ARTICLE
Centre for Biomaterials Research, University of Maastricht, PO Box 616, 6200 MD
Maastricht, The Netherlands
b
Department of Biomedical Engineering, Eindhoven University of Technology,
PO Box 513, 5600 MB Eindhoven, The Netherlands
c
Department of Mechanical Engineering, Eindhoven University of Technology,
PO Box 513, 5600 MB Eindhoven, The Netherlands
d
Department of Trauma Surgery, Uniklinik Aachen, Pauwelsstrasse 30 D-52074 Aachen,
Germany
Journal of
a
Materials
Chemistry
Catharina S. J. van Hooy-Corstjens,*a Yvette B. J. Aldenhoff,a
Menno L. W. Knetsch,a,b Leon E. Govaert,c Ece Arin,d Hans Erlid and Leo H. Koolea,b
www.rsc.org/materials
Radiopaque polymeric spinal cages: a prototype study{
Received 13th May 2004, Accepted 4th June 2004
First published as an Advance Article on the web 26th August 2004
Back pain, originating from degeneration of intervertebral discs, is often alleviated by the insertion of one or
more interbody fusion cages. The function of the cage is to restore the height between two adjacent vertebrae
and to mediate osseous fusion. Most commercial cages consist of titanium or a titanium alloy, while polymeric
cages, mostly consisting of polyether-etherketone (PEEK), are also in use. Titanium is known for its excellent
biocompatibility. Titanium cages can be located easily with imaging techniques based on X-ray absorption (e.g.
CT scans). However, they introduce artefacts in magnetic resonance (MR images). PEEK cages, on the other
hand, do not show up in CT images. For this reason, small metallic markers are usually incorporated. The
markers reveal the position of the cage, albeit indirectly. PEEK cages are clearly and integrally seen on MR
images, as they are essential free of water. There are no artefacts or disturbances; this feature, as well as its
strength, makes PEEK particularly attractive for the construction of cages. Here, we introduce new allpolymeric cages on the basis of an iodine-containing methacrylic copolymer (I-copolymer). This material has
been prepared from methylmethacrylate and 2-[4-iodobenzoyl]-oxo-ethylmethacrylate. Copolymerisation of
both monomers results in a high molecular weight material. Cytocompatibility experiments reveal that the
material contains no toxic leachables and that cells can well adhere to and proliferate on the I-copolymer.
Compression experiments at physiologically relevant strains disclose mechanical characteristics comparable to
PEEK. The advantage of cages prepared from this I-copolymer over commercially available cages is that the
present cage contains no metallic components, implying that it is compatible with MR imaging, and the
presence of the iodine atoms ensures X-ray visibility.
DOI: 10.1039/b407228f
1. Introduction
3008
The spinal column consists of 24 vertebrae that are separated
by intervertebral discs (IVD). Problems with one or more
intervertebral discs, being a frequent cause of back pain, are
often the result of degenerative conditions or trauma.1,2 Fig. 1
depicts a schematic view of an intervertebral disc, and the
underlying vertebra. The disc consists of the nucleus pulposus,
a central soft and gelatinous region, and the annulus fibrosus, a
tough fibrous ring that holds the nucleus in its position. In an
early stage of degeneration, when only the nucleus pulposus is
affected, or in cases where only a small part of the annulus
fibrosus is damaged by trauma, surgery can suffice with replacing the nucleus pulposus.3 In such cases, the use of a hydrogel
seems very promising, since most of the IVD’s functions can be
maintained. Though not many hydrogels are commercially
available yet, a lot of research in this direction is in progress.2–6
In more severe cases, where the annulus can not be preserved, the whole IVD has to be replaced. Then, interbody
fusion cages are often inserted.7 The aim of a cage is to restore
the disc height to decompress the neural structures in the
intervertebral foramina and to create stability in the spinal
segment by mediating osseous fusion of the neighbouring
{ Electronic supplementary information (ESI) available: colour
micrographs of cell-attachment to the I-copolymer and control
(glass). See http://www.rsc.org/suppdata/jm/b4/b407228f/
J. Mater. Chem., 2004, 14, 3008–3013
vertebrae. In those cases where osseous fusion can not be
achieved, the implant also functions as a load-carrying device
that provides stability to the spinal column. Besides cages, also
bone cement is often applied as spacer material after discectomy of the cervical spine, especially in Germany.8,9 There,
about 40% of the anterior cervical discectomies and fusions are
performed with bone cement, 32% with cages and 27% with
bone grafts.9 The difference between cages and bone cement is
that a cage possesses a cavity that can be filled by bone-graft
and facilitates bone ingrowth.
