Article
Iodinated glycidyl methacrylate
copolymer as a radiopaque material for
biomedical applications
Journal of Biomaterials Applications
28(1) 28–37
! The Author(s) 2012
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DOI: 10.1177/0885328211434090
jba.sagepub.com
S Dawlee and M Jayabalan
Abstract
Polymeric biomaterial was synthesized by copolymerizing 50:50 mol% of monomers, glycidyl methacrylate and methyl
methacrylate. Iodine atoms were then grafted to the epoxide groups of glycidyl methacrylate units, rendering the
copolymer radiopaque. The percentage weight of iodine in the present copolymer was found to be as high as 23%.
The iodinated copolymer showed higher glass transition temperature and thermal stability in comparison with unmodified polymer. Radiographic analysis showed that the copolymer possessed excellent radiopacity. The iodinated copolymer was cytocompatible to L929 mouse fibroblast cells. The in vivo toxicological evaluation by intracutaneous reactivity
test of the copolymer extracts has revealed that the material was nontoxic. Subcutaneous implantation of iodinated
copolymer in rats has shown that the material was well tolerated. Upon explantation and histological examination, no
hemorrhage, infection or necrosis was observed. The samples were found to be surrounded by a vascularized capsule
consisting of connective tissue cells. The results indicate that the iodinated copolymer is biocompatible and may have
suitable applications as implantable materials.
Keywords
Glycidyl methacrylate, iodinated copolymer, radiopacity, biocompatibility, tissue compatibility
Introduction
Methacrylate polymers are widely used for medical applications such as contact lenses, bone cements for partial
or total joint replacement, embolic materials and in dentistry as orthodontic and denture base materials.1–4
Methacrylate polymers are radiolucent due to the absence
of high electron density elements in their polymeric backbone. Thus, in order for these polymers to be used to
fabricate implants, they need to be radiopaque. Several
approaches have been made to introduce radiopacifying
elements to polymeric chain. One of the earliest methods
for imparting radiopacity to polymeric systems involved
the incorporation of radiopaque additives. Barium salts
have frequently been used although they have been implicated in the reduction of the mechanical strength and
fracture toughness of poly(methyl methacrylate) based
bone cement.5,6 Radiopaque additives, such as tantalum
or tungsten salts, have been incorporated with poly
(methyl methacrylate) used in percutaneous vertebroplasty.7,8 Poly(methyl methacrylate) containing an organobismuth radiopacifying additive such as triphenyl
bismuth have also been reported.9,10 The bismuth
compound reduced the glass transition temperature and
also slightly elevated cytotoxicity due to a reduction in
monomer conversion. Radiopaque miscible polymer
coordination complex of poly(methyl methacrylate) and
uranyl nitrate was also studied.11 However, the biocompatibility of the complex in vivo has not been established.
In spite of some promising results with these additiveloaded radiopaque polymers, a minimum of 23%
weight of radiopaque additive was necessary to obtain
the same radiopacity as that of the aluminium standard.
In such a situation, the physical and mechanical properties of the polymers were adversely affected making it
clinically unacceptable. Polymeric biomaterials with
inherent radiopacity were developed by covalently
Polymer Science Division, Biomedical Technology Wing, Sree Chitra
Tirunal Institute for Medical Sciences and Technology, Trivandrum,
Kerala, India
Corresponding author:
M Jayabalan, Polymer Science Division, Biomedical Technology Wing, Sree
Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum695012, Kerala, India.
Email: [email protected]
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Dawlee and Jayabalan
29
binding the radiopacifying elements with the monomers
prior to their polymerization or carrying out a post-polymerization halogenation of reactive groups. Physicomechanical and radiocontrast properties of the polymer
were not compromised by this method and the secondary
release of radiopacifying agent was also prevented. Over
the years, several studies on the copolymerization of
iodine containing monomers with other monomers for
various biomedical applications have been reported.12–17
Recently, we published a study on radiopaque iodinated poly(glycidyl methacrylate-co-methyl methacrylate) copolymer prepared with GMA and MMA
monomers (weight ratio 30:70).18 Since effective X-ray
absorption was associated with higher iodine content in
the polymers, the poly(glycidyl methacrylate-co-methyl
methacrylate) copolymer was modified with a higher
molar ratio of GMA in the present work. In the present investigation, synthesis, characterization, thermal
behavior and biocompatibility of iodinated poly(glycidyl
methacrylate-co-methyl methacrylate) having GMA
and MMA in the molar ratio 50:50 are reported. The
modified copolymer had higher iodine content and possessed excellent radiopacity than previously investigated
copolymer. Preliminary biocompatibility studies demonstrated in vitro cell compatibility and acceptable in vivo
tissue compatibility.
room temperature for 24 h. The copolymer of GMA
and MMA is represented as P(GMA-co-MMA).
