals, the age up to which this is true is a
relevant question. Products of non-enzymatic glycation
173

Review
(a)

Trends in Biotechnology March 2013, Vol. 31, No. 3

(b)

(c)

TRENDS in Biotechnology

Figure 2. Osteochondral repair in a horse using decellularized cartilage. (a) Macroscopic overview of osteochondral repair tissue after 8 weeks of implantation. Both (b)
glycosaminoglycan-rich (Safranin-O, Fast Green) and (c) collagen type II-rich neotissue was found after 8 weeks, with clear distinction between the cartilage and bone
phase. Scale bars represent 2 mm; the box approximates the osteochondral defect created.

such as pentosidine crosslinks start to accumulate linearly

in cartilage from approximately the age of 15 onwards [54].
This might be an indication of the cutoff age after which
ECM from young individuals can be supposed to have
acquired a mature metabolic rate. Xenogeneic use of cartilage has already been successful when implanting human
cartilage-derived scaffolds seeded with canine MSCs in
nude mice [48]. Cells showed good viability and the neocartilage contained both GAGs and collagen type II [48].
An important feature of the ultimate ECM scaffold is its
biomechanical behavior. This is an especially challenging
topic when considering the mechanical forces that are
exerted daily on the cartilage and underlying bone in a
human joint. Combining ECM with a stronger synthetic or
ceramic material could potentially enhance the biomechanical properties of an ECM scaffold, an approach
that may be especially attractive for the repair of osteochondral defects. Alternatively, a novel lyophilization
method has been used to control the orientation of collagen
fibers within a fabricated scaffold. This approach ultimately led to a Youngs modulus that was almost three times
higher than that in non-oriented scaffolds [55]. Moreover,
the chondrocytes that were seeded on these scaffolds
tended to align along these fibers, proliferated more rapidly, and produced similar amounts of GAG- and collagenrich neotissue compared to scaffolds without collagen fiber
alignment [55].
The repair of cartilage defects penetrating into the subchondral bone (osteochondral defects) poses additional challenges. First, bone regeneration should not extend beyond
the osseous phase of the defect, so there may be a need for
different biomaterials for the cartilaginous and osseous
phases. Second, integration between cartilage and bone is
challenging and depends on simultaneous maturation of
both tissues, which is influenced by the biomaterials chosen
for both tissue types. Similar to decellularized cartilage,
decellularized bone promotes tissue growth on subcutaneous implantation, even outperforming the bioactivity of
established biomaterials such as bioactive glass [56].
Attempts have been made to combine decellularized cartilage and decellularized bone to create biphasic constructs for
osteochondral defect repair [57]. Preculture of a biphasic
construct with MSCs for 4 days before implantation into an
174

osteochondral defect in canine knees led to full regeneration

after 6 months with near-hyaline cartilage repair [57]. In the
case of bone regeneration, decellularized tissue-engineered
ECM can also be used to enhance the biological interaction of
synthetic or ceramic biomaterials with cells [5860], and
may even aid in the controlled release of incorporated and
normally rapidly released growth factors such as bone morphogenetic protein 2 (BMP2) [59].
Current work by our group is focused on the use of
decellularized equine cartilage matrix for osteochondral
repair. We performed an equine pilot study in which an
osteochondral defect of critical size (11 mm 1  10 mm)
was created in the stifle (knee) joint of a horse. This defect
was treated using a decellularized cartilage matrix scaffold
and clear regeneration of both the bone and cartilage phase
was present after 8 weeks (Figure 2). The two tissues could
clearly be distinguished and the integration between the
two was satisfactory (Benders et al., manuscript under
review). This indicates that a biphasic construct might
not be a biological necessity for osteochondral regeneration, but may only serve as a biomechanical stabilizer
during the initial phases of tissue repair in a challenging
environment such as the joint. An issue that needs attention is assessment of the possible long-term ossification of
neocartilage tissue in vivo in long-term studies.
Future perspectives for ECM-based scaffolds for
osteochondral repair
The use of decellularized ECM is gaining ground within the
field of cartilage tissue engineering and may prove to be of
great potential because it allows for multifactorial mimicry
that has not yet been achieved by man-made biomaterials.
The approach is still relatively underexplored and extensive research is required to understand the biologic
responses to ECM scaffolds within the joint environment
and to optimize the decellularization techniques and ultimately the final repair tissue. There are several issues that
need to be addressed.
First, cartilage naturally consists of different zonal
layers that exert different functions due to differences in
matrix composition and chondrocyte phenotype [29,61,62].
The use of ECM sheets may offer a possibility to represent
this natural microenvironment by stacking ECM sheets

