ARTICLE IN PRESS

Biomaterials 26 (2005) 59835990

www.elsevier.com/locate/biomaterials

Review

Chitosan: A versatile biopolymer for orthopaedic tissue-engineering

Alberto Di Martinoa,b, Michael Sittingerc, Makarand V. Risbuda,
a

Department of Orthopaedic Surgery and Graduate Program in Tissue Engineering and Regenerative Medicine,
Thomas Jefferson University, Philadelphia, PA 19017, USA
b
Department of Orthopaedic and Trauma Surgery Campus Bio-Medico University, Rome, Italy
c
Tissue Engineering Laboratory, ChariteUniversity Medicine, Berlin, Germany
Received 11 February 2005; accepted 7 March 2005
Available online 13 May 2005

Abstract
Current tissue engineering strategies are focused on the restoration of pathologically altered tissue architecture by transplantation
of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable attention has been given to
chitosan (CS)-based materials and their applications in the eld of orthopedic tissue engineering. Interesting characteristics that
render chitosan suitable for this purpose are a minimal foreign body reaction, an intrinsic antibacterial nature, and the ability to be
molded in various geometries and forms such as porous structures, suitable for cell ingrowth and osteoconduction. Due to its
favorable gelling properties chitosan can deliver morphogenic factors and pharmaceutical agents in a controlled fashion. Its cationic
nature allows it to complex DNA molecules making it an ideal candidate for gene delivery strategies. The ability to manipulate and
reconstitute tissue structure and function using this material has tremendous clinical implications and is likely to play a key role in
cell and gene therapies in coming years. In this paper we will review the current applications and future directions of CS in articular
cartilage, intervertebral disk and bone tissue engineering.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Chitosan; Tissue engineering; Orthopaedics; Cartilage; Intervertebral disc; Bone

Contents
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. CS in cartilage tissue engineering . . . . . . . .
1.2. CS in intervertebral disc tissue engineering .
1.3. CS in bone tissue engineering . . . . . . . . . .
2. Future directions and conclusions . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
In recent years, functional biomaterial research has
been directed towards the development of improved
Corresponding author. Tel.: +215 955 1063; fax: +215 955 9159.

E-mail address: makarand.risbud@jefferson.edu (M.V. Risbud).

0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2005.03.016

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5983
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scaffolds and new drug delivery systems. In this regard,

considerable attention has been given to chitosan (CS)based materials [1]. CS is a deacetylated derivative of
chitin, a high molecular weight, second most abundant
natural biopolymer commonly found in shells of
marine crustaceans and cell walls of fungi. CS is a
linear polysaccharide, composed of glucosamine and

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A. Di Martino et al. / Biomaterials 26 (2005) 59835990