The copolymer was then converted to its inherently
radiopaque counterpart by regioselective ring opening
of epoxide groups of GMA units and subsequent covalent attachment of elemental iodine in the presence of a
catalytic amount of o-phenylenediamine. In a 100 mL
flask, 0.01 mol of P(GMA-co-MMA) was dissolved in
15 mL of dichloromethane. To this, 0.001 mol of o-phenylenediamine was added and 0.01 mol of iodine dissolved in 25 mL of dichloromethane was then added
through a pressure equalized addition funnel in drops.
The reaction was then kept at room temperature overnight with constant stirring. After the reaction, iodinated copolymer was precipitated out from methanol. It
was washed repeatedly with methanol, dried under
vacuum at room temperature for 24 h. The iodinated
copolymer was then dissolved in dichloromethane and
cast to form films of desired dimensions for further
characterization. The films were cleaned ultrasonically
and dried before the analyses. The samples for biocompatibility evaluation were also sterilized using ethylene
oxide gas. The radiopaque copolymer is represented as
IP(GMA-co-MMA).
Characterization techniques of the copolymers
1
Materials
GMA (Sigma-Aldrich) and MMA (Sigma-Aldrich)
were washed free of the inhibitor using sodium hydroxide solution followed by water, dried over anhydrous
sodium sulfate, and distilled under reduced pressure
prior to use. Iodine (Merck) and catalyst o-phenylenediamine (Merck) were used as such. The initiator, 2,2’azobis(isobutyronitrile) (AIBN) (Merck), was purified
through recrystallization from methanol before use.
Tetrahydrofuran (THF) (Merck) was refluxed over
sodium and distilled prior to use. Methanol (Merck)
and dichloromethane (Merck) were used as supplied
without any further purification.
Methods
Synthesis of inherently radiopaque GMA–MMA
copolymer
In an RB flask, a mixture of 0.02 mol of GMA and
0.0004 mol of AIBN were dissolved in 15 mL of THF.
Then, 0.02 mol of MMA was added to the mixture after
which it was flushed with nitrogen for 30 min. The reaction mixture was then kept in an oil bath maintained at
70 C for 24 h with constant stirring. After the reaction,
the copolymer was precipitated from methanol, washed
with methanol several times and dried under vacuum at
H NMR spectra were run on a Bruker 300 MHz instrument (Bruker AC-300, USA) with TMS as the internal
standard and CDCl3 as the solvent. Weight average
molecular weights (Mw) and the number average molecular weights (Mn) were determined by gel permeation
chromatography measurements on a Waters HPLC
system with 510 pump, 7725 Rheodyne Injector,
Styragel HR columns, Millenium 32 software and R401
Differential refractometer. THF was used as the mobile
phase at a flow rate of 1 mL/min. The instrument was
calibrated using polystyrene standards (Polysciences,
Warrington, PA, USA). Elemental iodine analysis was
performed at Service Central d’ Analyse, Centre
National de la Recherche Scientifique, Solaise, France.
Thermal stability was determined using a thermogravimetric analyzer (SDT-2960, TA Instruments Inc, USA),
with the measurements performed in nitrogen atmosphere at a heating rate of 10 C/min. Calorimetric measurements were made using a differential scanning
calorimeter (DSC-2960, TA Instruments Inc, USA),
with the measurements performed in nitrogen atmosphere at a heating rate of 10 C/min. The calorimeter
was calibrated with indium metal as a standard.