Fig. 1 Schematic view of an intervertebral disc with the underlying
vertebra.
This journal is ß The Royal Society of Chemistry 2004
To ensure that the cage stays properly positioned and to
study the bone fusion over time, follow-up after spinal surgery
is very important.
Common techniques, with different superiorities, for such
post-surgical evaluation are magnetic resonance (MR) imaging
and computed tomography (CT)/X-ray fluoroscopy. Whereas
MR imaging can very well differentiate between signal alterations in the vertebral bodies caused by oedema or inflammation, CT is highly suited for displaying bone ingrowth.10
Because of these differences it is very valuable to design a cage
that is compatible with both techniques.
Since the mid 80s, when Bagby11 introduced the use of
stainless steel cages, a variety of materials have been proposed
for the construction of such cages. Many commercially
available cages are composed of titanium or titanium alloys.
These cages have the disadvantage that MR images are
truncated. Also CT imaging of such titanium implants can
sometimes cause considerable artefacts (streaks).12 Besides
problems during follow-up evaluation, another drawback of
titanium cages is that vertebral body collapse may occur if the
endplate is degraded too much.13 This is caused by the large
mismatch in compressive elastic modulus between titanium
(E # 115 GPa) and cortical bone (E # 5 GPa).14 To decrease
stress shielding and to stimulate the formation of new bone
(which grows in response to mechanical stress), the best choice
would be to have a cage that has a modulus close to that of
cortical bone. In general, polymers can fulfil this requirement.
In literature, cages prepared from (carbon-fibre reinforced)
PEEK13,15 and poly-L-lactic acid16 have been introduced.
Studies with such polymeric cages give satisfactory results
concerning mechanical stability of the device.13 Also in terms of
post-operative evaluation several advantages exist over a
titanium cage. Both CT and MR imaging are not disturbed
by the cage material and bone fusion can well be studied. The
disadvantage however is that the polymeric cages are
radiolucent and therefore their position can not be verified
easily. To overcome the problem of radiolucency, routinely
markers are added to the polymeric cages. These markers will
be visible on the X-ray image, but it will be advantageous to
visualise the whole cage instead of only certain spots of the
implant.
In this study we explored the use of an iodine-containing
radiopaque methacrylic polymer as cage material. Such a cage
should be compatible with both imaging techniques. Like for
the other polymeric cages, the absence of metallic components
makes the cage suitable for MR imaging. The presence of
iodine accounts for the possibility of determining the exact
location of the cage by X-ray fluoroscopy.
The radiopaque material we chose is a copolymer of 80 wt.%
methylmethacrylate (MMA) and 20 wt.% 2-[4-iodobenzoyl]oxo-ethylmethacrylate (4-IEMA) (I-copolymer). This means
that the I-copolymer resembles the base material of bone
cement, which mainly consist of polymethylmethacrylate
(PMMA). A difference between the cement and our copolymer
can be found in the radiopacifier; whereas bone cement contains barium sulfate or zirconium dioxide as radiopacifier, the
cage material is intrinsically radiopaque owing to the covalently
bound iodine.
Here, the synthesis, the cytocompatibility and the mechanical characteristics of the I- copolymer will be described.
Furthermore, the results of MR and CT imaging of prototypes
of spinal cage will be presented.