N-acetyl glucosamine linked in a b(14) manner; the

glucosamine/N-acetyl glucosamine ratio being referred
as the degree of deacetylation. Depending on the source
and preparation procedure, its molecular weight may
range from 300 to over 1000 kD with a degree of
deacetylation from 30% to 95%. In its crystalline form,
CS is normally insoluble in aqueous solutions above pH
7; however, in dilute acids (pHo6.0), the protonated
free amino groups on glucosamine facilitate solubility of
the molecule [24].
The cationic nature of CS is primarily responsible for
electrostatic interactions with anionic glycosaminoglycans (GAG), proteoglycans and other negatively charged
molecules. This property is of great interest because a
large number of cytokines/growth factors are linked to
GAG (mostly with heparin and heparan sulphate), and a
scaffold incorporating a CSGAG complex may retain
and concentrate growth factors secreted by colonizing
cells [4]. Moreover, the presence of the N-acetylglucosamine moiety on CS also suggests related bioactivities. In
fact, CS oligosaccharides have a stimulatory effect on
macrophages, and both chitosan and chitin are chemoattractants for neutrophils both in vitro and in vivo.
Lysozyme is the primary enzyme responsible for in
vivo degradation of CS through hydrolysis of acetylated
residues. Although, other proteolytic enzymes have
shown low level of degradation activity on the molecule.
The degradation rate of CS is inversely related to the
degree of crystallinity, and thus on deacetylation. Highly
deacetylated forms may thus last several months in vivo;
eventual degradation products being CS oligosaccharides of variable length. Mao et al. [5] observed a direct
correlation between degree of deacetylation of the CS
and cell adhesion. Thus, careful selection of CS grade is
crucial while developing a scaffolds for tissue engineering. A number of researchers have examined the host
tissue response to CS-based implants. In general, these
materials evoke a minimal foreign body reaction, with
little or no brous encapsulation [6]. Formation of
normal granulation tissue associated with accelerated
angiogenesis, appears to be the typical course of the
healing response. This immunomodulatory effect has
been suggested to stimulate the integration of the
implanted material by the host [7].
One of the properties of CS is that it can be molded in
various forms [8]. CS possesses excellent ability to form
porous structures. Porous scaffolds are generated by
freezing and lyophilizing CS solutions [9] or by processes
such as an internal bubbling process (IBP) where
CaCO3 is added to chitosan solutions to generate
CSCaCO3 gels [10]. Ice removal by lyophilization
generates a porous material whose pore size and
orientation can be controlled by variation of the freezing
rate, the ice crystal size and the geometry of thermal
gradients during freezing. The obtained material can
then be molded as porous membranes, blocks, tubes and

beads. Pore size and orientation is shown to inuence

the mechanical properties of CS scaffolds. Tensile
testing of hydrated samples showed that porous
membranes have greatly reduced elastic moduli
(0.10.5 MPa) compared to non-porous membranes
(57 MPa). The extensibility (maximum strain) of
porous membranes varied from values similar to
nonporous CS (approximately 30%) to greater than
100% as a function of both pore size and orientation.
Porous membranes also exhibited a stressstrain curve
typical of composite materials with a low-modulus
region at low strains, together with a transition to a
23-fold higher modulus when high strains are applied.
Tensile strengths of the porous structures were reported
to be in the range of 3060 kPa [4,7]. In a recent study,
Geng et al. [11] have fabricated porous CS scaffolds
using a desktop rapid prototyping (RP) system, which
sequentially dispenses sodium hydroxide solution, and
CS dissolved in an acetic acid resulting in a gel-like CS
strand (Fig. 1).
Another interesting property of CS is its intrinsic
antibacterial activity. Studies have shown that CS can
reduce the infection rate of experimentally induced
osteomyelitis by Staphylococcus aureus in rabbits [12].
Its cationic amino group associates with anions on the
bacterial cell wall, suppressing biosynthesis; moreover,
CS disrupts the mass transport across the cell wall
accelerating the death of bacteria. Due to this antibacterial property it has been blended with other
polymers [13]. CS is also a preferred carrier for drug
delivery [12], thus combining its intrinsic antibacterial
activity with that of the bound antibiotic. When added
to HA and plaster of Paris to obtain a composite for
sustained vancomycin or fosfomycin release, the composite material was able to inhibit methicillin-resistant
S. aureus in vitro for as long as 3 months, a period
compatible with the treatment of most orthopedic
infections [14].
CS has been combined with a variety of delivery
materials such as alginate, hydroxyapatite, hyaluronic
acid, calcium phosphate, PMMA, poly-L-lactic acid
(PLLA), and growth factors for potential application in
orthopedics. Overall, CS offers a broad possibilities for
cell-based tissue engineering [8]. Possible matrix preparations for cell cultures include gels [15], sponges,
bers [16], or porous compositions of CS with ceramic
[17] or other polymeric materials such as collagen or
gelatin [18,19] to adjust cell seeding properties and
mechanical behavior of cell transplants for the intended
clinical application (Fig. 2).
1.1. CS in cartilage tissue engineering
Three-dimensional (3D) scaffolds are essential
for the development of engineered articular cartilage.
Ideal scaffolds are designed to be biocompatible,

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5985

Fig. 1. A desktop rapid prototyping (RP) system has been developed to fabricate scaffolds for tissue engineering applications (a). Scaffolds are built
by sequential dispensing of chitosan dissolved in acetic acid and sodium hydroxide solution. Neutralization of the acetic acid by the sodium
hydroxide results in a precipitate to form a gel-like chitosan strand. Chitosan scaffolds (b) fully hydrated (approximately 20
20
8.3 mm) and (c)
freeze-dried (approximately 14.8
15
5.8 mm). (Courtesy of Dr. Dietmar W. Hurmacher, National University of Singapore.)

