THE ROLE OF THREE-DIMENSIONAL SCAFFOLDS IN THE
REGENERATION OF JOINT CARTILAGE
J. L. Gómez Ribelles, M. Monleón Pradas
Centro de Biomateriales e Ingeniería Tisular, Universidad Politécnica de Valencia, 46022, Valencia, Spain
Regenerative Medicine Unit, Centro de Investigación Príncipe Felipe, Autopista del Saler 16, 46013 Valencia, Spain
CIBER en Bioingeniería, Biomateriales y Nanomedicina, Valencia, Spain
R. García Gómez
Regenerative Medicine Unit, Centro de Investigación Príncipe Felipe, Autopista del Saler 16, 46013 Valencia, Spain
F. Forriol, M. Sancho-Tello, C. Carda
Departamento de Patología, Facultad de Medicina y Odontología, Universidad de Valencia
Avda. Blasco Ibañez, 17, 46010 Valencia, Spain
Keywords: Three-dimensional scaffolds, Chondrocytes, Joint cartilage, Rabbit model.
Abstract: A variety of polymer scaffolds with pore architecture consisting of interconnected spherical pores with the
same architecture but varying mechanical properties (in particular elastic modulus), water sorption capacity,
pore surface characteristics (surface tension, presence of hydrophilic groups or electric charges) was
prepared and implanted in a 3mm diameter full thickness defects in the knee joint cartilage of rabbits in
order to show the influence of the scaffold properties on the histological characteristics of the regenerated
tissue.
1 INTRODUCTION
The poor self-regeneration of articular cartilage has
stimulated continued efforts to develop tissue
engineering techniques (Patrick 1998, Shoichet
1998). The clinical practice focused on either
transplantation or implantation of autologous
chondrocytes, a tissue engineering technique started
in 1987 and since then employed in the treatment of
around 10,000 patients all over the world (Brittberg
2003, Smith 2005, Marlovits 2006). In this
technique tissue regeneration is expected to be
originated in adult chondrocytes transplanted to the
site of the defect. Previously to the transplant to the
site of the defect chondrocytes must be expanded “in
vitro” to the cell numbers necessary to produce
sufficient extracellular matrix, since no further
mitosis is expected “in vivo”. “In vitro” culture has
been shown to de-differentiate cells, that loose the
phenotype of hyaline cartilage chondrocytes and
start expressing proteins such as type I collagen,
characteristic of other connective tissues. On the
other hand, microfracture of the subchondral bone
allows the recruitment of mesenchymal stem cells
from subchondral bone marrow whose
differentiation to chondrocytes is another way of
possible regeneration (Stradman 1999).
Whatever the cell source for regeneration, the
correct differentiation “in vivo” towards the hyaline
cartilage phenotype is addressed not only by the
presence of growth factors but also by the
biomechanical environment that the cells sense in
the joint cartilage (Nugent-Derfus 2007). Thus, the
success of a regenerative strategy depends to a high
extent on the way in which the medium that
surrounds the transplanted cells is able to transmit to
them the same compression forces to which cells are
subjected in a healthy joint cartilage. This is the role
of the scaffold in cartilage tissue engineering; cells
are seeded into a macroporous polymeric sponge
which is placed in the site of the cartilage defect.
The morphological and mechanical properties of the
229
L. Gómez Ribelles J., Monleón Pradas M., García Gómez R., Forriol F., Sancho-Tello M. and Carda C. (2010).
THE ROLE OF THREE-DIMENSIONAL SCAFFOLDS IN THE REGENERATION OF JOINT CARTILAGE .
In Proceedings of the Third International Conference on Biomedical Electronics and Devices, pages 229-234
DOI: 10.5220/0002765102290234
Copyright
c
SciTePress
material in addition to the interaction between cells
and pore surfaces, define to a large extent the
biomechanical environment that the cells experience
after being transplanted, and also the characteristics
of the tissue they are able to produce and organize
(Martínez-Díaz, 2009).