Evaluation of radiopacity
The radiopacity of the iodinated copolymer was
assessed by using a standard clinical X-ray instrument
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30
Journal of Biomaterials Applications 28(1)
(General Electric, USA). The relative X-ray opacity of
the iodinated copolymer was determined visually by
comparison with an aluminium step wedge (0.5–3 mm
thick in 0.5 mm steps) and noniodinated copolymer
(2 mm thickness). The copolymer film having 2 mm
thickness was radiographed and the resulting image
was compared with the opacity of 2 mm thick aluminium wedge, a widely used radiographic standard.19 The
grayness of the film in the resulting image was measured using Photoshop CS (Adobe Inc software). The
radiopacity of the film was then calculated as the ratio
of its grayness to that of aluminium (thickness 2 mm) as
described in literature.20
Cytocompatibility using in vitro culture
In vitro cytotoxicity testing of the iodinated copolymer
was evaluated using the direct contact method with the
iodinated copolymer based on ISO standards.21 MTT
(3 -(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide) assay was also done with material extracts to
evaluate cytotoxicity quantitatively.18
In vivo intracutaneous irritation test
In order to evaluate the local tissue response to the
extracts of IP(GMA-co-MMA) in rabbit; intracutaneous irritation test was conducted following ISO protocol.22 Albino Rabbit animal model was used for the
study. The physiological saline (PS) and cotton seed
oil (CSO) extracts of the IP (GMA-co-MMA) was
aseptically injected into 5 sites with the dosage of
0.2 mL/site on the upper left hand side and right hand
side of two rabbits. The control extracts (PS and CSO
alone) were injected into 5 sites on the lower left hand
side and right hand side of the same rabbits. The grading of erythema and edema of test and control sites of
all animals at 24, 48, and 72 h were recorded.
Tissue compatibility after in vivo implantation
Tissue compatibility of iodinated copolymer, IP(GMAco-MMA) was evaluated by subcutaneous implantation
in Wistar rat animal models for 12 weeks according
to ISO standards.23 Experiments with laboratory
animals were approved by the Institutional Animal
Ethics Committee constituted under the provisions of
Prevention of Cruelty to Animals (GOI). Six Wistar
rats aged 6 weeks (weighing 100–120 g) were used for
evaluating tissue compatibility. Surgery was performed
under anesthesia using xylazine (5 mg/kg body wt) and
ketamine (100 mg/kg body wt). Under aseptic precautions, incisions were made on the skin and subcutaneous pockets were made by blunt dissection for
introduction of the polymer. In each animal, three
radiopaque polymer specimens (IP(GMA-co-MMA))
and three control specimens (ultra high molecular
weight polyethylene, UHMWPE) were implanted. The
test and control specimens were of 10 mm in diameter
and 1 mm in thickness. The test and control were
inserted in the dorsal subcutaneous tissue of adult
rats proximally and distally. The subcutus were closed
and the skin was secured by a suture. The rats were
then caged and had free access to standard rat food
and water. Rats were sacrificed in groups of two periodically at 4, 8, and 12 weeks post-implantation.
Implants with surrounding tissues were immediately
removed, fixed by immersion in 10% buffered formalin
for 24 h, dehydrated in ethanol, clarified in xylene
and embedded in paraffin. Histological sections were
obtained and stained by hematoxylin and eosin. The
biological response was evaluated by documenting the
macroscopic and histopathological responses as function of time as per ISO standards.23 For subcutaneous
implantation, the interface between the tissue and the
material was studied. The scoring system used for histological evaluation had taken into account the extent
of area affected, semi-quntitatively. The biological
response parameters evaluated, were the extent of
fibrosis, the number and distribution as a function of
distance from the material/tissue interface of inflammatory cell types, namely, polymorphonuclear cells, lymphocytes, plasma cells, macrophages and giant cells, the
presence of necrosis, other tissue alterations such as
vascularization and fatty infiltration. The responses to
the test sample were compared to the responses
obtained at the control sites. The rats were also subjected to X-rays to evaluate the in vivo radiopacity of
the copolymer after 12 weeks of implantation.
Statistical analysis
Statistical analysis of data was evaluated by one-way
analysis of variance (ANOVA), assuming confidence
level of 95% (p < 0.05) for statistical significance.