Fig. 2. Illustration of selected examples of CS processing for use in tissue engineering: Cells may be encapsulated in gels or seeded in porous matrices
including sponge-like or brous structures. Combinations of CS with other biocompatible materials such as calcium phosphate or gelatin are applied
to modify biomechanical and cell-matrix-interaction properties. Different adaptations of CS may help to optimize cell and tissue differentiation and
tailor the transplant to different clinical cell delivery situations.

bioabsorbable and exhibit predictable porosity and

degradation rate. They provide a framework that
facilitates new tissue ingrowth; moreover, mechanical
characteristics are matched to those of the native tissue
increasing the chances that the reparative process will be
compatible with the hosts tissue physiology [3,20].
CS has been used as a scaffolding material in articular cartilage engineering [7,21], due to its structural
similarity with various GAGs found in articular

cartilage. This is of high importance because GAGs

are considered to play a pivotal role in modulating chondrocyte morphology, differentiation, and
function.
Alginate is another candidate biomaterial for cartilage
engineering but exhibits weak cell adherence. Iwasaki et
al. [22] reported an alginate-based CS hybrid polymer
bers which showed increased cell attachment and
proliferation in vitro compared to alginate. These hybrid

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A. Di Martino et al. / Biomaterials 26 (2005) 59835990

polymer bers showed increased tensile strength, implying a possible use in developing a 3D load bearing
scaffold for cartilage regeneration [22]. Chondrocytes
cultured on CS substrates in vitro maintained round
morphology and preserved synthesis of cell-specic
ECM molecules [19,21]. CS was used to improve
chondrocyte attachment to PLLA lms; the modied
substrate showed increased cell adhesion, proliferation
and biosynthetic activity [23]. CS was also conjugated
with hyaluronan to obtain a biomimetic matrix for
chondrocytes. Chondrocyte adhesion, proliferation, and
the synthesis of aggrecan and type II collagen were
signicantly higher on the hybrid ber than on CS [24].
Similarly, to increase the cellular adhesiveness of CS,
Hsu et al. have developed CSalginatehyaluronan
complexes with or without covalent attachment with
RGD containing protein. Cell-seeded scaffolds showed
neocartilage formation in vitro. When chondrocyte
seeded scaffolds were implanted into rabbit knee
cartilage defects, partial repair was observed after 1
month both in presence or absence of RGD indicating
potential of this composite material for cartilage
regeneration [25].
CS-based scaffolds can deliver growth factors in a
controlled fashion to promote the ingrowth and
biosynthetic ability of chondrocytes. Lee et al. [26]
reported porous collagen/CS/GAG scaffolds loaded
with TGF-b1. This scaffold exhibited controlled release
of TGF-b1 and promoted cartilage regeneration. Moreover, addition of CS to the collagen scaffold was seen to
improve mechanical properties [26] and stability of the
collagen network by inhibiting the action of collagenases
[27]. Kim et al. [28] used a porous freeze-dried CS
scaffold incorporating TGF-b1-containing microspheres, for the treatment of cartilage defects. TGF-b1
was released in a sustained fashion, and promoted
chondrocyte proliferation and matrix synthesis. MattioliBelmonte et al. [29] investigated the effect of BMP7
associated with N,N-dicarboxymethyl CS to induce
repair of femoral articular cartilage lesion in rabbit,
hypothesizing a synergism of their respective biological
effects [29]. Indeed, BMP-7 enhanced the in vivo
proliferation of chondrocytes, leading to partial healing
of the articular surface of the lesions. Whereas, CS was
linked to increased proliferation of brovascular tissue,
the initial process of ossication; and was crucial as a
BMP-7 carrier.
Hypothesizing that CS and some of its degraded
products could be involved in the synthesis of the
articular matrix components such as chondroitin,
chondroitin-sulfate, dermatane-sulfate, keratan-sulfate
and hyaluronic acid, Lu et al. studied the effect of intraarticular injection of CS on regeneration of articular
cartilage. An increase in epiphyseal cartilage in the tibial
and femoral joints was seen with an activation of
chondrocyte proliferation. Similarly, an intra-articular