Review
produced by the different zonal cell types. Recreation of the
zonal structure can be further stimulated through the use
of bioprinted 3D porous constructs to deposit zone-specific
matrices [63], combining hydrogels and strong synthetic
polymers, so that the mechanical properties can be tailored
[64]. The synthetic materials or hydrogels that are ideal for
bioprinting are often suboptimal in stimulating cell differentiation [65] and could be functionalized using ECM
particles to optimize cell responses to the biomaterial.
Second, current decellularization approaches have focused on decellularization of cartilage tissue [49,57] or
ECM produced by either stem cells or chondrocytes
[50,52,53]. However, the need to use a cartilage-specific
matrix may be questioned, and more readily available
tissues such as SIS and bladder matrices may have similar
effects. For example, SIS ECM has been successfully used
to regenerate other tissue types, such as cardiac and
vaginal tissues [66,67]. The use of non-cartilage-specific
matrix would have many advantages, because the scaffolds
can be produced via standardized protocols that have
already been established, the tissue is more easily accessible and available in larger volumes, and the use of SIS
ECM, for example, has already been evaluated both in vitro
and in vivo and is currently applied clinically.
Finally, repair of osteochondral defects remains a
huge orthopedic challenge owing to the complex combination of cartilage and bone, which frequently leads to
overgrowth of bone. Osteoinductive materials such as
tricalcium phosphate (TCP) and biphasic calcium phosphate (BCP) are already available and successful in the
regeneration of critical-size bone defects [68]. Therefore,
it seems logical to create a biphasic construct of such a
successful ceramic and combine it with bioactive decellularized cartilage, which on its own seems to drive
tissue regeneration in vivo. The use of ECM scaffolds
may even allow for a non-cell-laden approach to osteochondral repair because they can attract cells from the
implant site that will then differentiate into the appropriate cell type and elicit endogenous repair. Eventually,
this may lead to natural off-the-shelf products that can
be applied for a wide range of cartilage and osteochondral defects.
Concluding remarks
ECM scaffolds have shown great promise within the field of
tissue engineering and are now being developed specifically for cartilage repair. Decellularized ECM-based scaffolds
may solve many problems associated with the matrixbased approaches currently used for the repair of cartilage
or osteochondral defects, such as osteochondral allografting and mosaicplasty. This approach may lead to the
development of the ideal cartilage or osteochondral scaffold, providing the injured site with the right bioactive cues
that stimulate the regeneration of functional tissue that
resembles the healthy situation.
References
1 Orthoworld (20092010) The Orthopaedic Industry Annual Report,
Orthoworld Inc.
2 Brittberg, M. (2008) Autologous chondrocyte implantation technique
and long-term follow-up. Injury 39 (Suppl. 1), S40S49