Fig. 2 Structural formula of the iodine-containing methacrylate
4-IEMA.
from 4-iodobenzoyl chloride and purified hydroxyethylmethacrylate (HEMA); 1.88 mol (500 g) 4-iodobenzoylchloride was
dissolved in dried ether. Under constant stirring and icecooling, HEMA (1.97 mol; 256.5 g) and triethylamine
(1.97 mol; 199.4 g) were added dropwise. After complete
addition of the reagents and after running the reaction
overnight, the excess of HEMA and the formed salt were
removed by washing with distilled water. The ether was
removed from the product by rotor evaporation. To purify the
monomer, the residue was dissolved in hot ethanol and allowed
to crystallise. After crystallisation, the purity and identity of the
monomer were checked using 1H NMR spectroscopy: 1H
NMR (CDCl3): d 7.83 (2H, d, arom), 7.76 (2H, d, arom), 6.14
(1H, s, olef., trans to Me), 5.6 (1H, s, olef., cis to Me), 4.56 and
4.48 (4H, m, OCH2CH2O), 1.95 ppm (3H, s, Me).
To obtain a radiopaque polymer, the base material for the
cages, the purified 4-IEMA was copolymerised with MMA,
which was purchased from Acros and distilled before use. Both
monomers, with mass ratio MMA : 4-IEMA being 4 : 1, were
filled in a Teflon tube with an inner diameter of about 22 mm
and the polymerisation was performed in a temperaturecontrolled oil bath. To initiate the polymerisation reaction,
0.03 mol% dibenzoyl peroxide was added to the monomer
mixture. Furthermore, 1.8 6 1023 mol% Macrolex Violet B, a
non-toxic blue dye, was added to increase the visibility of the
cage. The polymerisation was performed by immersing the
Teflon tubes in an oil bath and running a programmed temperature profile (Fig. 3). A one-dimensional 1H NMR spectrum of the polymer was recorded at ambient temperature on a
Varian Unity-Plus (400 MHz). From the glassy rods several
prototypes of a cage (lumbar and cervical) and specimens for
mechanical testing were machined.
To study the influence of the network density on the
mechanical behaviour of the I-copolymer, crosslinked samples
were prepared by adding tetraethyleneglycol-dimethacrylate
(1–8 mol%) to the polymerisation mixture.
2.2. Imaging
To evaluate the X-ray and MR compatibility and visibility, a
prototype lumbar cage was implanted between two pieces of
marrowbone. An X-ray image of the cage was recorded by
2. Materials and methods
2.1. Synthesis of radiopaque cages
To prepare cages that are intrinsically radiopaque, an iodinecontaining methacrylic monomer was used. This monomer,
2-[4-iodobenzoyl]-oxo-ethylmethacrylate (Fig. 2), was prepared
Fig. 3 Temperature–time profile for the I-copolymer preparation.
J. Mater. Chem., 2004, 14, 3008–3013
3009
irradiating the sample at clinical conditions. More detailed
information was obtained by making a CT-scan, at a slice
thickness of 1 mm.
The X-ray visibility was further assessed by implanting a
cervical cage in-between two vertebrae of a cadaveric ovine
spinal column.
MR imaging was performed on a 1.5 Tesla MR scanner
(Philips Intera R 8.1). The images included sagittal
T 1-weighted spin-echo and T 1-weighted 3D fast field echo
sequences.
2.3. Cytocompatibility
Polymer discs (diameter 11 mm, thickness 2 mm) were sterilised
with Ultraviolet (UV) light for 15 min at each side and subsequently incubated for 3 days in culture medium at 37 uC. The
extracts were used to perform a MTT-cytotoxicity test.17 For
the direct contact assay, a drop of medium with approximately
30.000 3T3 mouse fibroblasts was placed on the washed discs
and the cells were allowed to adhere for 1 h at 37 uC. As a
positive control glass coverslips were used. Then additional
medium was added and the cells were incubated directly on the
discs for 20 h. Subsequently, cells were fixed with 2%
paraformaldehyde, 0.2% Triton X-100, 0.2% Glutaraldehyde,
in PHEM buffer (PHEM contains 60 mM PIPES, 25 mM
Hepes, 10 mM EGTA, 2 mM MgCl2, pH 6.4) for 15 min at
room temperature. The fixed cells on the discs were washed and
treated twice with 2 mg mL21 NaBH4 in PHEM for 10 min at
room temperature, in order to reduce background fluorescence.
After washing in PHEM the discs were blocked with Gel/BSA
buffer (20 mM Tris, 175 mM NaCl, 0.1% BSA, 0.02% gelatin)
for 1 h. F-actin was stained with 0.2 mM TRITC-phalloidin
overnight at 4 uC. After washing the discs were stained with
DAPI in order to visualise the nucleus. The discs were observed
sandwiched between a glass slide and a coverslip with a Nikon
Eclipse E800 fluorescence microscope equipped with a RS
Photometric CoolSnap camera.