brous tissue was observed for the 6 weeks of the

experiment, together with residual injected CS [30].
1.2. CS in intervertebral disc tissue engineering
Possible applications of CS in spine tissue engineering
encompass three different elds, namely spine fusion,
gene therapy and nucleus pulposus regeneration. When
a bone graft alternative is applied during spinal fusion
procedures, several local biomechanical factors are
considered, depending on the type and position of the
chosen graft. Anterior interbody grafts are exposed to
high compressive forces and need to possess loadbearing ability. On the contrary, a posterior applied
bone graft is placed along the tension side of the spinal
column in absence of local compressive stimuli, and thus
bone graft incorporation is less likely to be affected by
local biomechanical factors [31]. Materials such as PLA
or PLAPEG have been tested for spinal fusion, and are
considered good candidates [32] due to their plasticity,
stiffness, biodegradability and ability to support cells
and growth factor release such as BMPs in vivo [33]. A
possible application of CS could be in the development
of a composite graft material with a predictable
degradation rate and macroporous structure that would
allow linking of growth factors and support osteogenic
cells for spinal fusion.
Gene therapy represents a recent approach to facilitate vertebral body fusion, and is performed via the
transfer of the cDNA of osteoinductive genes to the
desired cells [34]. Nonviral vectors utilize physiochemical substrates to facilitate entry of the genetic material
into the cell. This method of delivery is advantageous
because the size of the genetic material that can be
introduced into the cell is not limited and therefore large
genes could be introduced with these vectors. However,
the efciency of transfection with nonviral vectors is
low, and the duration of expression of the protein
product tends to be short partly due to the episomal
nature of the transgene itself. The use of plasmids for
gene delivery is restricted because only a few cell types
will take up this naked form of DNA. This problem can
be partially overcome by combining the genetic material
with bioabsorbable scaffolds to form what has been
termed the gene-activated matrix (GAM). The scaffold serves as a substrate for cell adhesion and facilitate
the contact of naked DNA with the target cells. GAMs
have been successfully used to promote fracture healing
in rodents and canines [35,36]. Through its ability to
complex DNA [37,38] and its good bioabsorbability, CS
can be considered as an excellent candidate matrix for
nonviral gene therapy for spinal fusion applications
(Fig. 3).
Intervertebral discs possess different functional and
anatomic regions: the inner nucleus pulposus, a hydrated gelatinous tissue rich in proteoglycans, and the

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5987

Fig. 3. Schematic showing possible applications of chitosan (CS) in intervertebral disc tissue engineering. CS formulation may contain growth
factors for controlled release or bind DNA for gene delivery. Cells (nucleus pulposus or mesenchymal stem cells) would be mixed with a CS solution
(tailored to undergo thermo-gelling) that has been previously complexed with plasmid DNA (growth factor genes) or contains growth factors. This
solution would be injected into the disc space using a ne bore needle. Once inside the body, the chitosan solution mixed with cells would gel at 37 1C
and thus be retained in the disc space. Over a period of several months the injected cells would restore the biological function of the disc by deposition
of a proteoglycan-rich extracellular matrix. In another approach CS would be mixed with collagen and made into a brous scaffold. Cells would be
seeded onto brous CS scaffold and allowed to mature in vitro. After desired level of maturation these cell seeded scaffolds would be transplated in
vivo to repair annulus brosus.