Trends in Biotechnology March 2013, Vol. 31, No. 3

3 Zheng, M.H. et al. (2007) Matrix-induced autologous chondrocyte

implantation (MACI): biological and histological assessment. Tissue
Eng. 13, 737746
4 Hangody, L. et al. (1998) Mosaicplasty for the treatment of articular
cartilage defects: application in clinical practice. Orthopedics 21, 751
756
5 Bugbee, W. et al. (2012) Osteochondral allograft transplantation in the
knee. J. Knee Surg. 25, 109116
6 Bugbee, W.D. and Convery, F.R. (1999) Osteochondral allograft
transplantation. Clin. Sports Med. 18, 6775
7 Steadman, J.R. et al. (2002) Microfracture to treat full-thickness
chondral defects: surgical technique, rehabilitation, and outcomes. J.
Knee Surg. 15, 170176
8 Vinatier, C. et al. (2009) Cartilage engineering: a crucial combination of
cells, biomaterials and biofactors. Trends Biotechnol. 27, 307314
9 Tohyama, H. et al. (2009) Atelocollagen-associated autologous
chondrocyte implantation for the repair of chondral defects of the
knee: a prospective multicenter clinical trial in Japan. J. Orthop.
Sci. 14, 579588
10 Shin, H. et al. (2012) The mechanical properties and cytotoxicity of cellladen double-network hydrogels based on photocrosslinkable gelatin
and gellan gum biomacromolecules. Biomaterials 33, 31433152
11 Ahmed, T.A. et al. (2011) Fibrin glues in combination with
mesenchymal stem cells to develop a tissue-engineered cartilage
substitute. Tissue Eng. Part A 17, 323335
12 Jeong, C.G. et al. (2012) Three-dimensional polycaprolactone scaffoldconjugated bone morphogenetic protein-2 promotes cartilage
regeneration from primary chondrocytes in vitro and in vivo without
accelerated endochondral ossification. J. Biomed. Mater. Res. A 100,
20882096
13 Oshima, Y. et al. (2009) Variation of mesenchymal cells in polylactic
acid scaffold in an osteochondral repair model. Tissue Eng. Part C:
Methods 15, 595604
14 Badylak, S.F. et al. (2012) Engineered whole organs and complex
tissues. Lancet 379, 943952
15 Nelson, C.M. and Bissell, M.J. (2006) Of extracellular matrix, scaffolds,
and signaling: tissue architecture regulates development, homeostasis,
and cancer. Annu. Rev. Cell Dev. Biol. 22, 287309
16 DOnofrio, A. et al. (2011) Clinical and hemodynamic outcomes after
aortic valve replacement with stented and stentless pericardial
xenografts: a propensity-matched analysis. J. Heart Valve Dis. 20,
319325 discussion 326
17 Macchiarini, P. et al. (2008) Clinical transplantation of a tissueengineered airway. Lancet 372, 20232030
18 Ricchetti, E.T. et al. (2012) Scaffold devices for rotator cuff repair. J.
Shoulder Elbow Surg. 21, 251265
19 Martinello, T. et al. (2012) Successful recellularization of human tendon
scaffolds using adipose-derived mesenchymal stem cells and collagen gel.
J. Tissue Eng. Regen. Med. http://dx.doi.org/10.1002/term.1557
20 Meyer, T. et al. (2006) A new biocompatible material (Lyoplant) for the
therapy of congenital abdominal wall defects: first experimental
results in rats. Pediatr. Surg. Int. 22, 369374
21 Armitage, S. et al. (2012) Use of surgisis for treatment of anterior and
posterior vaginal prolapse. Obstet. Gynecol. Int. 2012, 376251
22 Badylak, S.F. (2007) The extracellular matrix as a biologic scaffold
material. Biomaterials 28, 35873593
23 Tottey, S. et al. (2012) The effect of source animal age upon
extracellular matrix scaffold properties. Biomaterials 32, 128136
24 Gilbert, T.W. (2012) Strategies for tissue and organ decellularization.
J. Cell. Biochem. 113, 22172222
25 Gong, J. et al. (2008) Effects of extracellular matrix and neighboring
cells on induction of human embryonic stem cells into retinal or retinal
pigment epithelial progenitors. Exp. Eye Res. 86, 957965
26 Sellaro, T.L. et al. (2007) Maintenance of hepatic sinusoidal endothelial
cell phenotype in vitro using organ-specific extracellular matrix
scaffolds. Tissue Eng. 13, 23012310
27 Pei, M. et al. (2011) A review of decellularized stem cell matrix: a novel
cell expansion system for cartilage tissue engineering. Eur. Cell Mater.
22, 333343 discussion 343
28 Vorotnikova, E. et al. (2010) Extracellular matrix-derived products
modulate endothelial and progenitor cell migration and proliferation
in vitro and stimulate regenerative healing in vivo. Matrix Biol. 29,
690700
175