In this work we show a procedure to obtain a
very versatile series of materials with varying
mechanical and other physical properties, and show
to what extent the mechanical properties of the
scaffold determine the histological characteristics of
the regenerated tissue in a rabbit knee model. This
study is performed using biostable polymers; thus,
there is no influence of degradation products in the
non-vascularised cartilage tissue, nor of the loss of
mechanical properties of the scaffold over time.
2 MATERIALS AND METHODS
2.1 Scaffold Preparation
In this work biostable polymeric scaffolds were
prepared using a template technique. Poly(methyl
methacrylate) microspheres with 90 microns
diameter in average (PMMA Colacryl DP 300,
Leucite International) were used as porogen. The
microspheres were introduced into a mold consisting
of two glass plates separated by a rubber ring. The
mold was placed into a hot press at 170 ºC to allow
PMMA microspheres to soften, and then compressed
to form a template, approximately 2 mm thick, the
interconnection points between porogen
microspheres creates the pore throats in the scaffold,
thus, pore interconnectivity is controlled by the
pressure applied when producing the template.
Monomer mixtures of ethyl acrylate, EA
(Aldrich, 99 %), and hydroxyethyl acrylate, HEA
(Aldrich, 96 %) or methacrylic acid, MAAc
(Aldrich, 99 %), were prepared adding different
amounts of triethyleneglycol dimethacrylate,
TrEGDMA (Aldrich 98% pure) as crosslinking
agent and 1 % of 98 % pure benzoin (Scharlau) as
photoinitiator. The templates were immersed in the
monomeric solutions and polymerized under
ultraviolet light at room temperature. After
polymerization, the template was dissolved with
acetone for approximately 48 h by means of a
Soxhlet extractor. Then, scaffolds were immersed in
a glass with a large excess of acetone and the solvent
was changed slowly by water to allow the uniform
contraction of the scaffolds. Replicas of the
scaffolds were cut in 3 mm diameter and washed
with water/ethanol for 6 hours in Soxhlet extractor.
Finally, scaffolds were dried in vacuo for 24 h at
room temperature and then another 24 h at 50 ºC.
The scaffold samples were sterilized with
gamma radiation, at a dose of 25 kGy before
implant.
The morphology of the scaffold was examined in
a cryogenic scanning electron microscope
(cryoSEM) (JEOL JSM 5410) equipped with a
cryounit (Oxford CT 1500). The samples were
mounted on copper stubs and gold coated using a
sputter coater. The microscope was used with an
acceleration tension of 15 kV.
2.2 Animals
Adult male New Zealand rabbits, weighing 1.5-2.0
kg were obtained from Granjas San Bernardo S.L.
(Tulebra, Spain) and kept under conventional
housing conditions. Quarantine lasted 7 days.
Animals were housed with appropriate bedding and
provided free access to drinking water and food.
Rabbits were kept in standard single cages under
controlled temperature and light conditions.
The study protocol was approved by the Ethics
Committee of the Universidad de Valencia
according to 86/609/EEC law and 214/1997 and
decree 164/1998 of the Generalitat Valenciana.
2.3 Scaffold Implant
Rabbits were preanaesthetized by subcutaneous
injection of 15 mg/kg Ketamine (Ketolar®, Pfizer
laboratories) and intramuscular injection of 0.1
mg/kg Medetomina (Domtor®, Pfizer laboratories),
and prepared before surgery (washed, shaved, etc.).
Then, general anaesthesia was induced by 4%
isofluorane using a specially designed mask and
maintained by administration of 1.5% isofluorane
with O
2
(2 l/min). The surgical site was sterilized
using iodine solution and rabbit non-sterile parts
were covered with sterile drapes. All surgeons wore
sterile coats and gloves, and all instruments were
sterilized and kept sterile during the operation.
Scaffolds were moistened with phosphate buffered
saline (PBS), and vacuum was applied to assure
liquid penetration into the porous cavities before
implanting. Two rabbits were used for each group.
An arthrotomy at the knee joint was performed
through a medial longitudinal parapatellar incision.