2.4. Mechanical characterisation
Intrinsic mechanical properties were determined by compression cylindrical rods (diameter and height 6 mm) at a true strain
rate of 3 6 1023 s21 on a MTS 810 servo-hydraulic tensilecompression tester. The resulting force versus displacement
data were transferred into a true stress-true strain curve. For
this, the force was divided by the actual surface area (assuming
an invariant volume) and the true strain was defined as e = ln
(h/h0). The mechanical behaviour of the I-copolymer was
compared to polymethylmethacrylate (PMMA), prepared
under similar conditions as the I-copolymer, and PEEK,
purchased from Eriks BV, Alkmaar, the Netherlands.
Dynamic mechanical thermal analyses were conducted to
determine the rubber-plateau modulus, which can be related to
the network density, and the glass-transition temperature.
Rectangular samples (thickness 1 mm, width 7 mm, length
15 mm) were mounted in a tension film clamp and a preload of
0.05 N was applied. The samples were loaded with a force of
0.001 N at a frequency of 1 Hz and a heating rate of 1 K min21.
Moduli can be transformed into network densities using the
following equation:
E0n = 3nekT
(1)
where ne is the network density, k is Boltzmann’s constant and
T is the absolute temperature.
Since the results obtained in triplicate experiments correspond very well, the data from the median curves are used to
relate the network density to the strain hardening modulus.
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J. Mater. Chem., 2004, 14, 3008–3013
3. Results and discussion
3.1. Cage preparation
During the last decade, considerable research into iodinecontaining methacrylic polymers has been performed in our
lab.18–23 Most of the time, 4-IEMA is copolymerised with
MMA. Implantation of rods of such copolymers in rats reveals
that (even after 2 years of implantation) no release of the iodine
occurs, meaning that the copolymer is very stable in vivo.24
Depending on the desired application, the radiopacity of the
polymer can be varied by altering the ratio between the two
monomers. Here we have chosen a MMA : 4-IEMA mass ratio
of 4 : 1, which corresponds to a mass fraction iodine of 7%.
This fraction is similar to the optimum amount of iodine in
radiopaque hydrogels which are designed for nucleus pulposus
replacement and which are clearly visible on an X-ray image.6
The copolymerisation of MMA and 4-IEMA has been
studied previously by NMR spectroscopy, using DMSO-d6 as
solvent.22 From the momentary copolymer composition (at a
conversion between 4 and 6%) and the monomer feed composition, the reactivity ratios have been determined: r4-IEMA =
0.93 ¡ 0.32, rMMA = 0.90 ¡ 0.13.22 These values denote the
formation of a random-type macromolecule.
Bulk copolymerisation of the two monomers results in a high
molecular weight glassy material. To verify the composition of
the I-copolymer, the glass transition has been determined from
DMTA measurements at the maximum of the phase angle.
For the I-copolymer the T g is 119 uC. For a random-type
copolymer, the theoretical T g can be predicted from the
monomer feed composition using the Fox equation. Based on a
T g of 130 uC (also measured with DMTA) for PMMA and
75 uC for P(4-IEMA) respectively, the predicted T g for the
I-copolymer is 118 uC. The resemblance between the measured
and predicted values corroborates that the polymer composition corresponds with the monomer feed composition, i.e.
20 wt.% 4-IEMA.
A 400 MHz NMR spectrum of the I-copolymer in CDCl3
has been recorded. The spectrum is shown in Fig. 4. The
spectrum clearly reveals the purity and identity of the
copolymer. The 1H NMR also shows the presence of residual
monomer traces (olefinic protons at 6.11 and 5.57 ppm). From
Fig. 4 1H NMR spectrum of the I-copolymer in CDCl3: peaks at 7.86
and 7.8 ppm correspond to the aromatic protons of the p-iodophenyl
rings in the polymer; peaks at 6.11 and 5.57 ppm are due to
unpolymerised monomer (content is less than 0.5 mol%); peaks at 4.53
and 4.29 arise from OCH2CH2O of built-in 4-IEMA; peak at 3.60 ppm
corresponds to the methoxy groups of built-in MMA; region 2.2–
1.5 ppm arises from the methylene groups in the polymer backbone
chain; region 0.6–1.2 ppm is due to the methyl groups attached directly
to the polymer backbone; * chloroform; ** TMS.
integration it is clear that the content of free monomer is less
than 0.5%.
