Radiopaque Bioactive Microspheres as
Radiopaque Bioactive Microspheres as Injectable Biomaterials
ISBN 978 90 5278 699 5
Printed by Datawyse / Universitaire Pers Maastricht
Copyright © K. Saralidze, Maastricht 2008
Radiopaque Bioactive Microspheres as
ter verkrijging van de graad van doctor aan de Universiteit Maastricht,
op gezag van de Rector Magnificus, Prof. Mr. G.P.M.F. Mols,
volgens het besluit van het College van Decanen,
in het openbaar te verdedigen
op donderdag 14 februari 2008 om 14.00 uur
Prof. dr. ir. L.H. Koole
Dr. M.L.W. Knetsch
Dr. ir. C.S.J. van Hooy-Corstjens
Prof. dr. Ph. Van Kerrebroeck (voorzitter)
Prof. dr. H. ten Cate
Dr. M. De Haan
Prof. dr. C. Jérôme (Université de Liège)
Prof. dr. D. Klee (Rheinisch-Westfälische-Technische-Hochschule Aachen)
This thesis-project was funded by Graduiertenkolleg 1035 “BioInterface – Detektion
und Steuerung grenzflächen-induzierter biomolekularer und zellulärer Funktionen” in
which the Rheinisch Westfälische Technische Hochschule Aachen, and the Universities
of Liège and Maastricht cooperate.
The research in this thesis was also supported by the Technology Foundation STW,
project 0.6777 entitled “Intrinsic radiopacity for embolic microspheres”.
Financial support for the printing of this thesis by the Dutch Society for Biomaterials
and Tissue Engineering is greatly acknowledged.
To my parents
eZRvneba Cem mSoblebs
General Introduction. 9
Injectable Polymeric Microspheres with X-ray visibility.
Preparation, properties, and potential utility as new
traceable bulking agents.
Development of new injectable bulking agents:
biocompatibility of radiopaque polymeric microspheres
studied in a mouse model.
Radio-opaque and surface-functionalized polymer
microparticles: potentially safer biomaterials for different
New acrylic microspheres for arterial embolization:
combining radiopacity for precise localization with
immobilized thrombin to trigger local blood coagulation.
Radiopaque microspheres containing acrolein for protein
attachment to improve cell adhesion.
New intrinsically radiopaque hydrophilic microspheres
for embolization: synthesis and characterization.
Chapter 3 47
Chapter 4 61
Chapter 5 77
List of publications
The number of applications of synthetic biomaterials continues to expand. One
particularly interesting new development concerns the use of injectable polymeric
biomaterials, e.g., to correct wrinkles and lips, to treat lipoatrophy in AIDS patients, or
to treat acne scars. Injection of synthetic biomaterials is also used to treat stress urinary
incontinence, or vesicoureteral reflux, for vocal cord augmentation, and in various
embolization strategies, such as those aimed at blockage of tumor-feeding arteries, or
uterine fibroids. The increase in use of injectable biomaterials has coincided with
improved imaging techniques, so that minimally invasive treatment of patients has
become more reliable and safe. Imaging of the injected biomaterials is important to
control the treatment procedure in real time, to avoid complications and to assess
therapeutic success. Minimally invasive approaches have set new standards for the
physico-chemical characteristics of the injectable materials, in so far that they have to
be easily detectable by clinically available imaging techniques, like X-ray or magnetic
resonance imaging (MRI). The standard polymeric biomaterials that have been used for
injection purposes are dominated by inert materials that are poorly visible with standard
This thesis focuses in particular on the development of new injectable biomaterials that
combine intrinsic X-ray visibility with a biofunctionality.
Stress Urinary Incontinence
In 2003, the International Continence Society committee on terminology presented a
well-considered set of definitions for lower urinary tract functions and dysfunctions.
The committee defined stress urinary incontinence as follows:
Stress urinary incontinence (SUI) is the complaint of involuntary leakage on effort or
exertion, or sneezing or coughing .
Incontinence, i.e., the complaint of involuntary leakage of urine, has been known as
long as written records are being made. Already in the 2nd millennium BC, the
Egyptians described a series of recipes and treatments for incontinence [2,3].
Nowadays, more than 200 million people worldwide live with incontinence. A major
subset of these patients, approximately 35%, suffers from so-called stress urinary
incontinence. SUI is a bladder storage problem in which the strength of the muscles
(urethral sphincter) that help to control urination is reduced. The sphincter is not able to
prevent urine flow when there is increased pressure from the abdomen. The disease is
usually caused by weakening of the pelvic floor muscles that support the bladder and
urethra or because of malfunction of the urethral sphincter (intrinsic sphincter
deficiency). The weakness may be caused by prior injury to the urethral area, child
birth, neurological injury, medications, or after surgery.
Figure 1. Front view of bladder. a) Strong sphincter and pelvic muscles keep the urethra closed.
b) Weak muscles result in urine leakage.
SUI is the most common type of urinary incontinence in women. Studies have shown
about 50% of all women have occasional urinary incontinence. At least 10% have
frequent incontinence, and approximately 20% of women over age 75 experience daily
urinary incontinence. One has to realize that these data represent only the portion of
women who chose to discuss their symptoms. Owing to embarrassment and other
factors, fewer than half of the women seek treatment or talk to a physician .
Therefore, SUI is an important medical and societal problem. More than 65% of the
treatment-seeking SUI patients in 14 European countries reported that they were
moderately to extremely bothered by the symptoms . For instance, the impact of SUI
on sexual activity in women is considerable, since the disease can lead to sexual anxiety
[6,7]. Furthermore, numerous women avoid sport activities .
The etiology of SUI is well investigated. The most important risks are: being female,
age [9-11], childbirth, smoking, obesity, and chronic coughing (e.g., as a result of
bronchitis and asthma). SUI is most prevalent in white Caucasian women [12,13].
Childbirth increases the risk of SUI drastically. The prevalence of SUI in parous women
is much higher than in nulliparous women, since pelvic floor injuries are common
during vaginal delivery [14,15]. Menopause is associated with aging of patients, and it
has been shown that postmenopausal women have higher frequency of SUI, compared
to peri- and pre-menopausal women [16,17]. Also, women whose mothers and older
sisters are incontinent have an increased risk of acquiring SUI themselves [18,19].
Smoking, which is associated with decreased collagen synthesis, is believed to cause
weakening of the pelvic floor’s supportive muscles . The association between SUI
and obesity is probably a consequence of the fact that excess weight places extra
pressure on the pelvic floor, compromising the outlet .
It seems logical to suppose, that physically fit woman have a strong pelvic floor as a
result of their regular training, thus have less chance of obtaining urinary incontinence
problems. But this does not correspond to the facts. Sports involving high impact
activities such as gymnastics, track and field, and some ball games also pose a
significant risk to develop urinary incontinence problems [22,23]. Tea drinkers are at
slightly higher risk for all types of incontinence. A decrease of fluid intake can result in
improvement of SUI. Intake of caffeine containing beverages has no important effect on
SUI . Diabetic and pre-diabetic woman have twice the prevalence of stress urinary
incontinence, compared with healthy woman .
Treatment of SUI
In 1948 Arnold Kegel introduced pelvic floor muscle training (PFMT) for management
of SUI . The aim of these exercises, which may actually root in Chinese Taoism, is
to strengthen the muscles which support the urethra, bladder sphincter muscles, via
permanent elevation of the levator plate into a higher location inside the pelvis,
increased muscle volume, strengthened connective tissue in the muscles, strengthened
bony connections, and more effective recruiting of motor neurons . Long-term
results of PFMT program are unclear. Symptoms tend to worsen on the long run and
women then prefer alternative treatments . Another considerable argument against
this method is the conviction that PFMT must be done permanently. But because of the
lack of risk and relatively small cost, PFMT is recommended by health professionals as
a first-line therapy .
Pharmacological treatment of SUI has been attempted, but with moderate success. The
mixed serotonin/noradrenaline reuptake inhibitor Duloxetine was used. Duloxetine was
believed to increase the strength of urethral sphincter contraction and to increase
bladder outlet resistance, thereby, prevent accidental urine leakage. However,
Duloxetine has a wide spectrum of severe side effects, which led to denial of approval
by the US Food and Drug administration [30-35].
There are also surgical interventions, which have been practiced for decades. In 1996
Ulmstein first introduced tension-free vaginal tape (TVT), used as a sling . This
procedure is performed through a small vaginal and two small abdominal incisions.
TVT involves placing a narrow strip of synthetic material around the middle of the
urethra. Surgical intervention of SUI has been associated with some serious
complications like perforation of the bladder , excessive bleeding , erosion of
the sling into the vagina or urinary tract [39-41], and infection. Urethral erosion is
dependent on the biomechanical and mesh properties of the tape, as well as on surgical
technique. Complications result in pain and worsening of SUI symptoms [42-45]. When
the implanted biomaterial meshes extrude from the tissue, infection is almost
unavoidable and the sling has to be removed. Furthermore, bone anchors used in SUI
surgery can be associated with pubic osteomyelitis and osteitis pubis. In such cases,
surgical removal and aggressive treatment with long-term antibiotics is required [46,47].
Modern minimally invasive therapies for SUI
During the last two decades, less invasive procedures, aimed at achieving high long-
term cure rates for SUI, have been developed. Most successful are peri-urethral
injections of a so-called bulking agent. This technique led to very good results,
especially for the elderly patients, for women who have undergone multiple failed
procedures, or after radiotherapy where the urethra may have become fixed and scarred.
From the perspective of biomaterials science, these developments are particularly
interesting. A variety of different bulking agents have been used; the most commonly
used materials are cross-linked bovine collagen (Contigen), polydimethylsiloxane
(PDMS, Macroplastique), ethylene vinyl alcohol copolymer (Tegress),
polytetrafluoroethylene (PTFE, Urethrin), carbon-coated zirconiumdioxide beads
(Durasphere), calcium hydroxylapatite (Coaptite), and dextranomer/hyaluronic acid
copolymer. Bulking agents are injected carefully in the periurethral tissue. The goal is
urethral coaptation during the storage of urine, maintenance of that coaptation during
periods of increased abdominal pressure, and improving sphincter closure.
Figure 2. Front view of bladder. a) Urethra with weak muscles results in urine leakage. b)
Complete coaptation of the urethra, which is achieved by injection of the bulking agent around
Degradable bulking agents such as autologous fat tend to relieve symptoms, but have
comparatively low efficiency and disappointing long-term results. In addition, some
side-effects were reported, such as granuloma formation, obstructive mass formation,
urine retention and fat embolism [48-55].
Ethylene vinyl alcohol copolymer suspended in DMSO (Tegress) was evaluated as
embolic agent, but was also approved as a bulking agent for treatment of SUI. Recent
studies not only demonstrated that Tegress may be less efficacious than reported in
FDA trials, but also that a significant percentage of patients experienced serious
complications like urethral erosion [56,57]. Injection of solid polydimethylsiloxane
(silicone rubber) particles proved to have moderate success, with approximately half of
the patients being cured after 24 months [58-60]. Furthermore, the misplacement or
injection of too many silicon particles leads to complications, caused by the invisibility
of the particles [61,62].