The medial capsule was incised and the patella
laterally dislocated. A 3-mm steel trephine was used
to create a chondral defect, 3 mm in diameter and 1
mm in depth, in the central articulating surface of
the trochlear groove. The defect was cleaned and
BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices
230
rinsed with sterile saline, and scaffolds were laid
into the defect and held in place by repositioning the
patella within the trochlear groove. Arthrotomy and
skin were sutured with continuous stitches of 4/0
Coated Vicryl® (Johnson-Johnson Intl) After
removal of the conformed anaesthesia mask, all
rabbits were returned to their cages and allowed free
cage activity. Postoperative analgesia consisted of
intramuscular injection of 3 mg/kg dexketoprofen
(Enantyum®, Menarini laboratories) on the surgery
day and the same dose every 24 h for 3 days. At the
end of surgery, 3 mg/kg intramuscular injection of
Gentamicine (Genta-Gobens®, Normon
laboratories) was administered as antibiotic
prophylaxis.
2.4 Animal Sacrifice and Tissue
Retrieval
Three months after scaffold implant, rabbits were
sacrificed with a lethal intravenous injection of
anesthetic overdose in the auricular vein (500 mg/iv
Tiopental; Tiobarbital®, Braun laboratories). A cut
of 10x10x5 mm was made in the articulations with
implanted scaffolds, and special care was taken in
order to keep the repaired defect at the centre of the
sample in order to asses cartilage repair by
histological and immunohistological analyses.
2.5 Histological Studies
Morphology was studied following standard
histological procedures. Briefly, rabbit articulation
specimens were rinsed with PBS and fixed with 4%
formaldehyde at room temperature for 5 days. Then,
samples were rinsed with PBS and decalcified with
Osteosoft decalcifier solution (Merck) during 5
weeks at room temperature. Finally, the specimens
were embedded in paraffin, and 5 μm thick serial
sections were obtained in order to localize the
middle part of the scaffolds (with a diameter of
approximately 3 mm), that were stained with
Haematoxylin-Eosin and Masson’s trichrome.
The ability of chondrocytes to synthesize
glycosaminoglycan (GAG) within the porous
scaffold was monitored by Alcian blue staining (pH
2.5), counterstained using Harris haematoxylin.
Stained sections were analyzed under Leica optical
microscope (Leica DM 4000B) and pictures were
taken using a camera (Leica DFC 420).
2.6 Immunohistochemistry
Standard immunohistochemistry techniques were
performed to detect the collagen type I and type II,
osteocalcin and Ki-67 expression. Anti Ki-67
antibody (MIB-1, DakoCytomation, Glostrup,
Denmark) at 1:50 dilution, incubated at RT during
60 min, was employed to detect proliferating cells.
Section of human neuroblastoma was used as a
positive control. A mouse anti-Collagen I antibody
(Sigma, Madrid, Spain) at 1:200 dilution and mouse
anti-Collagen II antibody (Calbiochem, Madrid,
Spain) at 1:200 dilution, incubated at -4ºC overnight,
were used to study the synthesis of Collagen type I
and type II. Rabbit cartilage and bone areas
surrounding the scaffold were used as positive
control. Osteocalcin detection was performed using
a mouse anti-osteocalcin antibody (R&D Systems,
Abingdon, UK) at 1:100 dilution and incubated at
-4ºC overnight. Rabbit subcondral bone was used as
positive control. As a negative control for each
specific staining, the preimmune serum was
substituted for the primary antibody.
Sections were deparaffinized and rehydrated
through graded ethanol, rinsed in distilled water and
treated with 0.3% H
2
O
2
and 10% normal horse
serum to block endogenous peroxidase and
nonspecific binding, respectively. Antigen retrieval
for collagen type I, II and Ki-67 was performed by
pressure cooker boiling for 3 minutes in 10 mmol/L
of citrate buffer (pH 6.0). For osteocalcin detection,
slides were permeabilized using 0.1% Triton X-100
for 5 min and antigen retrieval was performed using
0.5% trypsin for 30 min at 37°C in a humidified
chamber. Envision amplification system Dako
(Cytomation Envision+System labeled polymer-
HRP anti-mouse) was used, followed by revelation
with 3,3'-diaminobenzidine (DAB, Dako) as
chromogen according to the manufacturer's
instructions. Sections were finally counterstained
with Mayer's hematoxylin (Sigma).