Results and discussion
Synthesis and characterization of iodinated
copolymers
Polymers based on GMA have been widely investigated
for their potential applications in dentistry. Dental
composite resins are mostly composed of GMA monomers or some analogs of GMA. In the present work,
GMA and MMA were copolymerized by free radical
polymerization at 70 C using AIBN as an initiator to
give P(GMA-co-MMA) in good yield. Then the copolymer was modified to its radiopaque counter part by
the regioselective ring opening of the pendant epoxy
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Dawlee and Jayabalan
31
groups using elemental iodine in the presence of a catalytic amount of o-phenylenediamine as reported in our
previous publication.18
The average composition of the copolymer was
determined from the 1H NMR spectra. The 1H NMR
spectrum of the unmodified polymer is given in
Figure 1. The spectra showed two signals at 4.3 and
3.7 ppm due to splitting of methylene protons in the
CH2O–group attached to the carbonyl group of the
GMA unit by the methyne proton of the epoxy
group. The peak at 3.2 ppm corresponded to the
methyne proton of epoxy group. The peaks at 2.6 and
2.8 ppm were assigned to the methylene protons of the
epoxy group. The resonance signals at 3.59 ppm corresponded to the three methyl protons of –COOCH3 in
P(GMA-co-MMA). The peaks at 0.9–2.6 ppm were due
to the methylene groups in the polymeric chain and
other alkyl groups. Absence of signals around 5.00
and 5.30 ppm indicated the absence of protons corresponding to the methacrylic unsaturation. Thus, 1H
NMR data confirmed the incorporation of both monomeric units in the copolymer and the stability of the
epoxy groups to free radical polymerization. The
assignment of the resonance peaks in the 1H NMR
spectra resulted in the accurate evaluation of the content of each kind of monomeric units incorporated into
the copolymeric chains.24 The molar fraction of GMA
in the copolymer was calculated from measuring the
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
integrated peak area of the three resonances of epoxide
protons of the GMA unit and methoxyl protons of
MMA units. For the copolymer, the molar fractions
of GMA and MMA, as calculated from the NMR spectra, are about the same as the experimental ratio.
The molecular weights of the iodinated copolymers
were higher than those of the unmodified polymers
(Table 1) because of the presence of iodine in the
former materials. The GPC analyses showed that high
molecular weight polymers could be synthesized by
grafting iodine atoms into the polymeric backbone.
Iodine content in IP(GMA-co-MMA) was estimated
quantitatively by elemental analyses. It has been
reported that for clinically relevant X-ray visibility,
the biomaterial should contain at least 3–5 wt% of
iodine.25 The percentage weight of iodine in the present
iodinated copolymer was found to be as high as 23% in
comparison with 13.5% for the iodinated copolymer
prepared with GMA and MMA in the weight ratio of
30:70.18 From the iodine content value, the composition of GMA: MMA in IP(GMA-co-MMA) was theoretically calculated as 42:45. The partial iodination of
the copolymer could be due to the close packing of the
voluminous iodine moiety around the polymeric backbone resulting in a diminished reaction rate at higher
graft ratios as reported in literature.26
Thermal characteristics of the copolymer were
evaluated by DSC and TGA analysis (Table 2).
3.5
3.0
2.5
Figure 1. NMR spectra of the copolymer P(GMA-co-MMA).
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2.0
1.5
1.0
0.5
0.0
32
Journal of Biomaterials Applications 28(1)
Table 1. Composition, molecular weights, and yield of
copolymers.
P(GMA-co-MMA)
IP(GMA-co-MMA)
Molar ratio of GMA
and MMA (%)
Mw
Mn
Mw/Mn
Yield (%)
55: 45
42: 45
58,861
33,993
1.7
79
89,346
42,642
2
75
120
100
Radiopacity (%)
Copolymer
80
60
40
20
0
A
B
Figure 3. The graph illustrates the radiopacity of IP(GMA-coMMA) (A) evaluated as ratio of absorption relative to standard
2 mm thick Al wedge (B).
Figure 2. Positive print of a radiograph showing: (a) an aluminium step wedge 0.5–3 mm thick in 0.5 mm steps (right to left),
(b) IP(GMA-co-MMA), (c) P(GMA-co-MMA) (not visible in print).
making it good candidate materials for biomedical
applications.
Studies on radiopacity of iodinated copolymers
Table 2. Thermal characteristics of copolymers.
Copolymer
P(GMA-co-MMA)
IP(GMA-co-MMA)
Tg ( C)
Td1 ( C)
Weight remained
at Td1 (%)
Td1/2
44
112
98
99
238
97
334
316
Td1 – initial decomposition temperature, Td1/2 – temperature at 50%
decomposition.
The DSC scans of the copolymers showed that iodination caused the glass transition temperature (Tg) of
the copolymer to increase significantly. The reason
for which was attributed to the increase in rigidity
of the chains caused by the incorporation of the
iodine moiety into the copolymeric chains. TGA analysis has shown that the iodinated copolymer had
higher thermal stability than its noniodinated counter
part. This is because in the iodinated copolymer,
the highly sterically hindered iodine atoms decreased
the free volume of the polymeric chains, thereby
reducing its thermal decomposition.24 These thermal
characteristics would enable the iodinated copolymer
to be processed by any processing techniques that rely
upon plastic flow at elevated temperature, thereby
The X-ray visibility of the iodinated copolymer film
was compared with an aluminum step wedge and the
noniodinated copolymer (Figure 2). As expected, the
iodinated copolymer possessed excellent radiopacity
when compared to the reference material, aluminium.