Polytetrafluoroethylene paste is a resin with very high molecular weight and high
viscosity. The material is composed of small particles and has been used to treat SUI
since 1964. In spite of this, PTFE is not approved in the United States for treatment of
incontinence, because of dangerous complications, such as distant migration,
periurethral abscess, urethral diverticulum, urethral granuloma formation, and even
increased tumor risk [63-66].
Durasphere® is composed pyrolitic carbon-coated zirconium oxide beads suspended in
water–based carrier gel containing beta-glucan. Pyrolytic carbon is inert and
biocompatible, and is used in implantable medical devices, including replacement heart
valves . One of the potential advantages is its very low immunogenicity.
Durasphere® particles are relatively large, 200-500 µm, and radiopaque, which is useful
for tracing after injection. Durasphere® is non-degradable, therefore concerns were
raised over long-term durability . In spite of promising clinical data, Durasphere®
demonstrated some serious complications, among which migration from implantation
site is the most dangerous one. The obvious dislocation from the implantation site was
shown in animal studies . Six months after injection of carbon coated beads with
diameter of 251 to 300 µm, significant migration of these beads was observed into local
and distant lymph nodes, as well as into the urethral mucosa. The exact reason for bead
migration remains unclear . Durasphere® injection may result in long-term outlet
obstruction, which can cause voiding dysfunction, permanent urinary retention and
periurethral mass formation [71-73]. Urethral prolapse is an uncommon condition, but
some cases have been described . The long-term efficacy of Durasphere® injection
is low. At 18 months follow up in 70 patients only 13% of patients considered
themselves cured, 52.2% improved and 34.7% failed . Another study demonstrated
that Durasphere® remained effective in 35%, 33%, and 21% of patients at 12, 24, and 36
months respectively .
Synthetic calcium hydroxylapatite (CaHa) spheres suspended in a water-based gel
carrier is a sterile, radiopaque, non-pyrogenic, semi-solid, cohesive implant. It is known
as Coaptite and Radiesse. Coaptite is used for treatment of SUI and Radiasse is used for
the correction of facial lypoatrophy in HIV patients [77,78]. They differ in size range of
Theoretically, injected CaHa spheres have to form a scaffold for developing soft tissue
that will gradually replace the gel carrier. For integration in the surrounding tissue,
fibroblasts have to attach and grow on the surface of the spheres, resulting in anchoring
at the injection site. Coaptite had promising results beforehand . Nevertheless, the
implant also has shortcomings. The histology of injected CaHa microspheres
demonstrated, that microspheres become deformed, appearing irregular, and start being
adsorbed at 9 months, likely because of enzymatic breakdown of the calcium
hydroxylapatite . Furthermore, it looks as if CaHa microspheres migrate away from
the injection site. This migration may result in periureteral fibrosis, ureteric obstruction
and subsequent renal loss . Moreover, granulomatous reaction can lead to urethral
prolapse, as early as 3 months after the transurethral injection of calcium
Embolization is defined as “therapeutic introduction of various substances into the
circulation to occlude vessels, either to arrest or prevent hemorrhaging, to devitalize a
structure, tumor, or organ by occluding its blood supply, or to reduce blood flow to an
arteriovenous malformation” .
Closure of a target artery can be achieved by use of different embolic agents such as
synthetic microparticles, pellets, glues, or platinum coils [84,85]. This technique, also
known as embolotherapy, leads to obstruction of arterial blood flow. Injected particles
act in the same way as thrombotic emboli [86-90].
The field of interventional radiology routinely uses artificial microparticles and live-
imaging techniques to specifically block arteries and starve targeted tissues from
oxygen and nutrients. For this, a catheter is inserted and maneuvered to the target vessel
that has to be embolized, i.e., blocked. Subsequently, the embolic particles are injected
into the flowing blood and become stuck in the vessel, blocking the blood flow to the
Embolotherapy is used in a variety of treatments of: (i) tumors, varicoceles, and organ
ablation; (ii) hemorrhages, e.g., pelvic, posttraumatic, epistaxis, and hemoptysis; (iii)
vascular anomalies, e.g., venous, lymphatic, arteriovenous malformations. Also the
treatment of inoperable tumors is among the applications of embolotherapy. Tumors
that lie deep in the brain are an obvious target for embolotherapy although one has to
keep in mind that such interventions are often more palliative.
Figure 3. Overview of different applications of embolization therapy.
Furthermore, benign tumors like uterine fibroids can be treated by embolizing the
feeding artery as an alternative to the common hysterectomy. So in this case
embolotherapy is a minimally invasive alternative to surgery. Another application of
embolic particles is the treatment of hemoptysis, i.e., severe bleeding because of trauma
or of infections in the lungs, e.g., pneumonia can cause heavy bleeding. Embolization in
such cases is used to stop lethal blood loss of the patient [85, 91-98].
Embolization of tumors
Embolotherapy is a new minimally invasive technique for the treatment of solid tumors.
The embolization is performed pre-operatively, to shrink the tumor and prevent
excessive blood-loss, or as a palliative treatment of unoperable tumors. The benign
uterus tumors in women, called uterine fibroids (UF), will be discussed to explain tumor
embolization in further detail.
UF consist mainly of smooth muscles and large amounts of extracellular matrix
containing collagen, fibronectin, and proteoglycan. UF are also called fibromyomas,
leiomyomas or myomas. They are very common and clinically apparent in about 25% of
women, but because many commonly used imaging techniques lack resolution, the true
prevalence may be as high as 77% .
Figure 4. Front view of uterus: a) healthy uterus; b) uterus with fibroids.
UF can cause abnormal uterine bleeding, resulting in iron-deficiency anemia,
dysmenorrhea and non-cyclic pelvic pain [100-103]. The enlarging pelvic mass
contributes to urinary problems and constipation [104-106]. UF are also associated with
an increased risk of complications during pregnancy, and with infertility [107,108]. In
other words UF have significant impact on the quality of life of women .
Even there are no systematic estimates of the total healthcare and productivity-loss costs
attributable to uterine fibroid, the high cost of procedures used to treat (up $11,839 per
procedure) was reported .
Interestingly, race is an important risk factor for UF. Black women are more likely than
white women to have uterine fibroids, are more likely to have multiple fibroid tumors
too. Black women are also younger at diagnosis than white [111,112].
The prevalence of UF definitely increases with age with a peak around the menopause
and decreases after menopause. However, UF are present in the sixth, seventh, and even
the eighth decade [113,114]. First-degree relatives of women with UF have a 2.5 times
increased risk of developing myomas. Furthermore, cytogenetic, molecular and
epidemiological data strongly suggest a genetic component in the etiology of fibroids
[115,116]. Body mass index and weight gain exhibit a complex relation with risk of UF.
This can be supposed that women with greater upper body obesity have decreased sex
hormone-binding globulin levels, altered estrogen metabolism and some other such
factors, which may promote tumor development .
There are also several factors that surprisingly decrease the risk for UF among which
are smoking, pregnancy, and exercise . This does not mean that smoking is an
advised therapy in the prevention of UF, or can be used as an excuse for smoking.
Treatment of UF is relatively crude; surgical removal of the uterus, hysterectomy, is the
most common method. In the US only, approximately 600,000 hysterectomies are
performed annually, with uterine fibroids accounting for approximately 40% of all
hysterectomies . Especially for younger women, hysterectomy means that the
possibility for child birth is gone and this may have serious psychological effects. Also,
hysterectomy often does not relieve symptoms in several women, and some women
reported new symptoms, such as hot flashes, weight gain, and depression [119,120].
Myomectomy is the surgical removal of uterine fibroids with preservation of the uterus.
This is an invasive surgical technique, which can be performed in several different ways
depending upon the size, number and location of the UF. There are three approaches to
myomectomy: abdominal, laparoscopic, and hysteroscopic. Despite preserving fertility,
myomectomy has complications. Most common complications of myomectomy are
damage and infections of surrounding organs; damage, weakening and scaring of
womb, which can lead to complications during the pregnancy such as rupturing the
womb wall; bleeding, that can lead to full hysterectomy [121-123].
Medications for uterine fibroids do not eliminate fibroids, but may shrink them. Target
hormones regulate menstrual cycle, treating symptoms such as heavy menstrual
bleeding and pelvic pressure. The most commonly reported adverse effects of
progesterone receptor modulators were headache, abdominal pain, nausea, dizziness,
and metrorrhagia . Side effects such as muscle cramps, acne, fluid retention,
unwanted hair growth, weight gain, and deeper voice can occur during androgen therapy
Figure 5. Angiographic catheter is introduced into the femoral artery through the small opening
in the groin. Microspheres are injected into the artery that is supplying the fibroids and block
An alternative treatment of uterine fibroids is called embolotherapy. This means that the
feeding arteries to the fibroids are blocked by localized injection of microparticles. As a
consequence, the fibroids will become devoid of oxygen and nutrients. The mass of the
UF will diminish over time and the symptoms will thus be reduced. The interventional
radiologist has the choice of a number of synthetic microparticles that can be used for
These particles are however not ideal because: i) they are not visible under standard
clinical conditions (X-ray); ii) they are irregular in shape and size; iii) they are inert and
the interaction with the surrounding tissue is unpredictable. The lack of radiopacity, i.e.,
X-ray visibility forces the interventional radiologist to guess how much particles have
been injected and where these end up. Therefore the currently used microparticles are
mixed with liquid contrast agent to at least get some idea of where the injected particles
end up. Also, in this way reverse flow, and unwanted embolization distant from the
intended site, can be avoided. The current embolization-microparticles are irregular in
shape. This may lead to obstruction in the catheter and incomplete embolization, leaking
blood to the down-stream tissues. In practice microspheres are the ideal embolic
particles. After embolization the microparticles induce a reaction from the surrounding
tissue. Severe inflammatory reactions are in general undesirable, and also angiogenesis,
formation of collateral vessels is surely not wanted. The preferred tissue reaction would
be inertia, leaving the down-stream tissues devoid of oxygen and nutrients.
Aim of thesis
The research described in this thesis set out to achieve improvements over the injectable
polymeric biomaterials that are currently in clinical use. Most of the commercially
available particulate injectables are composed of inert polymers. These materials are
radiolucent, i.e., the particles are invisible on X-ray images. Obviously, this hampers
their retrieval once they have been injected in the body. Note that other imaging
techniques, notably magnetic resonance imaging, are also of limited use for localization
of small polymer particles. Some commercially available injectable particles are
radiopaque; the most important example is DuraSphere®. These particles consist of
ZrO2. For DuraSphere®, however, the advantage of the X-ray visibility is
counterbalanced by the fact that the particles have a high density (around 6 g/cm3).
Presumably, the high density of DuraSphere® particles is responsible for the fact that
these particles have a tendency to migrate from the site of injection . Our key
hypothesis was that methacrylate copolymers that contain covalently bound iodine
would offer a promising approach to injectable particles that uniquely combine:
adequate radiopacity, spherical shape, a high level of biocompatibility and a density
which is comparable to the density of soft tissues. This approach is a logical extension
of previous work from the laboratory on polymeric biomaterials featuring intrinsic
radiopacity, on the basis of incorporation of covalently bound iodine [126,127,128].