3 RESULTS AND DISCUSSION
3.1 Scaffold Morphology and
Properties
The pore architecture of the scaffold is determined
by the porogen template. As shown in Figure 1, the
pores have spherical form with a diameter that is
related to the size of the porogen microspheres, but
depends on the expansion or contraction of the
material during the template extraction and drying as
THE ROLE OF THREE-DIMENSIONAL SCAFFOLDS IN THE REGENERATION OF JOINT CARTILAGE
231
Figure 1: Scanning electron microscopy pictures of cross-
sections of PEA scaffolds cross-linked with 5% (a), 10%
(b) or 20 wt % (c) of TrEGDMA (see text).
well. We will further explain this point below. In
this way a broad interval of pore sizes is available,
since porogen microspheres made of different
materials (polymeric particles, sugar microspheres,
gelatine, paraffin and others) can be produced with
diameters ranging from the micrometer to the
millimetre scale (Brígido Diego 2005, Chen 2004,
Ma 2003). The pore interconnection is determined
by the compression of the porogen microspheres at
high temperatures followed by cooling under
pressure. Larger or smaller pore throats can thus be
obtained. Figures 1 to 3 show three polymeric
scaffolds made of coplymer networks in which one
of the comonomers is ethyl acrylate and the other
one is triethyleneglycol dimethacrylate, TrEGDMA,
a tetrafunctional cross-linker.
The effective extraction of the porogen and any
low molecular weight substance remaining in the
material after the polymer synthesis is a requirement
of any scaffold aimed as an implant or cell culture
support. In our materials, the fact that the polymer is
synthesized in the form of a copolymer network
allows swelling it in a suitable solvent of the
porogen, thus facilitating template extraction.
Nevertheless, simple drying of the scaffold after this
operation usually produces the collapse of the pore
structure (Brígido Diego 2007). In the example of
the scaffolds shown in Figure 1, when the PEA is
polymerized inside the template with low cross-
linking density and then immersed in acetone, it
absorbs a large amount of solvent, swells
significantly and the glass transition of the polymer
decreases to very low temperatures. Thus, when the
template completely disappears, the PEA network is
swollen in acetone and consequently very soft, and
in addition the pores are filled with acetone. If
acetone is evaporated in those conditions, the
scaffold collapses and the structure of the dry
polymer becomes non porous. Slow exchange of the
good solvent, acetone, by a bad solvent, water in this
case, allows the controlled contraction of the sponge
avoiding pore collapse. The properties of the
material from which the scaffold is made: cross-
linking density, solubility and glass transition
temperature highly affect the tendency of the
scaffold to close the pores (Brígido Diego 2005).
Figure 1c shows how a highly cross-linked polymer
network can be produced with an extremely porous
structure (up to 95% volume fraction of pores) while
maintaining mechanical consistency.
Physical and mechanical properties of the
scaffold can be tailored by combining different
monomers. For the animal model four series of
materials were prepared: poly(ethyl acrylate), PEA,
copolymers of ethyl acrylate and hydroxyethyl
acrylate, P(EA-co-HEA), containing 10 or 50% by
weight of HEA or copolymers of ethyl acrylate and
methacrylic acid, P(EA-co-MAAc) containing 10%
MAAc. Triethyleneglycol dimethacrylate at a ratio
of 5 wt% with respect to the rest of monomers was
used as cross-linking agent. PEA scaffold is
hydrophobous, P(EA-co-MAAc) 90/10 and P(EA-
a
)
b
)
c
)
BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices
232
co-HEA) 90/10 are slightly hydrophilic, and so the
bulk polymers are able to absorb 2.3 and 3.3% of
water (measured on dry basis) when immersed in
liquid water to equilibrium, and finally P(EA-co-
HEA) 50/50 is a hydrogel whose equilibrium water
content is 18.1% of water (Campillo-Fernandez
2007).