The noniodinated copolymer was not visible when
exposed on the same radiograph. It can also be seen
from the graph (Figure 3) that the radipacity of iodinated copolymer was much higher than 2 mm thick aluminum wedge. The X-ray opacity of the copolymer is
more than sufficient for clinical monitoring when used
as biomedical implants.
Studies on cytocompatibility of iodinated copolymers
Cytocompatibility studies showed that the iodinated
copolymer was noncytotoxic. Representative microphotograph of fibroblasts cells around iodinated copolymer film (scored as zero) is shown in Figure 4. The
cells retained their original spindle shaped morphology
and no detectable zone of cell lysis, vacuolization,
detachment or membrane disintegration was observed
around or under the specimens even after 24 h of contact. Cell viability was also determined by MTT assay
to quantitatively confirm the results (Figure 5). The
assessment of cytotoxicity by MTT assay of L929
cells after contact with extract of IP(GMA-co-MMA)
showed 82% metabolic activity. Statistical analysis of
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Dawlee and Jayabalan
33
Figure 4. Representative microphotograph of L929 mouse
fibroblast cells around iodinated copolymer IP(GMA-co-MMA).
1.8
Figure 6. (A)–(C) Representative X-ray image of a rat bearing
three test specimens of IP(GMA-co-MMA).
1.6
Absorbance (570 nm)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
PE
IP (GMA-co-MMA)
Phenol
Figure 5. Quantitative evaluation of cytotoxicity by MTT assay
of IP(GMA-co-MMA) extracts in comparison with negative control (polyethylene-PE) and positive control (dilute phenol).
absorbance values obtained for negative control (high
density polyethylene) and polymer sample showed
that there was no statistically significant difference
(p > 0.07). The absorbance values for positive control
(dilute phenol) and test sample were statistically significant (p < 0.001). Thus, the noncytotoxic nature makes
this copolymer a potential candidate as biomedical
implants.
Studies on biocompatibility of iodinated copolymers
The intracutaneous irritation reactivity of iodinated
copolymer was evaluated to assess the potential of the
material to produce irritation following intradermal
injection. The extracts of IP(GMA-co-MMA) in PS
and CSO did not exhibit adverse local response in
rabbits, such as erythema or oedema, when observed
for a period of 24 to 72 h and the results produced
an average irritation score of zero with the test extracts.
The in vivo toxicological screening has clearly indicated that the iodinated copolymer was free from irritant chemicals and could be used for biomedical
applications.
The rats with implanted iodinated copolymer
IP(GMA-co-MMA) specimens were radiographed
after 12 weeks of implantation. X-ray images of all
rats clearly showed the position of iodinated copolymer
films whereas the control samples (UHMWPE films)
were not visible. X-ray image of a rat with the test
specimens is shown in Figure 6. The shape and location
of iodinated copolymer films were clearly seen in the
X-ray image, implying that no loss of radiopacifier
occurred during the implantation.
To assess the tissue compatibility of the biomaterial,
the samples were implanted for up to 12 weeks and the
immediate short-term tissue response to the implants
were studied. In vivo subcutaneous implantation of the
candidate iodinated copolymer IP(GMA-co-MMA) in
rats and histopathological analyses of the tissues surrounding the implant showed acceptable in vivo biocompatibility. The photomicrographs of tissue sites for
negative control, UHMWPE and test material for postimplantation period of 12 weeks are given in Figure 7.
The biocompatible control material (UHMWPE) was
selected as per the ISO 10993 protocol. The general physical conditions of all the experimental animals were
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34
Journal of Biomaterials Applications 28(1)
Figure 7. Optical photomicrograph showing tissue response of test material IP(GMA-co-MMA) (A) and control UHMWPE (B).
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Dawlee and Jayabalan
35
normal. The increase in body weight and feed intake were
normal and none of the animals showed any abnormality
or behavioral changes during the experimental period.
The histopathological studies revealed that the tissue
response of test materials was similar to that of control
material at the end of 4, 8, and 12 weeks. The summary of
light microscopy observations on tissues around the test
and control material during the implantation period are
given in Table 3.