We chose to focus on poly(methacrylate) copolymers, since these already find
widespread use as implant biomaterials. Radiopacity was introduced through either
monomer 2-[4,-iodobenzoyloxy] ethyl methacrylate (4-IEMA), or monomer 2-
[2’,3’,5’,-triiodobenzoyloxy] ethyl methacrylate (2,3,5-TIEMA), shown in Figure 6 and
7, respectively. Note that these monomers have a reactive methacrylate group on one
side, and an iodine-bearing aromatic ring on the other side. Both monomers were
copolymerized with methyl methacrylate, 2-hydroxy ethyl methacrylate or N-
vinylpyrrollidinone, always in temperature-controlled free-radical polymerization
Figure 6. 4IEMA
Figure 7. 2,3,5-TIEMA.
We have become familiar with the copolymer materials that are produced in such
reactions. For instance, an animal study demonstrated that these radiopaque materials
are stable in vivo over a long period of time (2 years) . In one case, an official CE-
certification for an implant medical device that was based on an iodine-containing
copolymer was awarded. Through the use of different co-monomers, it proved possible
to manipulate the properties of the resulting copolymers. This is especially valuable
with regard to hydrophilicity an aqueous swelling. Completely new hydrogel
biomaterials, which combine radiopacity and controlled uptake of water,
biocompatibility, and surprisingly high resistance to fatigue, emerged form this work.
Preparation of microspheres with the desired properties proved to be possible. Two
routes of preparation were explored: solvent evaporation and suspension
polymerization; each proved effective after careful optimization of several essential
parameters. We further investigated the biocompatibility of these particles, both in vitro
and in vivo. Finally, we added and additional functionality through chemical
modification of the surface of the radiopaque spheres. A method was explored to attach
proteins, such as thrombin and collagen. In the case of thrombin, we found that the
enzyme retains its activity, at least partially, after immobilization.
Chapter 2 describes the first production of small microspheres using the 2,3,5-TIEMA
monomer. For this, copolymer was produced in bulk, dissolved in an organic solvent,
and dripped slowly in a stirred aqueous solution containing a stabilizing agent to obtain
small droplets. Upon evaporation of the solvent, small microspheres were obtained that
are visible under standard clinical X-ray conditions (mammography). A prerequisite for
all injectable biomaterials is their biocompatibility. The iodine-containing microspheres
were shown to be devoid of any cytotoxicity in vitro.
Biocompatibility was further investigated in Chapter 3 where we studied the behavior of
the microspheres in rats over a period of 3 months. Both subcutaneous and
intramuscular injection of the microspheres, suspended in collagen, demonstrated the
microspheres to be nicely biocompatible.
Chapter 4 describes a different method of obtaining X-ray visible microspheres.
Suspension polymerization results in microspheres that are more uniform and the
control over size and shape are improved. Furthermore, the problem of the inert surface
of the microspheres is tackled in this chapter. Heating of the microspheres in strong
alkaline environment results in the formation of carboxylic groups on the surface, which
can be used to anchor active protein. The addition of protein on the surface may be of
importance to anchor the spheres more strongly in the tissue, preventing the problem of
In Chapter 5 this biofunctionalization is improved by addition of methacrylic acid in the
suspension polymerization, resulting in microspheres with surface carboxylic groups.
This method is more controllable than the one described in Chapter 4. As an example,
active thrombin, the key enzyme in blood coagulation, was coupled to the spheres in
order to enhance embolization therapy. The clotting of blood upon mechanical closure
of e.g., a tumor-feeding artery or a severely bleeding artery, may lead to faster and more
stable closure of the blood vessel.
In Chapter 6 biofunctionalization was achieved by the synthesis of microspheres with
surface aldehyde groups. Coupling of collagen should result in improved adhesion of
cells and ultimately better anchoring of microspheres in surrounding tissue.
In Chapter 7, the synthesis of a different sort of microspheres is described. The X-ray
visible microspheres are designed to slowly swell in aqueous environment. This has
several advantages for embolotherapy. First of all these microspheres are compressible,
which can avoid microspheres getting stuck in the catheter. Also the microspheres could
potentially anchor themselves tightly in the artery by swelling in situ and blocking
Abrams P, Cardozo L, Fall M, Griffiths D, Rosier P, Ulmsten U, Van Kerrebroeck P, Victor A, Wein
A. Standardisation Sub-Committee of the International Continence Society. The standardisation of
terminology in lower urinary tract function: report from the standardisation sub-committee of the
International Continence Society. Urology. 2003;61(1):37-49.
Breasted JH: Edwin Smith surgical papyrus in facsimile and hieroglyphic transliteration with
translation and commentary. Chicago, University of Chicago Oriental Institute. 1930.
Joachim H: Papyrus Ebers, Berlin, G.Reimer.1890.
Shaw C, Tansey R, Jackson C, Hyde C, Allan R. Barriers to help seeking in people with urinary
symptoms. Fam Pract. 2001;18(1):48-52.
Monz B, Pons ME, Hampel C, Hunskaar S, Quail D, Samsioe G, Sykes D, Wagg A, Papanicolaou S.
Patient-reported impact of urinary incontinence--results from treatment seeking women in 14
European countries. Maturitas. 2005;52 Suppl 2:S24-34.
Salonia A, Zanni G, Nappi RE, Briganti A, Deho F, Fabbri F, Colombo R, Guazzoni G, Di Girolamo
V, Rigatti P, Montorsi F. Sexual dysfunction is common in women with lower urinary tract
symptoms and urinary incontinence: results of a cross-sectional study. Eur Urol. 2004;45(5):642-8.
Barber MD, Dowsett SA, Mullen KJ, Viktrup L. The impact of stress urinary incontinence on sexual
activity in women. Cleve Clin J Med. 2005;72(3):225-32.
Brown WJ, Miller YD. Too wet to exercise? Leaking urine as a barrier to physical activity in women.
J Sci Med Sport. 2001;4(4):373-8.
Hannestad YS, Rortveit G, Hunskaar S. Help-seeking and associated factors in female urinary
incontinence. The Norwegian EPINCONT Study. Epidemiology of Incontinence in the County of
Nord-Trondelag. Scand J Prim Health Care. 2002;20(2):102-7.
Hunskaar S, Burgio K, Diokno A, Herzog AR, Hjalmas K, Lapitan MC. Epidemiology and natural
history of urinary incontinence in women. Urology. 2003;62(4 Suppl 1):16-23.
Danforth KN, Townsend MK, Lifford K, Curhan GC, Resnick NM, Grodstein F. Risk factors for
urinary incontinence among middle-aged women. Am J Obstet Gynecol. 2006;194(2):339-45.
Sze EH, Jones WP, Ferguson JL, Barker CD, Dolezal JM. Prevalence of urinary incontinence
symptoms among black, white, and Hispanic women. Obstet Gynecol. 2002;99(4):572-5.
Thom DH, van den Eeden SK, Ragins AI, Wassel-Fyr C, Vittinghof E, Subak LL, Brown JS.
Differences in prevalence of urinary incontinence by race/ethnicity. J Urol. 2006;175(1):259-64.
Williams A, Herron-Marx S, Knibb R. The prevalence of enduring postnatal perineal morbidity and
its relationship to type of birth and birth risk factors. J Clin Nurs. 2007;16(3):549-61.
Chin HY, Chen MC, Liu YH, Wang KH. Postpartum urinary incontinence: a comparison of vaginal
delivery, elective, and emergent cesarean section. Int Urogynecol J Pelvic Floor Dysfunct.
Jackson SL, Scholes D, Boyko EJ, Abraham L, Fihn SD. Predictors of urinary incontinence in a
prospective cohort of postmenopausal women. Obstet Gynecol. 2006;108(4):855-62.
Steinauer JE, Waetjen LE, Vittinghoff E, Subak LL, Hulley SB, Grady D, Lin F, Brown JS.
Postmenopausal hormone therapy: does it cause incontinence? Obstet Gynecol. 2005;106(5 Pt
Hannestad YS, Lie RT, Rortveit G, Hunskaar S. Familial risk of urinary incontinence in women:
population based cross sectional study. BMJ. 2004;329(7471):889-91.
Ertunc D, Tok EC, Pata O, Dilek U, Ozdemir G, Dilek S. Is stress urinary incontinence a familial
condition? Acta Obstet Gynecol Scand. 2004;83(10):912-6.
Hannestad YS, Rortveit G, Daltveit AK, Hunskaar S. Are smoking and other lifestyle factors
associated with female urinary incontinence? The Norwegian EPINCONT Study. BJOG.
Subak LL, Whitcomb E, Shen H, Saxton J, Vittinghoff E, Brown JS. Weight loss: a novel and
effective treatment for urinary incontinence. J Urol. 2005;174(1):190-5.
Caylet N, Fabbro-Peray P, Mares P, Dauzat M, Prat-Pradal D, Corcos J. Prevalence and occurrence
of stress urinary incontinence in elite women athletes. Can J Urol. 2006;13(4):3174-9.
Bo K. Urinary incontinence, pelvic floor dysfunction, exercise and sport. Sports Med.
Swithinbank L, Hashim H, Abrams P. The effect of fluid intake on urinary symptoms in women. J
Brown JS, Vittinghoff E, Lin F, Nyberg LM, Kusek JW, Kanaya AM. Prevalence and risk factors for
urinary incontinence in women with type 2 diabetes and impaired fasting glucose: findings from the
National Health and Nutrition Examination Survey (NHANES) 2001-2002. Diabetes Care.
Kegel AH. Progressive resistance exercise in the functional restoration of the perineal muscles.
American Journal of Obstetrics and Gynecology. 1948;56:238-48.
Cammu H, Van Nylen M, Amy J. A ten-year follow-up after Kegel pelvic floor muscle exercises for
genuine stress incontinence. BJU Int. 2000;85:655–658
Hay-Smith EJ, Dumoulin C. Pelvic floor muscle training versus no treatment, or inactive control
treatments, for urinary incontinence in
Bo K. Pelvic floor muscle training is effective in treatment of female stress urinary incontinence, but
how does it work? Int Urogynecol J Pelvic Floor Dysfunct. 2004;15(2):76-84.
Oelke M, Roovers JP, Michel MC. Safety and tolerability of duloxetine in women with stress urinary
incontinence. BJOG. 2006;113 Suppl 1:22-6.
Guay DR. Duloxetine for management of stress urinary incontinence. Am J Geriatr Pharmacother.
Mariappan P, Ballantyne Z, N'Dow JM, Alhasso AA. Serotonin and noradrenaline reuptake inhibitors
(SNRI) for stress urinary incontinence in adults. Cochrane Database Syst Rev. 2005;(3):CD004742.
women. Cochrane Database Syst Rev.
Mariappan P, Alhasso A, Ballantyne Z, Grant A, N'Dow J. Duloxetine, a serotonin and noradrenaline
reuptake inhibitor (SNRI) for the treatment of stress urinary incontinence: a systematic review. Eur
van Kerrebroeck P, Abrams P, Lange R, Slack M, Wyndaele JJ, Yalcin I, Bump RC; Duloxetine
Urinary Incontinence Study Group. Duloxetine versus placebo in the treatment of European and
Canadian women with stress urinary incontinence. BJOG. 2004;111(3):249-57.