3.2 Animal Model
After implantation, scaffolds completely filled the 3
mm diameter chondral defect, with their external
surface aligned with the articular surface of the
trochlear grove. After sacrifice, macroscopic
observation at the implant zone revealed good
integration into the osteoarticular complex in all
animals.
As an example of tissue remodelling three
months after implantation, Figure 2 shows the cross
section of the implant site stained with hematoxylin-
eosin, where the material of the scaffold appears as
the white regions in these pictures. Figure 2a shows
that a layer of tissue has been formed on the surface
of the scaffold. This tissue contains cells isolated in
lacunae and ordered in columns, and histology and
inmunostaining show that it contains type II collagen
and glycosaminoglycans. The thickness of this layer
was very variable in the different animals. In some
cases a thin layer of hyaline cartilage was found (as
that shown in Figure 2a), while in other cases the
scaffold is much displaced in depth towards the
trabecular bone, penetrating the layer of subchondral
bone. On another hand, the stiffer scaffolds
maintained their original shape with the pore
structure filled with cartilaginous tissue showing
expression of type II collagen and
glycosaminoglycans, and with cells isolated in
lacunae, as shown in the detail of Figure 2b at high
magnification. Some cells are close to the pore
walls, but most of them appear in the centre of the
cavities. Interestingly enough, some of the cells were
Ki67 positive, indicating that they had proliferating
capacity. In the region of the scaffold touching the
bone tissue some capillaries were apparent and
positive staining for bone markers (osteocalcin and
type I collagen) was found. On the contrary, P(EA-
coHEA) 50/50 hydrogels suffered a high
deformation which closed the pore structure, in
which nearly no cells were found.
Since no cells were implanted with the scaffold,
all tissue regeneration must be assumed to come
from the messenchymal cells arriving to the site of
the implant, coming with blood flowing during
operation or from synovial fluids. Those cells placed
on top of the scaffold are subjected to compression
forces that lead to differentiation towards the hyaline
cartilage phenotype.
Figure 2: PEA scaffold integration in the surrounding
tissue three months after implant, stained with
haematoxylin-eosin.
These cells give rise to a layer of well
differentiated hyaline cartilage on top of the
scaffold, with chondrocytes isolated in lacunae
ordered in columns perpendicular to the joint
surface. In this way, the external surface of the
regenerated tissue acquires the characteristics that
resemble the original one. The growth of this tissue
layer moves the scaffold towards subchondral bone,
in a greater or lesser extent depending on the animal
a
b
THE ROLE OF THREE-DIMENSIONAL SCAFFOLDS IN THE REGENERATION OF JOINT CARTILAGE
233
and on the stiffness of the material. Other cells
invade the pore structure of the scaffold and
differentiate to chondrocytes as well, producing
sufficient extracellular matrix to fill the pores.
4 CONCLUSIONS
A broad series of biostable polymer networks can be
polymerized in the free spaces of a template made of
sintered microspheres. The dissolution of the
template allows producing the macroporous
scaffold, whose pore structure depends on that of the
template but also on the drying protocol and on the
properties of the material, in particular its glass
transition temperature and solubility in the solvents
employed to extract the template. When scaffolds
made with this procedure were implanted in a knee
joint model, without any pre-seeded cells, good
integration of the scaffold in the host cartilage tissue
was observed in all animals after three months, with
an even surface in the zone of the defect. Tissue
regeneration comes from the differentiation of
mesenchymal cells arriving to the site of the implant
and invading the scaffold. These cells are able to
produce neotissue with the characteristics of hyaline
cartilage. This work shows the important role of the
scaffold as the support that allows mechanical
stresses to be transferred to cells during cartilage
regeneration “in vivo”, and the possibility of
regenerating joint cartilage without the artificial
supply of autologous chondrocytes or pluripotential
cells to the site of the implant.