To investigate the compatibility of the I-copolymer with the
different imaging techniques prototypes of a lumbar and a
cervical cage have been machined from the glassy rods (Fig. 5).
3.2 Imaging
Fig. 6a and b show how a lumbar cage is implanted between
two pieces of marrowbone. On the resulting X-ray image
(Fig. 6c) the cage can clearly be distinguished. Also the sagittal
sequence of the CT scan clearly shows the cage, without any
artefacts, such as the occurrence of streaks.
Implanting a cervical cage in a cadaveric ovine spine results
in an X-ray image as depicted in Fig. 7. From this figure it
is clear that the amount of iodine that is present in the
I-copolymer provides sufficient contrast to visualise the cage in
a realistic situation.
Also with MR imaging the cage can clearly be visualised
without the occurrence of artefacts. Fig. 8 shows MR images
recorded via the T1-SE and 3D-FFE mode. Both methods
provide a clear view of the cage.
Fig. 7 X-ray image of a cervical cage implanted between two
vertebrae of a cadaveric ovine spinal column.
Fig. 5 Photographs of a prototype of a lumbar cage (a, b) side view,
(c) top view and a cervical cage (d) top view, (e) side view.
Fig. 8 MRI sagittal sequences of a lumbar cage placed between two
pieces of bone marrow (a) T1-SE, (b) 3D-FFE.
3.3 Cytocompatibility
Fig. 6 Lumbar cage placed between two pieces of marrow bone (a, b)
photograph of set-up, (c) X-ray image, (d) CT-scan.
To determine whether the I-copolymer contains cytotoxic
leachables, a MTT assay has been performed. The number of
living cells in the extract of the I-copolymer is equal to the
number of cells in culture medium (positive control) (data not
J. Mater. Chem., 2004, 14, 3008–3013
3011
Fig. 9 Cell-attachment to (a) I-copolymer, (b) control; scale bar ~
10 mm.1{
shown). This means that no cytotoxic leachables were present
in the I-copolymer.
Representative micrographs of the direct cell contact assay
are depicted in Fig. 9. On discs of the I-copolymer the cells are,
like for the glass control, nicely spread over the surface. This
indicates that the cells readily attach to the surface and also
the proliferation of the cells is by no means hindered by the
material.
Both results together indicate that the I-copolymer is a
cytocompatible material, for which no toxic effects are observed.
Combined with earlier results which indicate that for in vivo
experiments no signs of chronic inflammatory response, nor
tissue necrosis are observed in the vicinity of the iodinecontaining methacrylic implants,24 the I-copolymer proves a
biocompatible candidate as base material for spinal cages.
3.4 Mechanical characterisation
To ensure that cages prepared from the I-copolymer have
sufficient stability upon loading in the human body, it is
important to know the mechanical characteristics of the base
material. For PEEK cages it is known that they reveal excellent
resistance to crushing and are able to withstand forces that are
normally applied to the cervical spine.13 We therefore compared the behaviour of the I-copolymer to that of PEEK. Since
the I-copolymer resembles PMMA, which is also used in spinal
fusion in the form of bone cement, also a comparison with this
polymer has been made. To determine the intrinsic material
properties of the polymers, uniaxial compression experiments
have been performed. During the experiment, where the
samples are compressed from a height of 6 mm to approximately 2 mm, all samples stay intact and no cracks are
observed. The resulting true stress–true strain behaviour is
shown in Fig. 10.
The initial elastic modulus, which is equal to the slope of the
first part of the compression curve, is 2.43 ¡ 0.03 GPa for the
I-copolymer, 2.28 ¡ 0.05 GPa for PMMA and 2.39 ¡ 0.07 GPa
for PEEK. These moduli are of the same order of magnitude as
that of cortical bone, which is favourable for the formation and
ingrowth of new bone. The maximum that is observed around a
compressive strain of 0.08 is called the yield stress and it defines
the stress and strain beyond which the material will deform
plastically. For the I-copolymer the yield stress is 123.9 ¡
0.2 MPa, whereas for PMMA it is 116.1 ¡ 0.5 MPa and for
PEEK a value of 129.6 ¡ 0.2 MPa is observed. It is of course
important that loading of the cage will not extend these stresses
because in that case irreversible plastic deformation will take
place.