Lenzer J. FDA warns that antidepressants may increase suicidality in adults. BMJ.
Ulmsten U, Henriksson L, Johnson P, Varhos G. An ambulatory surgical procedure under local
anesthesia for treatment of female urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct.
Comiter CV. Surgery insight: management of failed sling surgery for female stress urinary
incontinence. Nat Clin Pract Urol. 2006;3(12):666-74.
Flock F, Reich A, Muche R, Kreienberg R, Reister F. Hemorrhagic complications associated with
tension-free vaginal tape procedure. Obstet Gynecol. 2004;104(5 Pt 1):989-94.
Amundsen CL, Flynn BJ, Webster GD Urethral erosion after synthetic and nonsynthetic pubovaginal
slings: differences in management and continence outcome. J Urol. 2003;170(1):134-7.
Madjar S, Tchetgen MB, Van Antwerp A, Abdelmalak J, Rackley RR. Urethral erosion of tension-
free vaginal tape. Urology. 2002;59(4):601
Abdel-Fattah M, Sivanesan K, Ramsay I, Pringle S, Bjornsson S. How common are tape erosions? A
comparison of two versions of the transobturator tension-free vaginal tape procedure. BJU Int.
Yamada BS, Govier FE, Stefanovic KB, Kobashi KC. High rate of vaginal erosions associated with
the mentor ObTape. J Urol. 2006;176(2):651-4.
Siegel AL. Vaginal mesh extrusion associated with use of Mentor transobturator sling. Urology.
Comiter CV, Colegrove PM. High rate of vaginal extrusion of silicone-coated polyester sling.
Govier FE, Kobashi KC, Kuznetsov DD, Comiter C, Jones P, Dakil SE, James R Jr. Complications of
transvaginal silicone-coated polyester synthetic mesh sling. Urology. 2005;66(4):741-5.
Frederick RW, Carey JM, Leach GE. Osseous complications after transvaginal bone anchor fixation
in female pelvic reconstructive surgery: report from single largest prospective series and literature
review. Urology. 2004;64(4):669-74.
Goldberg RP, Tchetgen MB, Sand PK, Koduri S, Rackley R, Appell R, Culligan PJ. Incidence of
pubic osteomyelitis after bladder neck suspension using bone anchors. Urology. 2004;63(4):704-8.
Mayer RD, Dmochowski RR, Appell RA, Sand PK, Klimberg IW, Jacoby K, Graham CW, Snyder
JA, Nitti VW, Winters JC Multicenter prospective randomized 52-week trial of calcium
hydroxylapatite versus bovine dermal collagen for treatment of stress urinary incontinence. Urology.
Chapple CR, Haab F, Cervigni M, Dannecker C, Fianu-Jonasson A, Sultan AH. An open, multicentre
study of NASHA/Dx Gel (Zuidex) for the treatment of stress urinary incontinence. Eur Urol.
van Kerrebroeck P, ter Meulen F, Larsson G, Farrelly E, Edwall L, Fianu-Jonasson A. Efficacy and
safety of a novel system (NASHA/Dx copolymer using the Implacer device) for treatment of stress
urinary incontinence. Urology. 2004;64(2):276-81.
Abdelwahab HA, Ghoniem GM. Obstructive suburethral mass after transurethral injection of
dextranomer/hyaluronic acid copolymer. Int Urogynecol J Pelvic Floor Dysfunct. 2007;18(11):1379-
Castillo-Vico MT, Checa-Vizcaíno MA, Payà-Panadés A, Rueda-García C, Carreras-Collado R.
Periurethral granuloma following injection with dextranomer/hyaluronic acid copolymer for stress
urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct. 2007;18(1):95-7.
Lee PE, Kung RC, Drutz HP. Periurethral autologous fat injection as treatment for female stress
urinary incontinence: a randomized double-blind controlled trial. J Urol. 2001;165(1):153-8.
Andersen RC. Long-term follow-up comparison of durasphere and contigen in the treatment of stress
urinary incontinence. J Low Genit Tract Dis. 2002;6(4):239-43.
Chrouser KL, Fick F, Goel A, Itano NB, Sweat SD, Lightner DJ. Carbon coated zirconium beads in
beta-glucan gel and bovine glutaraldehyde cross-linked collagen injections for intrinsic sphincter
deficiency: continence and satisfaction after extended followup. J Urol. 2004;171(3):1152-5.
Hurtado E, McCrery R, Appell R. The safety and efficacy of ethylene vinyl alcohol copolymer as an
intra-urethral bulking agent in women with intrinsic urethral deficiency. Int Urogynecol J Pelvic
Floor Dysfunct. 2007;18(8):869-73.
Erekson EA, Sung VW, Rardin CR, Myers DL. Ethylene vinyl alcohol copolymer erosions after use
as a urethral bulking agent. Obstet Gynecol. 2007;109(2 Pt2):490-2.
Maher CF, O'Reilly BA, Dwyer PL, Carey MP, Cornish A, Schluter P. Pubovaginal sling versus
transurethral Macroplastique for stress urinary incontinence and intrinsic sphincter deficiency: a
prospective randomised controlled trial. BJOG. 2005;112(6):797-801.
Radley SC, Chapple CR, Mitsogiannis IC, Glass KS. Transurethral implantation of macroplastique
for the treatment of female stress urinary incontinence secondary to urethral sphincter deficiency. Eur
Tamanini JT, D'Ancona CA, Netto NR. Treatment of intrinsic sphincter deficiency using the
Macroplastique Implantation System: two-year follow-up. J Endourol. 2004;18(9):906-11.
Kulkarni S, Davies AJ, Treurnicht K, Dudderidge TJ, Al-Akraa M. Misplaced Macroplastique
injection presenting as a vaginal nodule and a bladder mass. Int J Clin Pract Suppl. 2005;(147):85-6.
Peeker R, Edlund C, Wennberg AL, Fall M. The treatment of sphincter incontinence with
periurethral silicone implants (macroplastique). Scand J Urol Nephrol. 2002;36(3):194-8.
Malizia AA, Reiman HM, Myers RP, Sande JR, Barham SS, Benson RC, Dewanjee MK, Utz WJ.
Migration and granulomatous reaction after periurethral injection of polytef (Teflon). JAMA.
Kiilholma PJ, Chancellor MB, Makinen J, Hirsch IH, Klemi PJ. Complications of Teflon injection
for stress urinary incontinence. Neurourol Urodyn. 1993;12(2):131-7.
Dewan PA, Owen AJ, Byard RW. Long-term histological response to subcutaneously injected
Polytef and Bioplastique in a rat model. Br J Urol. 1995;76(2):161-4.
Dewan PA. Is injected polytetrafluoroethylene (Polytef) carcinogenic? Br J Urol. 1992;69(1):29-33.
Beavan A. Material properties and applications of Pyrolite™ Carbon. Materials Engineering.
Lightner D, Calvosa C, Andersen R, Klimberg I, Brito CG, Snyder J, Gleason D, Killion D,
Macdonald J, Khan AU, Diokno A, Sirls LT, Saltzstein D. A new injectable bulking agent for
treatment of stress urinary incontinence: results of a multicenter, randomized, controlled, double-
blind study of Durasphere. Urology. 2001;58(1):12-5.
Lemperle G, Morhenn VB, Pestonjamasp V, Gallo RL. Migration studies and histology of injectable
microspheres of different sizes in mice. Plast Reconstr Surg. 2004;113(5):1380-90.
Pannek J, Brands FH, Senge T. Particle migration after transurethral injection of carbon coated beads
for stress urinary incontinence. J Urol. 2001;166(4):1350-3.
Hartanto VH, Lightner DJ, Nitti VW. Endoscopic evacuation of Durasphere. Urology.
US Food and Drug Administration, Center for Devices and Radiological Health: Summary of safety
and effectiveness data: Durasphere™ injectable bulking agent, 1999. Available at:
.www.fda.gov/cdrh/pdf/p980053.html. Accessed April 17, 2007.
Madjar S, Sharma AK, Waltzer WC, Frischer Z, Secrest CL. Periurethral mass formations following
bulking agent injection for the treatment of urinary incontinence. J Urol. 2006;175(4):1408-10.
Ghoniem GM, Khater U. Urethral prolapse after durasphere injection. Int Urogynecol J Pelvic Floor
Madjar S, Covington-Nichols C, Secrest CL. New periurethral bulking agent for stress urinary
incontinence: modified technique and early results. J Urol. 2003;170(6 Pt 1):2327-9.
Chrouser KL, Fick F, Goel A, Itano NB, Sweat SD, Lightner DJ. Carbon coated zirconium beads in
beta-glucan gel and bovine glutaraldehyde cross-linked collagen injections for intrinsic sphincter
deficiency: continence and satisfaction after extended followup. J Urol. 2004;171(3):1152-5.
Food and Drug Administration, Center for Devices and Radiological Health: Summary of safety and
effectiveness data: Coaptite®, injectable implant for soft tissue augmentation, November 10, 2005.
Available at: http://www.fda.gov/cdrh/PDF4/p040047b.pdf Accessed April 17, 2007.
Food and Drug Administration, Center for Devices and Radiological Health: Summary of safety and
effectiveness data: Radiesse®, injectable dermal filler, December 22, 2006. Available at:
http://www.fda.gov/cdrh/pdf5/p050037.html Accessed April 18, 2007.
Mayer R, Lightfoot M, Jung I. Preliminary evaluation of calcium hydroxylapatite as a transurethral
bulking agent for stress urinary incontinence. Urology. 2001;57(3):434-8.
Lemperle G, Morhenn V, Charrier U. Human histology and persistence of various injectable filler
substances for soft tissue augmentation. Aesthetic Plast Surg. 2003;27(5):354-66.
Onol FF, Tarcan T, Tinay I, Kotiloglu E, Simsek F. Kidney loss due to periureteral fibrosis and
ureteral obstruction secondary to migration of subureterically injected calcium hydroxylapatite. J
Pediat Urol. 2006;2:503-508.
Palma PC, Riccetto CL, Martins MH, Herrmann V, de Fraga R, Billis A, Netto NR Jr. Massive
prolapse of the urethral mucosa following periurethral injection of calcium hydroxylapatite for stress
urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct. 2006;17(6):670-1.
Stedman T. Stedman’s medical dictionary. 27th ed. Lippincott Williams & Wilkins. 2000.
Coldwell DM, Stokes KR, Yakes WF. Embolotherapy: agents, clinical applications, and techniques.
Ramsey DE, Geschwind JF. New interventions for liver tumors. Semin Roentgenol. 2002;37(4):303-
Agnelli G, Becattini C. Venous thromboembolism and atherosclerosis: common denominators or
different diseases? J Thromb Haemost. 2006;4(9):1886-90.
Eliasson A, Bergqvist D, Björck M, Acosta S, Sternby NH, Ogren M. Incidence and risk of venous
thromboembolism in patients with verified arterial thrombosis: a population study based on 23,796
consecutive autopsies. J Thromb Haemost. 2006;4(9):1897-902.