outer annulus brosus made of concentric collagenic

lamellae. Loss of proteoglycans and water content in the
inner nucleus pulposus initiates degenerative spinal
disease. Biologic disc regeneration is considered as a
promising approach to restore biological integrity and
function of a pathologically altered disc [39,40]. Several
strategies can be employed for different stages of disc
degeneration that utilize direct drug/growth factor
delivery to the disc, as well as gene transfer to disc cells
cultured in vitro [41]. CS gel has been used as a scaffold
for nucleus pulposus cells, and GFs were used to
modulate matrix synthesis in an attempt to produce a
tissue with a similar molecular composition to native
nucleus pulposus tissue. However, in one published
study, the in vitro formation of nucleus pulposus tissue
did not appear to be facilitated by using of a articial
3D scaffold, although nucleus pulposus cells implanted
in CS gel synthesized aggrecan, small proteoglycans as
well as Type I and II collagen, retention of matrix
molecules within the scaffolds was low and synthetic
levels did not exceed 35% of the native nucleus pulposus
tissue. However, CS gels were superior to other
scaffolds, such as collagen and hyaluronan, with
increased matrix synthesis and stable cell phenotype,
suggesting possible applications after improvement of
this technique in the future [42]. Another idea is to
complex cationic CS to DNA forming CS nanoparticles
that could be transfected into nucleus pulposus cells to
promote cell differentiation and matrix synthesis in both
in vitro and in vivo studies (Fig. 3).
1.3. CS in bone tissue engineering
CS has been extensively used in bone tissue engineering since it was shown to promote growth and mineral

rich matrix deposition by osteoblasts in culture [43].

Also CS is biocompatible (minimizes additional local
inammation), biodegradable, and can be molded into
porous structures (allows osteoconduction) [44]. Several
studies have focused on the use of CScalcium
phosphate (CP) composites for this purpose [45,46]. A
3D macroporous CP bioceramic embedded with porous
CS sponges is developed by Zang et al. [47]. In this
scaffold, a nested CS sponge enhanced the mechanical
strength of the ceramic phase via matrix reinforcement
and preserved the osteoblast phenotype [48]. Similarly,
gentamycin-conjugated macroporous CS scaffolds reinforced with beta-tri calcium phosphate bTCP and
CP have been developed for bone engineering [46].
Macroporous CS scaffolds incorporating hydroxyapatite (HA) or CP glass with an interconnected porosity of
approximately 100 mm have been synthesized [48].
Overall composites of CSCP appear to have a
promising clinical application in the future.
Ge et al. [49] reported a HAchitin material that was
osteoinductive and exhibited rapid degradation and
neovascularization in vivo during a 3-month period.
Kawakami et al. studied the in vivo effect of a CSHA
paste when applied on the surface of the tibia after
periosteum removal. Formation of new bone was
observed after 1 week and continued during a 20-week
follow-up indicating suitability of this paste for further
clinical studies as a bone lling material [50]. The issue
of mechanical resistance of CS-based composites was
addressed by Hu et al. [8], who reported a CSHA
multilayer nanocomposite with high strength and
bending modulus rendering the material suitable for
possible application for internal xation of long bone
fractures. A macroporous CS-gel/b-TCP composite
scaffold for bone tissue engineering using freeze-drying

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process is developed [51]. This study investigated the