Review
29 Martel-Pelletier, J. et al. (2008) Cartilage in normal and osteoarthritis
conditions. Best Pract. Res. Clin. Rheumatol. 22, 351384
30 Engler, A.J. et al. (2006) Matrix elasticity directs stem cell lineage
specification. Cell 126, 677689
31 Turner, N.J. et al. (2010) Xenogeneic extracellular matrix as an
inductive
scaffold
for
regeneration
of
a
functioning
musculotendinous junction. Tissue Eng. Part A 16, 33093317
32 Turner, N.J. et al. (2012) Biologic scaffold remodeling in a dog model of
complex musculoskeletal injury. J. Surg. Res. 176, 490502
33 Penolazzi, L. et al. (2012) Human mesenchymal stem cells seeded on
extracellular matrix-scaffold: viability and osteogenic potential. J. Cell.
Physiol. 227, 857866
34 Badylak, S.F. (2004) Xenogeneic extracellular matrix as a scaffold for
tissue reconstruction. Transpl. Immunol. 12, 367377
35 Schwarz, S. et al. (2012) Processed xenogenic cartilage as innovative
biomatrix for cartilage tissue engineering: effects on chondrocyte
differentiation and function. J. Tissue Eng. Regen. Med. http://
dx.doi.org/10.1002/term.1650
36 Crapo, P.M. et al. (2011) An overview of tissue and whole organ
decellularization processes. Biomaterials 32, 32333243
37 Chun, S.Y. et al. (2007) Identification and characterization of bioactive
factors in bladder submucosa matrix. Biomaterials 28, 42514256
38 Han, E. et al. (2011) Contribution of proteoglycan osmotic swelling
pressure to the compressive properties of articular cartilage. Biophys.
J. 101, 916924
39 Keane, T.J. et al. (2012) Consequences of ineffective decellularization of
biologic scaffolds on the host response. Biomaterials 33, 17711781
40 Voytik-Harbin, S.L. et al. (1997) Identification of extractable growth
factors from small intestinal submucosa. J. Cell. Biochem. 67, 478491
41 Badylak, S.F. et al. (2008) Macrophage phenotype as a determinant of
biologic scaffold remodeling. Tissue Eng. Part A 14, 18351842
42 Cole, B.J. et al. (2011) Outcomes after a single-stage procedure for cellbased cartilage repair: a prospective clinical safety trial with 2-year
follow-up. Am. J. Sports Med. 39, 11701179
43 Chen, C.C. et al. (2012) Cartilage fragments from osteoarthritic knee
promote chondrogenesis of mesenchymal stem cells without exogenous
growth factor induction. J. Orthop. Res. 30, 393400
44 Ghanavi, P. et al. (2012) The rationale for using microscopic units of a
donor matrix in cartilage defect repair. Cell Tissue Res. 347, 643648
45 Peretti, G.M. et al. (2006) Tissue engineered cartilage integration to
live and devitalized cartilage: a study by reflectance mode confocal
microscopy and standard histology. Connect. Tissue Res. 47, 190199
46 Elder, B.D. et al. (2009) Extraction techniques for the decellularization
of tissue engineered articular cartilage constructs. Biomaterials 30,
37493756
47 Schwarz, S. et al. (2012) Decellularized cartilage matrix as a novel
biomatrix for cartilage tissue-engineering applications. Tissue Eng.
Part A 18, 21952209
48 Yang, Q. et al. (2008) A cartilage ECM-derived 3-D porous acellular
matrix scaffold for in vivo cartilage tissue engineering with PKH26labeled chondrogenic bone marrow-derived mesenchymal stem cells.
Biomaterials 29, 23782387
49 Yang, Z. et al. (2010) Fabrication and repair of cartilage defects with a
novel acellular cartilage matrix scaffold. Tissue Eng. Part C: Methods
16, 865876
50 Lu, H. et al. (2011) Cultured cell-derived extracellular matrix scaffolds
for tissue engineering. Biomaterials 32, 96589666
51 Lu, H. et al. (2011) Autologous extracellular matrix scaffolds for tissue
engineering. Biomaterials 32, 24892499
52 Gong, Y.Y. et al. (2011) A sandwich model for engineering cartilage
with acellular cartilage sheets and chondrocytes. Biomaterials 32,
22652273