Prandoni P, Ghirarduzzi A, Prins MH, Pengo V, Davidson BL, Sørensen H, Pesavento R, Iotti M,
Casiglia E, Iliceto S, Pagnan A, Lensing AW. Venous thromboembolism and the risk of subsequent
symptomatic atherosclerosis. J Thromb Haemost. 2006;4(9):1891-6.
Turpie AG. Preventing thromboembolism in patients with prosthetic heart valves. Cardiol Clin.
Al-Mubarak N, Roubin GS, Vitek JJ, Iyer SS. Microembolization during carotid stenting with the
distal-balloon antiemboli system. Int Angiol. 2002;21(4):344-8.
Liapi E, Geschwind JF. Transcatheter and ablative therapeutic approaches for solid malignancies. J
Clin Oncol. 2007;25(8):978-86.
Lupattelli T, Basile A, Garaci FG, Simonetti G. Percutaneous uterine artery embolization for the
treatment of symptomatic fibroids: current status. Eur J Radiol. 2005;54(1):136-47.
Helmberger TK, Jakobs TF, Reiser MF. Embolization of uterine fibroids. Abdom Imaging.
Yoon W. Embolic agents used for bronchial artery embolization in massive haemoptysis., Expert
Opin Pharmacother. 2004;5(2):361-7.
Funaki B. Superselective embolization of lower gastrointestinal hemorrhage: a new paradigm. Abdom
Hsu AA. Thoracic embolotherapy for life-threatening haemoptysis: a pulmonologist's perspective.
Enting D, van der Werf TS, Prins TR, Zijlstra JG, Ligtenberg JJ, Tulleken JE. Massive haemoptysis:
primary care, diagnosis and treatment. Ned Tijdschr Geneeskd. 2004;148(32):1582-6.
Abe Y, Nakamura M, Suzuki K, Hashizume T, Tanigaki T, Saito T, Fujino T, Kikuchi K. Massive
hemoptysis due to Mycobacterium fortuitum infection controlled with bronchial artery embolization -
a case report. Clin Imaging. 1999;23(6):361-3.
Parker WH. Etiology, symptomatology, and diagnosis of uterine myomas. Fertil Steril.
Munro MG, Lukes AS; Abnormal Uterine Bleeding and Underlying Hemostatic Disorders Consensus
Group. Abnormal uterine bleeding and underlying hemostatic disorders: report of a consensus
process. Fertil Steril. 2005;84(5):1335-7.
Wegienka G, Baird DD, Hertz-Picciotto I, Harlow SD, Steege JF, Hill MC, Schectman JM,
Hartmann KE. Self-reported heavy bleeding associated with uterine leiomyomata. Obstet Gynecol.
Lippman SA, Warner M, Samuels S, Olive D, Vercellini P, Eskenazi B. Uterine fibroids and
gynecologic pain symptoms in a population-based study. Fertil Steril. 2003;80(6):1488-94.
Dick ML. Chronic pelvic pain in women: assessment and management. Aust Fam Physician.
Hosokawa Y, Kishino T, Ono T, Oyama N, Momose H. Two cases of female acute urinary retention
caused by an impacted pelvic mass. Int J Urol. 2005;12(12):1069-70.
Barnacle S, Muir T. Intermittent urinary retention secondary to a uterine leiomyoma. Int Urogynecol
J Pelvic Floor Dysfunct. 2007;18(3):339-41.
Iavazzo C, Myriokefalitaki E, Vorgias G, Akrivos T, Lekka I, Katsoulis M. Giant solid abdominal
mass with cystic lesions: a case report and diaphorodiagnostic approach. Bratisl Lek Listy.
Vilos GA. Uterine fibroids: relationships to reproduction. Minerva Ginecol. 2003;55(5):417-23.
Casini ML, Rossi F, Agostini R, Unfer V. Effects of the position of fibroids on fertility. Gynecol
Williams VS, Jones G, Mauskopf J, Spalding J, DuChane J. Uterine fibroids: a review of health-
related quality of life assessment. J Womens Health (Larchmt). 2006;15(7):818-29.
Mauskopf J, Flynn M, Thieda P, Spalding J, Duchane J. The economic impact of uterine fibroids in
the United States: a summary of published estimates. J Womens Health (Larchmt). 2005;14(8):692-
Day Baird D, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine
leiomyoma in black and white women: ultrasound evidence. Am J Obstet Gynecol. 2003;188(1):100-
Wise LA, Palmer JR, Stewart EA, Rosenberg L. Age-specific incidence rates for self-reported uterine
leiomyomata in the Black Women's Health Study. Obstet Gynecol. 2005;105(3):563-8.
Borgfeldt C, Andolf E. Transvaginal ultrasonographic findings in the uterus and the endometrium:
low prevalence of leiomyoma in a random sample of women age 25-40 years. Acta Obstet Gynecol
Lurie S, Piper I, Woliovitch I, Glezerman M. Age-related prevalence of sonographicaly confirmed
uterine myomas. J Obstet Gynaecol. 2005;25(1):42-4.
Schwartz SM, Marshall LM, Baird DD. Epidemiologic contributions to understanding the etiology of
uterine leiomyomata. Environ Health Perspect. 2000;108 Suppl 5:821-7.
Ligon AH, Morton CC. Leiomyomata: heritability and cytogenetic studies. Hum Reprod Update.
Wise LA, Palmer JR, Spiegelman D, Harlow BL, Stewart EA, Adams-Campbell LL, Rosenberg L.
Influence of body size and body fat distribution on risk of uterine leiomyomata in U.S. black women.
Myers ER, Barber MD, Gustilo-Ashby T, Couchman G, Matchar DB, McCrory DC. Management of
uterine leiomyomata: what do we really know? Obstet Gynecol. 2002;100(1):8-17.
Kjerulff KH, Langenberg PW, Rhodes JC, Harvey LA, Guzinski GM, Stolley PD. Effectiveness of
hysterectomy. Obstet Gynecol. 2000;95(3):319-26.
Hartmann KE, Ma C, Lamvu GM, Langenberg PW, Steege JF, Kjerulff KH. Quality of life and
sexual function after hysterectomy in women with preoperative pain and depression. Obstet Gynecol.
West S, Ruiz R, Parker WH. Abdominal myomectomy in women with very large uterine size. Fertil
Muñoz JL, Jiménez JS, Hernández C, Vaquero G, Pérez Sagaseta C, Noguero R, Miranda P,
Hernández JM, De la Fuente P. Hysteroscopic myomectomy: our experience and review. JSLS.
Hurst BS, Matthews ML, Marshburn PB. Laparoscopic myomectomy for symptomatic uterine
myomas. Fertil Steril. 2005;83(1):1-23.
Chwalisz K, Perez MC, Demanno D, Winkel C, Schubert G, Elger W. Selective progesterone
receptor modulator development and use in the treatment of leiomyomata and endometriosis. Endocr
Chavez NF, Stewart EA. Medical treatment of uterine fibroids. Clin Obstet Gynecol. 2001;44(2):372-
Boelen EJ, van Hooy-Corstjens CS, Bulstra SK, van Ooij A, van Rhijn LW, Koole LH. Intrinsically
radiopaque hydrogels for nucleus pulposus replacement. Biomaterials. 2005;26(33):6674-83.
van Hooy-Corstjens CS, Govaert LE, Spoelstra AB, Bulstra SK, Wetzels GM, Koole LH. Mechanical
behaviour of new acrylic radiopaque iodine-containing
Kruft MA, Benzina A, Blezer R, Koole LH. Studies on radio-opaque polymeric biomaterials with
potential applications to endovascular prostheses. Biomaterials. 1996;17(18):1803-12.
Aldenhoff YB, Kruft MA, Pijpers AP, van der Veen FH, Bulstra SK, Kuijer R, Koole LH. Stability
of radiopaque iodine-containing biomaterials. Biomaterials. 2002;23(3):881-6
bone cement. Biomaterials.
Injectable Polymeric Micropsheres with X-ray visibility.
Preparation, Properties, and potential utility as New
Traceable Bulking Agents
Ketie Saralidze, Yvette B.J. Aldenhoff, Menno L.W. Knetsch, and
Leo H. Koole
Biomacromolecules 4 (2003), 793-798
The copolymer of methyl methacrylate (MMA) and 2-[2’,3’,5’-triiodobenzoyl]oxoethyl
methacrylate (1), ratio 3:1 (mass/mass) was prepared via a free-radical polymerization
in bulk. The copolymer (Mw = 97.9 kD and Mn = 41.5 kD) was dissolved in chloroform
and subsequently transformed into beads with a diameter in the micrometer range, using
a solvent evaporation technique. The resulting microbeads were characterized by
different techniques, including NMR spectroscopy, differential scanning calorimetry,
gel permeation chromatography, and scanning electron microscopy. The latter technique
was used as the basis for statistical analysis of the bead size. Typically, an average
diameter of 96 µm and a standard deviation of 21 µm were obtained. The beads were
also subjected to some preliminary test regarding cytotoxicity. The copolymer of MMA
and 1 contains covalently bound iodine. Therefore, the material is intrinsically
radiopaque, i.e., capable of adsorbing X-radiation while no contrast additive is needed.
Our interest in these microspheres stems primarily from their possible utility as
injectable and afterward traceable (radiopaque) bulking agents, e.g., for use in urology
for the treatment of female stress incontinence due to sphincter deficiency. As a first test
into this direction, a sample of the microbeads was mixed with ethylene glycol, and the
resulting suspension was studied with respect to injectability and radiopacity. The
results suggest that the radiopaque microbeads may provide access to improved bulking
agents. Further modification of the surface may be necessary in order to suppress the
migratory aptitude of the radiopaque polymeric microspheres in vivo.
Polymeric beads with a diameter in the micrometer range find use in various medical
applications, such as bone dements (powder fraction) , in local delivery of drugs ,
and in treatment of female stress urinary incontinence [3,4]. Medical microspheres
usually consist of poly(methyl methacrylate) (PMMA), poly(lactic acid-co-glycolic
acid), poly(tetrafluoroethylene) (PTFE), or silicone rubbers. A particularly important
application of microspheres relates to their use as so-called bulking agents; the
microspheres are injected via a syringe, usually as a suspension in a collagen solution. It
is known that injected microspheres have a tendency to migrate, depending on their
size. It is believed that beads with a diameter > 80 µm are unlikely to migrate , but it
Injectable Polymeric Microspheres with X-ray visibility
remains unclear how other factors (such as the exact site of the injection) might
influence their migratory aptitude.
Herein, we report the preparation of new polymeric microspheres with clear X-ray
visibility (radiopacity). In principle, this feature offers the advantage that injected beads
can be detected through X-ray fluoroscopy, not only shortly after the injection but also
on the long term . Microspheres consisting of PMMA, PTFE, or silicon rubbers are
radiolucent, i.e., invisible on the X-ray image. The practical utility of microspheres with
X-ray visibility lies in the fact that the clinician can assess possible migration in a direct
manner. This information may provide guidance in cases where a second injection of
bulking agent is necessary (repeated treatment).
The microspheres which are described in this work are made out of a polymer that
belongs to the poly(methacrylate) family. A copolymer of methyl methacrylate (MMA)
and a methacrylate that contains covalently bound iodine in the side chain was used.