ACKNOWLEDGEMENTS
The support of the Spanish Ministry of Science
through projects No. MAT2007-66759-C03-01 and
MAT2007-66759-C03-03, including the FEDER
financial support, with complementary funding of
Generalitat Valenciana with project
ACOMP/2009/112 and Universidad Politécnica de
Valencia with 2911-2008 project is acknowledged.
JLGR and MMP acknowledge funding in the Centro
de Investigación Principe Felipe in the field of
Regenerative Medicine through the collaboration
agreement from the Conselleria de Sanidad
(Generalitat Valenciana), and the Instituto de Salud
Carlos III (Ministry of Science and Innovation).
REFERENCES
Brígido Diego R., Pérez Olmedilla M., Serrano Aroca A.,
Gómez Ribelles J.L., Monleón Pradas M., Gallego
Ferrer G., Salmerón Sánchez M. Acrylic scaffolds with
interconnected spherical pores and controlled
hydrophilicity for tissue engineering. J.Mat. Sci. Mat.
Medicine (2005) 16, 693-698
Brígido Diego R., Gómez Ribelles J.L., Salmerón Sánchez
M. On the pore collapse during the fabrication
process of rubber-like polymer scaffolds. J. Appl.
Polym. Sci. (2007) 104, 1475-1481.
Brittberg M., Peterson L., Sjögren-Jansson E,, Tallheden
T., Lindahl A. Articular cartilage engineering with
autologous chondrocyte transplantation. A review of
recent developments.The Journal of bone and joint
surgery. American volume (2003) 85-A,109-115
Chen V.J., Ma P.X. Nano-fibrous poly(L-lactic acid)
scaffolds with interconnected spherical macropores.
Biomaterials (2004) 25, 2065-2073.
Ma Z., Gao C., Gong Y., Shen J. Paraffin spheres as
porogen to fabricate poly(L-lactic acid) scaffolds with
improved cytocompatibility for cartilage tissue
engineering. Journal of biomedical materials research.
Part B, Applied biomaterials (2003) 67, 610-617.
Marlovits S., Zeller P., Singer P., Resinger C., Vilmos
Vécsei V. Cartilage repair: Generations of autologous
chondrocyte transplantation. European Journal of
Radiology (2006) 57, 24–31
Martinez-Diaz S., Garcia-Giralt N., Lebourg M., Gomez
Tejedor J.A., Vila G, Caceres E., Benito P, Monleon
Pradas M., Nogues X, Gomez Ribelles J.L., Monllau
J.C. In vivo evaluation of three-dimensional PCL
scaffolds for cartilage repair in rabbits. The American
Journal of Sports Medicine, In press
Nugent-Derfus G. E., Takara T., O’Neilly J. K., Cahill S.
B., Gortz S., Pong T., Inoue H., Aneloski N. M., Wang
W. W., Vega K. I., Klein T. J., Hsieh-Bonassera N. D.,
Bae W. C., Burke J. D., Bugbee W. D. Sah R. L.,
Continuous passive motion applied to whole joints
stimulates chondrocyte biosynthesis of
PRG4,OsteoArthritis and Cartilage (2007) 15, 566-
574. Patrick Jr C.W., Mikos A.G., McIntire L.V. (ed)
Frontiers in Tissue Engineering Pergamon Press
(1998).
Shoichet M.S., Hubbell J.A. (ed) Polymers in Tissue
Engineering. VSP, Utrech (1998)
Smith G.D., Knutsen G., Richardson J.B., A clinical
review of cartilagerepair techniques J. Bone and Joint
Surgery (2005) 87, 445-449.
Steadman J.R., Rodkey W.G., Briggs K.K., Rodrigo J.J.
The microfracture technic in the management of
complete cartilage defects in the knee joint. Orthopade
(1999) 28, 26-32.
BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices
234