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

 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 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

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-

)

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

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