It can be concluded that at small compressive deformations,
a situation realistic for physiological loading of cages, the
behaviour of all three polymers is virtually identical. A
difference between the methacrylic polymers and PEEK can
3012
J. Mater. Chem., 2004, 14, 3008–3013
Fig. 10 Intrinsic mechanical behaviour of I-copolymer, PMMA,
PEEK.
be found at larger deformations, in the post-yield behaviour. In
general, yielding of the material is followed by intrinsic strain
softening and strain hardening. Intrinsic softening induces
localisation of strain and the evolution of the localised plastic
zone depends on the stabilising effect of the strain hardening. A
higher strain hardening modulus implies a better stabilisation
of localised plastic zones. So, for a material to be ductile in
tension, the intrinsic behaviour should demonstrate limited
strain softening and pronounced strain hardening.
PEEK is known to be a ductile material, which reaches a
strain of about 50% before fracture occurs. Accordingly, the
intrinsic behaviour shows hardly any strain softening and
moderate strain hardening, implying that strain localisation
will barely occur upon loading the material in tension. On the
other hand, the intrinsic behaviour of the I-copolymer is very
similar to that of PMMA, showing considerable strain softening
and limited strain hardening. As a result of the strain softening
the material will fail in tension at relatively low strains.
For the application as cage material, where mainly compressive forces play an important role, the post-yield behaviour
is not relevant. For other applications, tension forces may play
an important role and therefore a few notes will be made on
altering the post-yield behaviour of the methacrylic polymers.
We attempted to increase the contribution of the strain
hardening, which has a stabilising effect on the deformation
behaviour, by crosslinking the material. DMTA experiments
were performed to determine the influence of the amount of
crosslinker on the network-density of the polymer and
compression experiments were performed to check the
influence of the strain hardening modulus (GR). According to
the strain energy function, first proposed by Mooney, the latter
is determined from the slope of the curve true stress versus
(l2 – 1/l) at large strains.25 Fig. 11 reveals that the strain
hardening modulus is influenced by the amount of crosslinker.
Table 1 gives the strain hardening modulus and network
density for different amounts of crosslinker. It is clear that both
strain hardening modulus and network density increase with
the amount of crosslinker. Plotting the network density versus
the strain hardening modulus (Fig. 12) shows that the modulus
is proportional to the network density, similar to rubber-elastic
behaviour.25 From these experiments is it concluded that
crosslinking the methacrylic polymers will result in a more
homogeneous deformation of the material in tension.
Conclusions
Cage prototypes prepared from the I-copolymer are compatible with both CT-imaging and MR imaging, resulting in
clear appearance of the cage, without the occurrence of
mechanical characteristics as for PEEK, a commercial cage
material, are obtained.
The material described in this article is unique in the sense
that it can be visualised in an integral way by both MR and
X-ray imaging.
The current results prompt us to further develop these cages
towards a medical device for clinical use. Future work encompasses extensive biocompatibility tests in vitro and in vivo
(conform ISO standards), development and evaluation of
protocols for packaging and sterilisation (it is known that these
materials can be sterilised effectively and without side effects
through treatment with ethylene oxide), as well as Phase I–III
clinical trials.
References
1
Fig. 11 Influence of crosslinking on the intrinsic mechanical behaviour of the I-copolymer. Numbers indicate mol% tetraethyleneglycoldimethacrylate.
Table 1 Relationship between crosslinking of the I-copolymer and the
network density (ue) and strain hardening modulus (GR)
mol% crosslinker
0
2.5
6
8
ue/molecules m23
0.92
2.10
3.94
5.15
6
6
6
6
26
10
1026
1026
1026
2
3
4
5
6
GR/MPa
7
42
65
97
112
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Fig. 12 Relationship between the network density and the strain
hardening modulus for crosslinked I-copolymer.
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23
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