176

Trends in Biotechnology March 2013, Vol. 31, No. 3

53 Xue, J.X. et al. (2012) Chondrogenic differentiation of bone marrowderived mesenchymal stem cells induced by acellular cartilage sheets.
Biomaterials 33, 58325840
54 Bank, R.A. et al. (1998) Ageing and zonal variation in posttranslational modification of collagen in normal human articular
cartilage. The age-related increase in non-enzymatic glycation
affects biomechanical properties of cartilage. Biochem. J. 330, 345351
55 Jia, S. et al. (2012) Oriented cartilage extracellular matrix-derived
scaffold for cartilage tissue engineering. J. Biosci. Bioeng. 113, 647653
56 Gerhardt, L.C. et al. (2012) Neocellularization and neovascularization
of nanosized bioactive glass-coated decellularized trabecular bone
scaffolds. J. Biomed. Mater. Res. A http://dx.doi.org/10.1002/jbm.a.
34373
57 Yang, Q. et al. (2011) Evaluation of an extracellular matrix-derived
acellular biphasic scaffold/cell construct in the repair of a large
articular high-load-bearing osteochondral defect in a canine model.
Chin. Med. J. (Engl.) 124, 39303938
58 Sadr, N. et al. (2012) Enhancing the biological performance of synthetic
polymeric materials by decoration with engineered, decellularized
extracellular matrix. Biomaterials 33, 50855093
59 Kang, Y. et al. (2011) Creation of bony microenvironment with CaP and
cell-derived ECM to enhance human bone-marrow MSC behavior and
delivery of BMP-2. Biomaterials 32, 61196130
60 Thibault, R.A. et al. (2010) Osteogenic differentiation of mesenchymal
stem cells on pregenerated extracellular matrix scaffolds in the
absence of osteogenic cell culture supplements. Tissue Eng. Part A
16, 431440
61 Klein, T.J. et al. (2009) Tissue engineering of articular cartilage with
biomimetic zones. Tissue Eng. Part B: Rev. 15, 143157
62 Malda, J. et al. (2012) Comparative study of depth-dependent
characteristics of equine and human osteochondral tissue from the
medial and lateral femoral condyles. Osteoarthritis Cartilage 20, 1147
1151
63 Klein, T.J. et al. (2009) Strategies for zonal cartilage repair using
hydrogels. Macromol. Biosci. 9, 10491058
64 Schuurman, W. et al. (2011) Bioprinting of hybrid tissue constructs
with tailorable mechanical properties. Biofabrication 3, 021001
65 Khalil, S. and Sun, W. (2009) Bioprinting endothelial cells with
alginate for 3D tissue constructs. J. Biomech. Eng. 131, 111002
66 Padalino, M.A. et al. (2012) Extracellular matrix graft for vascular
reconstructive surgery: evidence of autologous regeneration of the
neoaorta in a murine model. Eur. J. Cardiothorac. Surg. 42, e128
e135
67 Geoffrion, R. et al. (2011) Vaginal paravaginal repair with porcine
small intestine submucosa: midterm outcomes. Female Pelvic Med.
Reconstr. Surg. 17, 174179
68 Yuan, H. et al. (2010) Osteoinductive ceramics as a synthetic
alternative to autologous bone grafting. Proc. Natl. Acad. Sci. U.S.A.
107, 1361413619
69 Jin, C.Z. et al. (2007) In vivo cartilage tissue engineering using a cellderived extracellular matrix scaffold. Artif. Organs 31, 183192
70 Revell, C.M. and Athanasiou, K.A. (2009) Success rates and
immunologic responses of autogenic, allogenic, and xenogenic
treatments to repair articular cartilage defects. Tissue Eng. Part B:
Rev. 15, 115
71 Guilak, F. (2011) Biomechanical factors in osteoarthritis. Best Pract.
Res. Clin. Rheumatol. 25, 815823
72 Kheir, E. et al. (2011) Development and characterization of an acellular
porcine cartilage bone matrix for use in tissue engineering. J. Biomed.
Mater. Res. A 99, 283294
73 Badylak, S.F. et al. (2009) Extracellular matrix as a biological scaffold
material: structure and function. Acta Biomater. 5, 113