At 4 weeks post implantation, mild to moderate
inflammatory response (represented as " in Figure 7)
was noticed in test and control groups. The inflammatory response was marked by the presence of polymorphonuclear cells, lymphocytes, plasma cells, and
macrophages. Polymeric biomaterials are known to
act as foreign bodies when they are implanted and
have been reported to elicit initial inflammatory reaction.27,28 Inflammation is initiated when surgical injury
causes migration of cells from the circulating blood to
the site of polymer implantation. Monocytes that
migrated from the blood to the implant site may
adhere to the surface of the implant. As time progresses, these adherent monocytes differentiate into
macrophages, which can then fuse together to form
foreign body giant cells.29,30 The histopathological
analysis revealed no evidence of foreign body giant
cells invasion in tissues surrounding both test and control materials. The tissues around the test and control
material showed good healing response at 4 weeks post
implantation. There was also evidence of repair in the
tissue around both test and control implants as early as
4 weeks post implantation. Fibrous tissue capsules with
fibroblasts and collagen were observed around test and
control implants. Statistical analysis showed that there
was no significant difference (p > 0.05) in thickness of
fibrous capsule in both test and control groups. The
inflammatory response decreased (shown as " in
Figure 7) after 8 weeks post implantation, in tissues
around both test and control implants. Except for the
presence of a few lymphocytes and macrophages, histopathological analysis revealed considerable reduction
in inflammation. Moreover, evidence of repair was
noted with fibrous tissue capsule and neovascularization
in both groups. At the end of 12 weeks of implantation,
only few inflammatory cells were seen surrounding the
implants. The histopathological analysis at 12 weeks
post implantation also revealed evidence of repair with
fibrous tissue capsule consisting of fibrocytes and collagen (designated as ) in Figure 7) around the implant site
and neovascularization (angiogenesis, represented as :
in Figure 7) in both groups of the polymers. Statistical
analysis showed that there was no significant difference
(p > 0.05) in fibrous capsule quality of both test and
control materials, 12 weeks after implantation. Fatty
infiltration was absent in both test and control implants
during implantation. The implant material was stable
Table 3. Summary of light microscopy observations around IP(GMA-co-MMA) (Test) and control.
4 weeks
8 weeks
12 weeks
Parameters
Test
Control
Test
Control
Test
Control
Polymorphonuclear cells
Lymphocytes
Plasma cells
Macrophages
Foreign body type of giant cells
Necrosis
Neovascularization
Fibrosis
Fatty infiltration
1
1
0
1
0
0
2
4
0
1
1
0
1
0
0
2
4
0
0
1
0
1
0
0
2
4
0
0
1
0
1
0
0
2
4
0
1
1
0
1
0
0
2
4
0
0
1
0
0
0
0
2
4
0
Inflammatory cells – Polymorphonuclear cells, Lymphocytes, Plasma cells, and Macrophages – Grading based on the number and distribution of cells
(0 ¼ 0 cells, 1 ¼ 1-5 cells, 2 ¼ 6-10 cells, 3 ¼ heavy infiltration, 4 ¼ packed cells, average of five fields at a magnification of 400).
For giant cells (0 ¼ 0 cells, 1 ¼ 1-2 cells, 2 ¼ 3-5 cells, 3 ¼ heavy infiltration, 4 ¼ packed cells, average of five fields at a magnification of 400).
Neovascularization – Measurements under magnification of 400 (0 ¼ no capillaries, 1 ¼ 1–3 capillaries, 2 ¼ 4–7 capillaries, 3 ¼ broad blood vessels,
4 ¼ extensive vascularization).
Fibrosis – Measurement under magnification of 400 determined by fibrous capsule thickness (0 ¼ absent, 1 ¼ <5 mm, 2 ¼ 6-15 mm, 3 ¼ 16–30 mm,
4 ¼ >30 mm).
Necrosis – Grading as determined by cell debris and inflammation (0 ¼ not present, 1¼ minimally present, 2 ¼ mild degree, 3 ¼ moderate degree,
4 ¼ severe degree).
Fatty infiltrate – Grading as determined by the amount of fat tissues (0 ¼ not present, 1 ¼ minimally present, 2 ¼ mild degree, 3 ¼ moderate degree,
4 ¼ severe degree).
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36
Journal of Biomaterials Applications 28(1)
throughout the implantation period, thereby confirming
its nondegradable nature. Tissue incompatibility signs
are usually associated with necrosis, calcification and
tumorigenesis.31 None of the implantation sites showed
any macroscopic abnormalities such as hemorrhage,
necrosis, discoloration or infection during the implantation period. Thus, the histopathological analysis of tissue
surrounding implanted IP(GMA-co-MMA) suggest that
the copolymer exhibits reasonable biocompatibility.
