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
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(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