effects of concentration of composite suspension and the
freezing temperature on the ability to resist compression
by the scaffold [52].
CS is used as an adjuvant with bone cements to
increase their injectability while keeping the chemicophysical properties suitable for surgical use (e.g. setting
time and mechanical properties). The rationale of using
CS for this purpose is based on the property that
chitosan solutions gel in response to a pH change from
slightly acidic to physiological; in fact, the CSCP
composites address the need to develop bone llers that
set in response to physiological conditions, but not while
mixing the components in vitro. Likewise, when CS is
added to calcium phosphate cements (CPC), octocalcium phosphate is obtained; a material that is shown to
improve injectability and strength [52]. Many of these
CS gel composites are proposed mainly for nonloadbearing bony defects [53].
Wang et al. added phosphorylated CS to CPC and
used it to ll bony defects in the radius and tibia in vivo.
The composite material was biocompatible, osteoinductive, bioresorbable and remodeled through a creeping
substitution process [54]. Xu et al. studied the feasibility
of creating macropores in CPC using chitosan and/or
absorbable mesh. A synergistic effect of adding CS and
bioabsorbable mesh to CPC was seen. This injectable,
bioabsorbable composite material possessed interconnected macropores (osteoconductive) and provided
strength to the implant during tissue regeneration [55].
The intramolecular hydrogen bonds of CS provide
interacting macromolecules with a good resistance to
heat. To exploit this property, composites of CS with
poly methyl-methacrylate (PMMA) were developed that
exhibited lower exothermic curing temperatures [56].
This composite material possessed a high interconnectedporosity with a pore size suitable for osteoconduction
and better anchorage to the surrounding bone. It was
observed that the pore size of this composite material
increased with time due to biodegradation of the CS [56].
The ability of CS to bind growth factors is suitable for
bone tissue engineering [5759]. Due to its cationic
nature and predictable degradation rate, CS-based
materials bind growth factors and release them in a
controlled fashion. The cationic nature of CS can be
further enhanced by the introduction of a covalently
linked imidazole group. The biochemical signicance of
imidazole addition is that this group inhibits thromboxane synthetase, acts as antioxidant and facilitates
intracellular buffering for the tissue healing process.
This imidazole-linked CS material promoted mineralization, induced bone formation and lled critical size
bone defects with the apposition of trabecular bone [60].
When applied in rat calvarial defect, CS sponges
incorporating PDGF induced new bone formation
[57,61]. Kim et al. studied the effect of CS on early

bony consolidation to evaluate osteogenesis after a

(mandibular) distraction in a dog model. This study
showed the ability of CS to promote osteogeneic
progenitor cell recruitment and attachment, facilitating
bone formation [62]. Effectiveness of CS combined with
CP to obtain early bony consolidation in distraction
osteogenesis in a dog model was also reported [63].
CS has been used to modify the surface properties of
prosthetic materials for enhancing the attachment of
osteoblasts [58]. Titanium (Ti) surface coated with CS
via silaneglutaraldehyde chemistry exhibited increased
osteoblast attachment and proliferation [64]. The bond
strength of CS coating with Ti was in the range of
1.51.8 MPa and it degraded slowly over 8 weeks. These
results supported the hypothesis that CS coatings could
promote osseointegration of Ti implant devices commonly used for orthopedic and craniofacial implants,
although CS bond strength was found to be less
compared to CP coatings [64,65]. Wang et al. used CS
to increase the biocompatibility of electrolytically
deposited apatite coatings on Ti alloys. A hybrid
calcium phosphate/CS coating was developed through
electrodeposition. This coating exhibited an increased
dissolution rate in both acidic and neutral simulated
physiologic solution. Moreover, the calcium phosphate/
CS coating showed an improved bone marrow stromal
cell attachment [66].

2. Future directions and conclusions

At present, CS is one of the most promising
biopolymers for tissue engineering and possible orthopedic applications. In particular, the possibility to
generate structures with predictable pore sizes and
degradation rates make CS a suitable material as a bone
graft alternative in orthopedic procedures. However,
efforts to improve the mechanical properties of CS-based
composite biomaterials are essential for this type of
application. Of great importance is the ability of CS to
bind anionic molecules such as GFs, GAGs and DNA.
In fact, the combination of good biocompatibility,
intrinsic antibacterial activity, ability to bind to growth
factors and to be processed in a variety of different
shapes makes CS a promising candidate scaffold
material for cartilage, intervertebral disc and bone tissue
engineering in clinical practice. Moreover, the ability to
link CS to DNA molecules renders this material a good
potential as a substrate for gene activated matrices in
gene therapy applications in orthopedics.

Acknowledgements
Authors wish to thank Dr. Dietmar Hutmacher,
National University of Singapore for generously

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A. Di Martino et al. / Biomaterials 26 (2005) 59835990

providing pictures of chitosan scaffolds used in Fig. 1

and sharing his unpublished results. We thank Mrs
Johana Golla for her help with illustrations. This work
was supported by grant 1RO1-AR050087-01A2.

[19]

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