Conclusion
Radiopaque copolymer of GMA with MMA was synthesized by introducing iodine atoms via the regioselective ring opening reactions of epoxide groups. The
percentage weight of iodine in the present copolymer
was found to be as high as 23%. The iodinated copolymer showed higher glass transition temperature and
thermal stability in comparison with unmodified
polymer. The presence of bulky iodine atoms in the
polymer backbone decreased the flexibility of the
macromolecule and created modified polymer with
novel properties. Radiographic analysis showed that
the copolymer possessed excellent radiopacity. The
iodinated copolymer was cytocompatible. The toxicological studies and in vivo implantation of candidate
IP(GMA-co-MMA) have shown that the material was
nontoxic and tissue compatible. In vivo tissue compatibility coupled with in vivo radiopacity of IP(GMA-coMMA) offer many potential biomedical applications as
denture base, bone cement for percutaneous vertebroplasty or orthopedic applications.
Acknowledgements
Thanks are due to Prof. K. Radhakrishnan, Director,
SCTIMST and Dr G.S. Bhuvaneshwar, Head, BMT Wing,
SCTIMST for providing the facilities to carry out this work.
S.D. acknowledges the financial support from Council for
Scientific and Industrial Research, New Delhi.
References
1. Michel V. Polymeric biomaterials: strategies of the past
vs. strategies of the future. Progr Polym Sci 2007; 32:
755–761.
2. Yang JM. Polymerization of acrylic bone cement using
differential scanning calorimetry. Biomaterials 1997; 18:
1293–1298.
3. Horak D, Metalova M and Rypacek FJ. New radiopaque
poly HEMA-based hydrogel particles. J Biomed Mater Res
1997; 34: 183–188.
4. Saralidze K, Aldenhoff YB, Knetsch ML and Koole LH.
Injectable polymeric microspheres with X-ray visibility.
Preparation, properties, and potential utility as new traceable bulking agents. Biomacromolecules 2003; 4(3):
793–798.
5. Molino LN and Topoleski LD. Effect of BaSO4 on the
fatigue crack propagation rate of PMMA bone cement.
J Biomed Mater Res 1996; 31(1): 131–137.
6. Lewis G, Van Hooy-Corstjens CSJ, Bhattaram A and
Koole LH. Influence of the radiopacifier in an acrylic
bone cement on its mechanical, thermal, and physical
properties: barium sulfate-containing cement versus
iodine-containing cement. J Biomed Mater Res B Appl
Biomater 2005; 73(1): 77 –87.
7. Jensen ME, Evans AJ, Mathis JM, Kallmes DF, Cloft HJ
and Dion JE. Percutaneous polymethylmethacrylate vertebroplasty in the treatment of osteoporotic vertebral
body compression fractures: technical aspects. Am J
Neuroradiol 1997; 18: 1897– 1904.
8. Cotten A, Boutry N, Cortet B, Assaker R, Demondion X,
Leblond D, et al. Percutaneous vertebroplasty: state of
the art. Radiographics 1998; 18: 311–320.
9. Rawls HR, Granier RJ, Smid J and Cabasso I.
Thermomechanical investigation of poly(methylmethacrylate) containing an organobismuth radiopacifying
additive. J Biomed Mater Res 1996; 31: 339–343.
10. Smid J, Delaviz Y and Cobasso, I. Homogeneous radiopaque polymer-organobismuth composites. US Patent
5256334.
11. Cabasso I, Smid J and Suresh S. Radiopaque miscible
systems composed of poly(methyl methacrylate) and
transition and nontransition metal salts: Spectroscopic,
thermal, and radiographic characterization. J Appl
Polym Sci 1989; 38(9): 1653–1666.
12. Zaharia C, Zecheru T, Marculescu B, Filmon R,
Mabilleau G, Cincu C, et al. Chemical structure
of methylmethacrylate-2-[2’,3’,5’-triiodobenzoyl]oxoethyl
methacrylate copolymer, radio-opacity, in vitro and
in vivo biocompatibility. Acta Biomat 2008; 4: 1762–1769.
13. Van Hooy-Corstjens CSJ, Saralidze K, Knetsch MLW,
Emans PJ, de Haan MW, Magusin PCMM, et al.
New intrinsically radiopaque hydrophilic microspheres for embolization: synthesis and characterization.
Biomacromolecules 2008; 9(1): 84–90.
14. Lakshmi S, James NR and Jayakrishnan A. Synthesis
and polymerization of a new iodine-containing monomer.
J Appl Polym Sci 2003; 88: 2580–2584.