The copolymer features intrinsic radiopacity; i.e., it has the capacity to absorb X-
radiation while no radiopaque additive is used. Biomaterials of this type have proven
biocompatibility and stability in vivo , which may explain the increased interest in
these biomaterials over the last years . The copolymer used in this work was
synthesized from MMA and 2-[2’,3’,5’-triiodobenzoyl]oxoethyl methacrylate (1), via a
free-radical polymerization in bulk. We report synthesis and physicochemical analysis
of the copolymer with ratio MMA:1 = 3:1 (mass:mass).
Furthermore, we describe a reproducible procedure, based on a solvent evaporation, for
transformation of the crude materials into microspheres with a mean diameter of
approximately 96 µm and a standard deviation of 21 µm. Size and size distribution
could be varied, and the most important process parameters to accomplish this were
identified. In addition, it is shown that the microspheres have clear X-ray visibility after
injection in a realistic model system. Furthermore, the microspheres were tested with
respect to their biocompatibility in vitro. Finally, it is discussed that these microspheres
are potentially useful as bulking agents which can be traced by X-ray fluoroscopy, after
their implantation, e.g., for treatment of stress urinary incontinence.
Materials and Methods
All materials were purchased from Sigma/Aldrich/Fluka chemicals, Acros Organics,
and Invitrogen. MMA was distilled at atmospheric pressure and stored at -20 ºC. 2-
Hydroxyethyl methacrylate (HEMA) was distilled under reduced pressure (13 mbar)
and stored at -20 ºC. Tetrahydrofuran (THF), dichloromethane, and triethylamine (TEA)
were distilled from calcium hydride and stored over either 3 Å molecular sieves (THF,
dichloromethane) or potassiumhydroxide pellets (TEA).
Synthesis of 2-[2’,3’,5’-triiodobenzoyl]oxoethyl methacrylate (1)
At room temperature, thionyl chloride (19.00 g, 159,70 mmol) was added dropwise to a
magnetically stirred solution of 2,3,5-triiodobenzoic acid (40.00 g, 80.13 mmol) in 300
mL of anhydrous THF. After completion of the addition, the reaction mixture was
heated, refluxed (30 min), and then allowed to cool to ambient temperature. All volatiles
were removed under reduced pressure (stench!). The residue, 2,3,5-triiodobenzoyl
chloride, was dissolved in 450 mL of anhydrous dichloromethane. The solution was
magnetically stirred and cooled to -5 ºC, and a solution of HEMA (11.47 g, 88.14
mmol) and TEA (32.43 g, 320.52 mmol) in 50 mL of anhydrous dichloromethane was
added dropwise. After the addition was completed, the ice bath was removed, and
stirring was continued for 1 h at room temperature. Then, the reaction mixture was
again cooled -5 ºC, and water (approximately 150 mL) was added carefully. The
reaction mixture was transferred into a separatory funnel, and the organic layer was
separated. The organic layer was washed with 0.1 M sodium bicarbonate (once) and
with brine (twice), dried over MgSO4, filtered, and concentrated. The crude product was
recrystallized from hexane/ethanol to afford 1 as a white solid in 77% yield, mp 95.5 ºC.
1H NMR (CDCl3): δ 1.95 (s, 3H, CH3), 4.48 (d, 2H, CH2CH2), 4.57 (d, 2H, CH2CH2),
5.61 (s, 1H, CH2=C), 6.16 (s, 1H, CH2=C), 7.73 (s, 1H, arom), 8.29 (s, 1H arom). 13C
NMR (CDCl3): δ 18.67, 62.24, 64.23, 94.02, 106.96, 113.78, 126.75, 135.99, 137.44,
141.15, 149.13, 165.85, 167.24. 1H and 13C NMR spectra were recorded at 399.6 and
Injectable Polymeric Microspheres with X-ray visibility
100.6 MHz, respectively, on a Varian Unity-Plus spectrometer using deuterated
chloroform as the solvent. Tetramethylsilane was used as the internal standard (δ = 0.00
A 500 mL round-bottom flask was charged with 1 (10.00 g, 16.34 mmol) and MMA
(30.00 g, 299.64 mmol), dibenzoyl peroxide (BPO, 1.2 mol%, 0.92 g, 3.80 mmol) as
initiator, and N,N-dimethyl-p-toluidine (0.13 mL, 0.91 mmol) as accelerator. Monomers
1 and MMA were miscible at room temperature in the mass:mass ratio 1:3; further
increase of the content of 1 resulted in a inhomogeneous mixture and, hence, a
nonhomogeneous copolymer. The flask was placed in a thermostated oil bath, equipped
with a programmable time-temperature control system (PM LAUDA, Könogshausen,
Germany). The temperature profile as show in table 1 was run. The procedure afforded
the copolymer as an amber-like solid. The procedure involves relatively slow
polymerization at 60 ºC, and subsequent thermal treatment at 80 and 100 ºC. It is our
experience that this temperature profile results in high conversions (typically >95%).
Presently, a conversion of approximately 96% was achieved, as judged by 1H NMR of
the crude polymer. Remnants of unreacted MMA and 1 were still present, vide infra.
Table 1. Temperature Profile for the Polymerization
temperature (ºC) time (h) temperature (ºC) time (h)
1) heat to 60 0.5 5) heat to 100 1.0
2) maintain at 60 8.0 6) maintain at 100 4.0
3) heat to 80 1.0 7) cool down to 40 2.0
4) maintain at 80 4.0
Preparation of microspheres
The copolymer was dissolved in chloroform (150 mg/mL). The solution was added
dropwise to a mechanically stirred (500 rpm) solution of 12 g of detergent (Dubro,
Proctor & Gamble) in 2 L of distilled water. Other detergents tried were Triton, Tween
(both nonionic detergents) and sodium dodecyl sulfate (anionic).
Figure 1. Overall proton NMR spectrum (δ 9.0 to -0.5 ppm) of a sample of the radiopaque
microspheres, after redissolution in deuterated chloroform. The spectrum provides evidence for
the composition and purity of the material. A and B are expansions of the spectral regions δ 6.65-
4.20 and 8.65-7.55 ppm. The two broadened singlet peaks in A correspond with the methylene
protons of iodine-containing elements in the polymer chain. The two broadened singlet peaks in B
correspond with the aromatic protons in the same elements. Assignments of the resonances in the
overall spectra are as follows: a, sharp singlet of CHCl3 traces in the solvent; b, pendant OCH3
groups; c, methylene protons in the polymer chain; d, singlet due to trace of water in the solvent;
e, pendant CH3 groups; f, tetramethylsilane (reference, δ = 0 ppm).
Injectable Polymeric Microspheres with X-ray visibility
No microspheres were obtained with Triton X-100 (poly-(oxyehtylene)(10)-
isooctylhexyl ether (CAS # 92046-34-9), 4-(C8H17)C6H4C6H10(OCH2CH2)nOH (n =
10)), or Tween 20 (poly(oxyethylene)(20) sorbitan monolaureate (CAS # 9005-64-5)),
and sodium dodecyl sulfate performed less satisfactorily, as compared to Dubro.
Stirring was continued overnight at room temperature to remove the chloroform (fume
hood). Then, stirring was stopped and the microspheres were allowed to precipitate. The
microspheres were thoroughly washed with water (2x), ethanol (3x) and water (3x). The
microspheres were transferred into a 100-mL round-bottom flask and dried by
lyophilization. Yield: 76%. 1H NMR (CDCl3): δ 0.71-2.10 (aliphatic H), 3.59 (OMe),
5.30 and 4.52 (methylene H), 7,81 and 8.36 (aromatic H) see Figure 1.
Scanning Electron Microscopy (SEM)
Morphology and size of the microspheres were characterized using SEM (RJ Lee
PSEM75, Goffin Meyvis, Etten Leur, The Netherlands). The microspheres were stuck
on an aluminum stub with double-face adhesive carbon tape. The stubs were coated
under vacuum for 2 min with a thin layer of gold and examined at an accelerating
voltage of 20 kV. Three different windows of view were chosen, and approximately 300
spheres were included in each determination of average size and size distribution.
Differential Scanning Calorimetry (DSC)
Samples (approximately 14 mg) placed in aluminum pans were analyzed with a Perkin-
Elmer-DSC-7 instrument at a heating rate of 10 deg/min. Each sample was heated
beyond the glass transition temperature, cooled rapidly, and heated again. The data
presented were collected during the second heating scan. The glass transition
temperature was taken as the midpoint of the heat capacity change. Indium and gallium
were used as standards for temperature calibration.
Gel Permeation Chromatography (GPC)
GPC was performed using a Water Wisp autoinjection apparatus (Millipore Corp.,
Milford, MA), equipped with 105, 104, and 103 µ-Styragel (Alltech, Deerfield, IL). THF
was used as the mobile phase at a flow rate of 1.0 mL/min. Detection was based on UV
(UV 440, ambient conditions) at 254 nm and reflective index (RI 410, 40 ºC).
Calibration was against a series of polystyrene standards (580 to 6 x 106 Da). We realize
that use of these standards does not provide an accurate measure of MW and MN of our
copolymer, which will have a different “molecular density” as compared to polystyrene
(i.e., different Mark-Houwink constant). Nonetheless, the experiment values for MW and
MN, as derived from the GP chromatograms and using the polystyrene standards, allow
us to conclude that the copolymer was a mixture of genuine macromolecules (vide
The radiopacity of the microspheres was tested in a realistic model, under routine
hospital conditions. First, a suspension of microspheres in ethylene glycol was made
(500 mg of microspheres/1.00 mL of ethylene glycol). The suspension was transferred
into a 2-mL syringe. A chicken leg, purchased in a local supermarket, was used as a
model. The microsphere suspension was injected into the soft tissue, and the model was
examined with a Philips Optimus Z75C radiodiagnostic system.
Microspheres were sterilized with UV light for 15 min and subsequently incubated for
48 h in culture medium at 37 ºC. Culture medium used was Dulbecco’s modification of
eagle medium/F-12 nutrient mix containing Glutamax-I and supplemented with 10%
fetal bovine serum and antibiotic/antimycotic solution (1x). Mouse 3T3 fibroblasts were
inoculated into a 96-well tissue culture plate (TCP) at a density of approximately 103
cells/well. The cells were cultured to semi-confluence, in an incubator at 37 ºC and 5%
CO2 at high (near 100%) humidity for 3 days. The incubated microspheres were placed
in the wells in such a way that estimated 15-20% coverage of the total area by the
spheres was achieved. The remaining extract medium, 100 µL, was separately added to
the cells. TCP was used as a negative control, and latex was used as a positive control.
Cells were cultured for another 3 days. Then, the medium was removed and replaced
with culture medium containing thiazolyl blue (MTT, 0.5 mg/mL). The cells were
cultured for 1-2 h, the medium was removed, and the formed precipitated formazan was
dissolved in dimethyl sulfoxide (DMSO). The absorbance of the samples was
determined using a microtiter plate reader at 550 nm.