15. Van Hooy-Corstjens CSJ, Govaert LE, Spoelstra AB,
Bulstra SK, Wetzels GM and Koole LH. Mechanical
behaviour of a new acrylic radiopaque iodine-containing
bone cement. Biomaterials 2004; 25(13): 2657–2667.
16. Aviv H, Bartling S, Kieslling F and Margel S. Radiopaque
iodinated copolymeric nanoparticles for X-ray imaging
applications. Biomaterials 2009; 30(29): 5610–5616.
17. Zaharia C, Ruzen E, Marculescu B, Filmon R, Gavan M,
Constantin N, et al. New acrylic radio-opaque cement.
J Optoelectr Adv Mater 2007; 9: 2143–2146.
18. Dawlee S, Jayakrishnan A and Jayabalan M. Studies on
novel radiopaque methyl methacrylate: glycidyl methacrylate based polymer for biomedical applications.
J Mater Sci Mater Med 2009; 20: 243–250.
19. Mottu F, Rufenacht DA and Doelkar E. Radiopaque
polymeric materials for medical applications. Current
aspects of biomaterial research. Invest Radiol 1999; 34:
323–335.
Downloaded from jba.sagepub.com at PENNSYLVANIA STATE UNIV on September 16, 2016
Dawlee and Jayabalan
37
20. Boelen EJ, Lewis G, Xu J, Slots T, Koole LH and Van
Hooy-Corstjens CSJ. Evaluation of a highly-radiopaque
iodine-containing acrylic bone cement for use in augmentation of vertebral compression fractures. J Biomed
Mater Res 2008; 86A(1): 76–88.
21. ISO. Biological evaluation of medical devices. Part 5. Tests
for cytotoxicity: In vitro methods. ISO-10993-5. Geneva:
ISO, 2009.
22. ISO. Biological evaluation of medical devices. Part 10.
Test for irritation and delayed type hypersensitivity. Intracutaneous Reactivity Test. ISO-10993-10. Geneva: ISO,
2002.
23. ISO. Biological evaluation of medical devices. Part 6. Tests
for local effects after implantation. ISO-10993-6. Geneva:
ISO, 2007.
24. Safa KD and Nasirtabrizi MH. Ring opening reactions of
glycidyl methacrylate copolymers to introduce bulky
organosilicon side chain substituents. Polym Bull 2006;
57: 293–304.
25. Vasquez B, Ginebra MP, Gil FG, Planell JA, López
Bravo A and San Román J. Radiopaque acrylic cements
prepared with a new acrylic derivative of iodo-quinoline.
Biomaterials. 20 1999; 2047–2053.
26. Navarro-Rodriguez
D,
Rodriguez-Gonzalez
FJ,
Romero-Garcia J, Jimenez-Regalado EJ and Guillon D.
Chemical modification of glycidyl methacrylate polymers
27.
28.
29.
30.
31.
with 4-hydroxy-40 -methoxybiphenyl groups. Eur Polym J
1998; 34: 1039–1045.
Pereira IH, Ayres E, Patrı́cio PS, Góes AM, Gomide VS,
Junior EP, et al. Photopolymerizable and injectable
polyurethanes for biomedical applications: synthesis
and biocompatibility. Acta Biomater 2010; 6(8):
3056–3066.
Velayudhan S, Anilkumar TV, Kumary TV, Mohanan
PV, Fernandez AC, Varma HK, et al. Biological evaluation of pliable hydroxyapatite-ethylene vinyl acetate copolymer composites intended for cranioplasty. Acta
Biomater 2005; 1(2): 201–209.
Kao WJ, Hiltner A, Anderson J and Lodoen GA.
Theoretical analysis of in vivo macrophage adhesion
and foreign body giant cell formation on strained poly
(etherurethane urea) elastomers. J Biomed Mater Res
1994; 28(7): 819–829.
Zhao OH, Anderson JM, Hiltner A, Lodoen GA and
Payet CR. Theoretical analysis on cell size distribution
and kinetics of foreign-body giant cell formation in vivo
on polyurethane elastomers. J Biomed Mater Res 1992;
26(8): 1019–1038.
Ihova B. Biocompatibility of biomaterials: hemocompatibility, immunocompatiblity and biocompatibility of solid
polymeric materials and soluble targetable polymeric carriers. Adv Drug Deliv Rev 1996; 21(2): 157–176.
Downloaded from jba.sagepub.com at PENNSYLVANIA STATE UNIV on September 16, 2016