Injectable Polymeric Microspheres with X-ray visibility
Direct Cell Contact Assay
Mouse 3T3 cells were inoculated into a 24-well TCP at a density of approximately 25 x
103 cells/well. The same culture medium as described above was used. The cells were
incubated at 37 ºC and 5% CO2 overnight. When the cells were semi-confluent, a
suspension of the microspheres in culture medium (50 µg/mL) was added to the cells.
Cells were cultured for another 4 days and examined with light microscopy. Pictures
were taken. Light microscopy was performed on a Leica DM-IL inverted microscope
equipped with a Sony DSC-70 digital camera.
Results and Discussion
The two-step synthesis of 1, starting from 2,3,5-triiodobenzoic acid and HEMA,
proceeded smoothly and with satisfactory overall yield (77%). The subsequent free-
radical copolymerization of 1 and MMA was run in bulk, using dibenzoyl peroxide and
N,N-dimethyl-p-toluidine as radical initiator and accelerator, respectively. 1H NMR
analysis of the crude product revealed that the conversion was approximately 95%. Both
unreacted MMA and 1 were present, as judged by four singlet resonances in the spectral
region δ 6.2-5.5 ppm.
The key step in the preparation of the microspheres was the dropwise addition of the
copolymer-in-chloroform solution to the stirred solution of detergent in water. Upon
falling in the turbulent aqueous medium, each drop of the copolymer solution is split
into numerous smaller droplets. These are stabilized by the detergent. Continuous
stirring results in evaporation of the chloroform. Several experimental parameters were
found to influence the average size and the size distribution of the microspheres. The
concentration of the polymer-in-chloroform solution was set at 15 g/100mL. This
resulted in microspheres with average diameter of 96 µm, and a standard deviation of 21
µm (vide infra). At lower concentrations, the average diameter became smaller (e.g., 25
µm (6 µm standard deviation) at 5 g/100mL). At higher concentrations, the procedure
failed in most cases; a clump of polymer then attached to the rotor blade. Another
important parameter, although less sensitive, was the speed of stirring. The optimal
speed was found to be 500 rpm. Slower stirring resulted in broader size distributions,
whereas faster stirring was practically impossible. A third important parameter was the
height from which each drop fell into the detergent solution: this height was fixed at
approximately 10 cm. With respect to the detergent, it was found that the most
satisfactory results were obtained with the commercial household detergent Dubro, at a
concentration of 6 g/L. After a series of test experiments, the other experimental
parameters were chosen as follows: 2 L of detergent solution in a 4-L conical glass
vessel, addition of 100 mL of the copolymer solution, time of addition 30 min.
The microspheres precipitated readily when stirring was stopped (typically 18 h after
the addition). Workup consisted of several washing steps, including three washings with
alcohol to remove monomer remnants, and lyophilization. 1H NMR analysis of the
microspheres showed the absence of free monomer (Figure 1). GPC analysis showed
that the microspheres consist of genuine macromolecular structures with MW = 97.8 kD
and MN = 41.5 kD, i.e., polydispersity = 2.4. We realize that these numbers must be
treated with caution, as a series of polystyrenes was used for GPC calibration, and the
Mark-Houwink constants for our copolymer and polystyrene are most likely different.
Figure 2. Typical scanning electron micrograph of microspheres as obtained by the solvent
Nonetheless, we can safely conclude that the copolymer consists of a mixture of
genuine macromolecules with a relatively small polydispersity. DSC showed a clear
glass transition at 104 ºC. Figure 2 shows a SEM micrograph of a typical batch of
microspheres. Size distributions of all different batches were derived from the SEM
pictures, by measuring all microspheres in several arbitrarily chosen windows of view.
Approximately 300 microspheres were incorporated in each analysis. The data were
sorted into 10 µm intervals. Figure 3 shows a histogram that compiles the results of a
Injectable Polymeric Microspheres with X-ray visibility
typical size analysis, showing an average diameter of 96 µm, and a standard deviation of
Figure 3. Size distribution, as measured for a typical batch of microspheres. The data were
derived from several independent scanning electron micrographs of the same synthetic batch
A pilot experiment was conducted in order to check whether the microspheres have
adequate radiopacity. A suspension of microspheres (500 mg) in ethylene glycol (1.0
mL) was made and transferred into a 2-mL syringe.
Figure 4. Demonstration of the X-ray visibility of injected microspheres, in a representative
model, a chicken leg as purchased in a local supermarket: (A) X-ray image before the injection
(filled syringe); (B) X-ray image after the injection. Note that the injected suspension is imaged as
clearly as the femur and tibia.
A fresh chicken leg, purchased in a local supermarket, was used as a realistic model
which allowed us to compare the X-ray visibility of (i) the microspheres after injection
in the soft tissue, and (ii) the chicken femur and tibia. Figure 4 shows the X-ray images
of the chicken leg before (left) and after (right) injection of the microspheres. The
images of Figure 4 were recorded under normal clinical conditions in equipment that is
routinely used for the detection of breast tumors.
The injected microspheres show excellent visibility, as judged by an experienced
radiologist. It was also established that comparable microspheres of PTFE, PMMA, or
silicone rubber are practically invisible under the same experimental conditions.
Two different experiments were done in order to investigate the in vitro
biocompatibility of the microspheres.
Figure 5. Survival percentage of the mouse 3T3 fibroblast cells in the MTT test.
First, the MTT test was performed, using mouse 3T3 fibroblast cells. Defining adhered
cell density onto tissue-culture-polystyrene as 100%, it was found that the adhered cell
density is 88% and 95%, in contrast with the extract and microspheres, respectively
Second, the growth of mouse 3T3 cells in direct contact with microspheres was
monitored. After 4 days, the cells formed a confluent layer on the bottom of the well, as
is shown in Figure 6. Moreover, cells appear to cover parts of the surface of the
microspheres (arrows), which may be interpreted as additional proof for the non-
cytotoxicity of the material.
Injectable Polymeric Microspheres with X-ray visibility
Figure 6. Light microscopic image of microspheres, after 4 days of incubation with 3T3 mouse
fibroblast cells. The cells adhere not only to the bottom of the well (polystyrene) but also to some
of the microspheres, indicating that the material is not cytotoxic.
In 1992, Jayakrishnan and Thanoo already described polymeric iodine-containing
radiopaque microspheres which are similar to ours [7a,b]. They reported on copolymers
of (i), the HEMA-ester of iothalamic acid (IEH) and HEMA, and (ii), triiodophenyl
methacrylate (TIPM) and HEMA [7a]. These copolymers were converted into beads
with large diameter (mostly >1 mm), also through the solvent evaporation method. The
monomer IEH has a bulky side chain, which was held responsible for the observation
that copolymerization of IEH and MMA did not produce a high-molecular-weight
copolymer. The monomer TIPM has its iodine-containing aromatic ring very close to
the reactive double bond, which may explain why the copolymerization TIPM + MMA
also failed. It is for this reason, that Jayakrishnan and Thanoo restricted themselves to
copolymers based on HEMA [7a]. These microspheres have a slightly hydrophilic
nature, and it must be assumed that they will absorb some water and become softer after
implantation. This is undesirable as far as the application as a bulking agent is
concerned. While Jayakrishnan and Thanoo observed that copolymerization of IEH or
TIPM with MMA is troublesome, we have now found that the copolymerization MMA
+ 1 proceeds smoothly. The resulting microspheres consist of hydrophobic building
blocks exclusively. Therefore, they will retain their hardness after implantation.
We anticipate that the present microspheres can be helpful in the search for an improved
treatment of female stress urinary incontinence, caused by intrinsic sphincter deficiency.
Several injectable bulking agents have been developed specifically for this purpose, but
their use is still associated with significant drawbacks and complications. Microparticles
of PTFE, formulated in a paste, have been used with reasonable success, but the safety
of this material is uncertain for two reasons: (i) the tendency of PTFE microbeads to
migrate away from the implantation site to lymph nodes, lungs, and brain  and (ii) the
risk for granuloma formation (sometimes called “Teflonoma”) . Silicone rubber
particles, suspended in hydrogel, appear to work better than PTFE. Distant migration
was found, but only for particles smaller than 70 µm [5,10]. The long-term risks of
silicone microbeads are unknown [3a]. Biological materials, such as cross-linked bovine
collagen or autologous fat, are biodegradable, and therefore the initial bulking effect is
usually lost on the long-term. A very promising new injectable bulking agent, consisting
of biocompatible and non-immunogenic zirconium dioxide beads with a coating of
pyrolytic carbon (Durasphere®) was introduced recently [3b,11]. However, clear
evidence of their migration was found . This is a puzzling observation, since
Durasphere® beads have a large diameter (ranging from 251 to 300 µm); this diameter is
at least three times larger than the reported threshold for particle size migration (80 µm)
. A factor that may be responsible in part for the apparent migratory aptitude of
Durasphere in vivo is the density of the bulk material (zirconium dioxide, 5.89 g/cm3
density), which is much higher than the density of the surrounding soft tissue
(approximately 1 g/cm3) . It must be noted that migration of Durasphere® particles
could be monitored in a straightforward manner, due to their excellent X-ray visibility
[3b]. Migration of the other polymeric beads (PTFE, silicone) is much more difficult to
assess, since these polymers are radiolucent. Our new polymeric microspheres share the
advantage of having clear X-ray visibility with Durasphere®. We envisage two
additional advantages of the new radiopaque microbeads: (i) a better match of the
density of the microbeads (approximately 1.35 g/cm3) with the surrounding soft tissue
and (ii) the possibility for further modification of the surface, e.g., to promote anchoring
in the surrounding tissue in order to minimize the risk of migration. We have started to
work on this type of surface engineering of radiopaque polymeric microspheres,
combined with testing of biocompatibility, functionality, and migratory aptitude in vivo.
Injectable Polymeric Microspheres with X-ray visibility
The microbeads described in this work are potentially useful as injectable and afterward
traceable bulking agents. This expectation is based on (i) the combined physical
properties of the copolymer and the beads, (ii) their non-cytotoxicity, and (iii) their cell
friendliness, at least with respect to fibroblasts in vitro. With respect to clinical utility, it
is mandatory that migration of the microbeads, after injection, is prevented. In vivo
animal experiments to test this point are ongoing. It is of interest that surface
modification of the microbeads may be required in order to achieve sufficient anchoring
in the soft tissues of the implantation site.
(a) Lewis GJ. Biomed. Mater. Res. 1997;38:155-182. (b) Artola A, Goni I, Ginebra P, Manero JM,
Gurruchaga MJ. Biomed. Mater. Res. 2003;64:44-55. (c) Jang JS, Lee SH, Jung SK. Spine
2002;27:416-418. (d) Espehaug B, Fumes O, Havelin LI, Engesaeter LB, Vollset SE. J. Bone Jt. Surg.
Br. 2002;84:832-838. (d) Lewis GJ. Biomed. Mater. Res. 2002;63:455-466.
(a) Tsapis N, Bennett D, Jackson B, Weitz DA, Edwards DA. Proc. Natl. Acad. Sci. U.S.A.
2002;99:12001-12005. (b) Brandau T. Int. J. Pharm. 2002;242:197-201. (c) Hickey T, Kreutzer D,
Burgess DJ, Moussy F. Biomaterials 2002;23:1649-1656. (d) Stenekes RJ, de Smedt SC, Demeester J,
Sun G, Zhang Z, Hennink WE. Biomacromolecules 2002;1:696-703. (e) Kim HD, Valentini RF.
Biomaterials 1997;18:1175-1181. (f) Emerich DF, Snodgrass P, Lafreniere D, Dean RL, Salzberg H,
Marsh J, Perdomo B, Arastu M, Winn SR, Bartus RT. Pharm. Res. 2002;19:1052-1060. (g) Du J, Jasti
B, Vasavada RC. J. Controlled Release 1997;43:223-230.
(a) Herschorn S. Can. J. Urol. 2001;8:1281-1289. (b) Dmochowski RR, Appell RA. Urology
2000;56:328-340. (c) Dmochowski RR, Appell RA. Tech. Urol. 2001;2;110-117. (d) Lightner D,
Calvosa C, Andersen R, Klimberg I, Brito CG, Snyder J, Gleason D, Killion D, Macdonald J, Khan
AU, Diokno A, Sirls LT, Saltzstein D. Urology 2001;58:12-15. (e) Barranger E, Fritel X, Kadoch O,
Liou Y, Pigne A. J. Urol. 2000;164:1619-1622. (f) Bidmead J, Cardozo L. Lancet 2000;355:2183-
(a) Peeker R, Edlund C, Wennberg AL, Fall M. Scand. J. Urol. Nephrol. 2002;36:194-198. (b)
Duckett JRA. Br. J. Obstet Gynecol. 1998;105:390-396. (b) Herschorn S, Glazer AA. J. Urol.
(a) Malizia AA Jr, Reiman HM, Myers RP, Sande JR, Barham SS, Benson RC Jr, Dewanjee MK, Utz
WJ. JAMA, J. Am. Med. Assoc. 1984;251:3277-3281. (b) Henly DR, Barrett DM, Weiland TL,
O’Connor MK, Malizia AA, Wein AJ. J. Urol. 1995;153:2039-2043.
Aldenhoff YBJ, Kruft MAB, Pijpers AP, van der Veen FH, Bulstra SK, Kuijer R, Koole LH.
(a) Jayakrishnan A, Thanoo BC. J. Appl. Polym. Sci. 1992;44:743-748. (b) Jayakrishnan A, Thanoo
BC, Rathinam K, Mohanty M. J. Biomed. Mater. Res. 1990;24:993-1000. (c) Davy KWM, Anseau
MR, Odlyha M, Foster GM. Polym. Int. 1997;43:143-154. (d) Moszner N, Salz U, Klester AM,
Rheinberger V. Angew. Makromol. Chem. 1995;224:115-123. (e) Kruft MAB, Benzina A Ba¨r
FHMW, van der Veen FH, Bastiaansen CWM, Blezer R, Lindhout T, Koole LH. J. Biomed. Mater.
Res. 1994;28:1259-1265, and 1996;32:459-466. (f) Kruft MAB, Benzina A, Blezer R, Koole LH.
Biomaterials 1996;17:1803-1810. (g) Davy KWM, Anseau MR, Berry C. J. Dent. 1997;25:499-505.
(h) Horak D, Metalova M, Rypacek F. J. Biomed. Mater. Res. 1997;34:183-188. (i) Kruft MAB, van
der Veen FH, Koole LH. Biomaterials 1997;18:31-38. (j) Koole LH, Kruft MAB, Aldenhoff YBJ,
van’t Oost NE, van Kroonenburgh MJPG, van der Veen FH. Nat. Biotechnol. 1998;16:172-176. (k)
Mottu F, Rufenacht DA, Doelker E. InVest. Radiol. 1999;5:323-335. (l) Ginebra MP, Aparicio C,
Albuixech L, Fernandez-Barragan E, Gil F J, Planell JA, Morelon L, Vazquez B, San Roman J. J.
Mater. Sci., Mater. Med. 1999;10:733-737.
(a) Aaronson IA, Rames RA, Greene WB, Walsh LG, Hasal UA, Garen PD. Eur. J. Urol.
1993;23:394-399. (b) Rames RA, Aaronson IA. Ped. Surg. Int. 1991;6:239-240.
(a) Aragona F, Artibani W. Eur. Urol. 1995;4:146-151. (b) Kiiholma P, Makinen J. Eur. J. Urol.
Harriss DR, Iacovou JW, Lemberger RJ. Br. J. Urol. 1996;78:722-728.
Lightner D, Calvosa C, Andersen R, Klimberg I, Gilberto Brito C, Snyder J, Gleason D, Killion D,
MacDonald J, Khan AU, Diokno A, Sirls LT, Saltzstein D. Urology 2001;58:12-15.
Pannek J, Brands FH, Senge Th. J. Urol. 2001;166:1350-1353.
Handbook of Chemistry and Physics, 70th ed.; CRC Press: Boca Raton, FL, 1989; p B-145.
Development of new injectable bulking agents:
Biocompatibility of radiopaque polymeric microspheres
studied in a mouse model
Pieter J. Emans, Ketie Saralidze, Menno L. W. Knetsch, Marion J. J.
Gijbels, Roel Kuijer and Leo H. Koole
Journal of Biomedical Materials Research 73A (2005), 430-436
Radiopaque polymeric microspheres have a potential as new bulking agents for
treatment of stress urinary incontinence (SUI). The advantage over existing bulking
agents lies in their X-ray visibility in situ; other polymeric bulking agents (e.g., PTFE or
silicone rubbers) are practically radiolucent (i.e., incapable of absorbing X-radiation).
Radiopacity is useful in practice because of the high spatial accuracy of X-ray imaging.
For instance, X-ray fluoroscopy can be used to assess possible migration of the bulking
agent over time or to provide guidance in cases in which a second injection of a bulking
agent is necessary (repeated treatment of SUI). Biocompatibility of injected radiopaque
microspheres was investigated in vivo by using the mouse as a model. Microspheres
were injected subcutaneously (9 animals) or intramuscularly (9 animals), and follow-up
was 8 days or 3 months. X-ray fluoroscopy gave clear images of the microspheres as an
ensemble, and it was found that no migration occurred during 3 months. Histopathology
confirmed that all microspheres stayed close to the site of the injection. The
microspheres appeared to be well tolerated; only a few giant cells, manifesting a mild
inflammatory reaction, were encountered. At 3 months, capillary blood vessels were
observed throughout the microsphere beds, and macrophages and fibroblast cells were
seen in between the microspheres. This is encouraging with respect to the intended
application, although it must be acknowledged that the data refer merely to a mouse
model. Further experiments with larger, more representative models (rabbit and goat)
are in progress.
Polymeric spheres with a diameter in the micrometer range find use in several
therapeutic strategies. Degradable polymer microspheres can be applied as vehicles for
controlled local administration of drugs. This principle is used in numerous drug
1. oral administration of low-molecular-weight heparin [1,2],
2. development of better delivery systems in antisense therapy,
3. controlled local release of cytostatic agents [4,5],
4. treatment of Heliobacter pylori through mucoadhesive gastric retention [6,7],
5. oral delivery of insulin ,
6. production of systemic and mucosal antibodies in mice , and
Biocompatibility of radiopaque polymeric microspheres in a mouse model
7. sustained release of drugs (gancyclovir) to the vitreous body of the eye .
Nondegradable polymeric microspheres find use in totally different applications; two
examples may serve to illustrate this. First, microspheres of poly(methylmethacrylate)
(PMMA) suspended in collagen or dextran microbeads suspended in hyaluronic acid
gels are used by plastic surgeons and ophthalmologists as soft tissue fillers for facial
rejuvenation . These materials provide reduction of wrinkles, volume augmentation,
and contour improvement when injected into lines and furrows. Second, stable
polymeric microspheres play a role in an emerging technique to treat recurrent stress
urinary incontinence (SUI) in women [12–14]. We focus in this article on new
polymeric microspheres that are potentially useful for SUI treatment. SUI is a
complicated phenomenon that is associated with weakening of the soft tissue around the
bladder neck. This may result from local denervation after childbearing, estrogen
deficiency, and/or congenital factors. Periurethral injection of microspheres is a
promising therapeutic option ; the microparticles are believed to provide more
stiffness to the soft tissue, thus compensating deficiency of the sphincter muscle.
Periurethral injection therapy for SUI was first described in 1938 by Murless, who used
sodium morrhuate . The first use of polymer microspheres in SUI treatment dates
back to 1973 when Berg used poly(tetrafluoroethylene) (PTFE) . Clinical
experience showed, however, that PTFE is by no means the ideal biomaterial for SUI
treatment. It is stable, biocompatible, and nonantigenic, but it induces granuloma
formation . Moreover, PTFE microparticles tend to migrate away from the injection
site , even to distant sites (e.g., lungs, brain, kidneys, and choroids plexus). Other
biomaterials did not provide an adequate solution, so far. Poly(dimethylsiloxane)
microspheres were also found to migrate, and collagen-based agents rapidly lost their
augmenting effect due to degradation in situ . Durasphere®, a new agent consisting
of microspheres of zirconium dioxide with a coating of pyrolytic carbon, was also found
to migrate, despite the relatively large diameter of the microbeads (251–300 µm) .
We recently described the preparation, properties, and biocompatibility in vitro of new
polymeric microspheres, consisting of a copolymer of methyl methacrylate (MMA) and
a methacrylate monomer that contains covalently bound iodine . We advocated that
these microspheres are potentially useful for SUI treatment; they offer an important
advantage over the existing polymeric agents. Our microspheres can be detected and
Chapter3 Download full-text
visualized directly through X-ray fluoroscopy, not only shortly after the injection but
also on the long term. In this way, the clinician can assess possible migration in a direct
manner. This information may be very useful in cases in which a second injection of
bulking agent is necessary (repeated treatment). Here, we report a study on the in vivo
biocompatibility of the new radiopaque microspheres by using a mouse model. Sterile
suspensions of the new microspheres in collagen were injected subcutaneously or
intramuscularly. The animals were sacrificed after 8 days or 3 months.
Materials and Methods
All chemicals were purchased from Sigma/Aldrich/Fluka or Acros Organics. MMA was
distilled at atmospheric pressure and stored for a maximum of 24 h at -20° C. The
iodine-containing monomer, 2-[2’,3’,5’-triiodobenzoyl]oxoethyl methacrylate (1) was
synthesized as described previously .
1H and 13C NMR spectra were recorded at 400 and 100.6 MHz, respectively, on a
Varian Unity-Plus spectrometer using deuterated chloroform as the solvent. Tetramethyl
silane (TMS) was used as the internal chemical shift reference (δ = 0.00 ppm).
Morphology and size of the microspheres were characterized by scanning electron
microscopy (SEM) with use of an RJ Lee Personal SEM75 system (Goffin Meyvis,
Etten-Leur, The Netherlands).
Differential scanning calorimetry (DSC) was performed with a PerkinElmer DSC-7
instrument. Samples (~14 mg) were placed in aluminum pans. The heating rate was 10
degrees/min. Each sample was heated beyond the glass transition, cooled rapidly, and
heated again. The glass transition temperature was measured during the second heating
scan and taken as the midpoint of the heat capacity change. Indium and gallium were