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University of Groningen
Low-cost antibiotic delivery system for the treatment of osteomyelitis in developing countriesRasyid, Hermawan Nagar
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Low-cost antibiotic delivery
system for the treatment of
osteomyelitis in developing
countries
STELLING EN behorende bij het proefschrift
"Low-cost Antibiotic Delivery System for the Treatment of Osteomyelitis in Developing Countries"
1. Research can be an effective method to optimize behavior of personnel involved in biomaterial-associated surgery.
2. Orthopedic surgeons in developing countries are faced with diverse challenges posed by the complications resulting from management of fractures by the traditional bone setters. This
thesis
3. Clinically, the adagium "once osteomyelitis, always osteomyelitis" is still valid for patients with osteomyelitis and the disease remains difficult to treat by conventional means. This thesis
4. Biofilms form the key in the development and persistence of chronic bone infections. This thesis
5. Release kinetics, antibacterial spectrum, and mode of action of antibiotics (bacteriostatic or bactericidal) are of importance in the development of drug-releasing systems. This thesis
6. The elution characteristics of specific antibiotics from bone cement beads vary depending on the type of cement and the preparation of the beads. This thesis
7. Handmade beads must be uniform in size, possess sufficient porosity at their surface, and have an optimal area-to-volume ratio to obtain maximal release rates. This thesis
8. Application of the principles of percolation theory is useful to design bead systems with an optimal antibiotic release. This thesis
9. A semi-manual beads template system can successfully produce antibiotic loaded beads with uniform size, optimal shape and porosity. This thesis
10. In vitro efficacy of handmade, fosfomycin-loaded Indonesian beads is unacceptably low in
comparison with Septopal® beads. This thesis
11. Gentamicin-loaded beads prepared with half the amount of prescribed monomer and with a biodegradable filler added are promising as an alternative in clinical practice for developing countries, mainly because of economical reasons. This thesis
12. Cheap solutions are sometimes the best.
13. Developing countries should set up their own biomedical technological industries in order to make these technologies available to their own people.
14. Collaboration between institutes in developing and developed countries should be mutually beneficial.
15. Tolerance of public smoking is a reprehensible disregard of public health.
16. Innovation requires new ideas and strong competition. ------------
Hermawan Nagar Rasyid Groningen, 9 February 2009
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Cover design by Yanti, Donny Danudirdjo and Hermawan Nagar Rasyid.
The front cover photograph shows a sequence of osteomyelitis treatment using handmade antibiotic-loaded PMMA beads.
The back cover represents a picture of handmade beads made by scanning electron microscopy.
Ph.D. Thesis, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
Printed by PrintPartners lpskamp, Enschede Copyright© 2008 by Hermawan Nagar Rasyid, Groningen, The Netherlands
ISBN (book)
ISBN (digital document)
/ rijlcsuniversiteit gron1ngen
Low-cost antibiotic delivery system for the treatment of osteomyelitis in developing
countries
Proefsch rift
ter verkrijging van het doctoraat in de Medische Wetenschappen
aan de Rijksuniversiteit Groningen op gezag van de
Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op
maandag 9 februari 2009 om 16.15 uur
door
Hermawan Nagar Rasyid
geboren op 22 december 1957 te Jakarta, Indonesia
Ci:ntrale
Medische
Bibliotheek
Groningen
u
M C
G
Promotores: Prof.dr.ir. H.J. Busscher Prof.dr. H.C. van der Mei Prof.dr. J.R. van Horn Prof.dr. Soegijoko Soegijardjo
Leescommissie:
Paranimfen:
Prof.dr. J.M.M. Hooymans Prof.dr. W.M. Molenaar Prof.dr.ir. G.J. Verkerke
Drs.ing. Marten Koetsier Dyah Ekashanti Octorina Dewi, ST, MT
Contents
Chapter 1 Introduction and aim of this thesis 1
Chapter 2 Efficacy of handmade fosfomycin-loaded 25 PMMA -beads in vitro
Chapter 3A Concepts for increasing the gentamicin 47 release from bone cement beads
Chapter 3B Addition of soluble fillers to achieve 63 percolation in antibiotic-loaded PMMA beads
Chapter 4 A template to produce antibiotic-loaded 75 PMMA beads in the operating room
Chapter 4- Gentamicin-release from template made 91 appendix beads including PVP17
Chapter 5 In vitro evaluation of hand-made gentamicin-93 loaded PMMA beads to prevent biofilm
formation
Appendix 101
General discussion 105
Summary 113
Samenvatting 119
Ringkasan 127
Acknowledgements 135
Short CV 141
Chapter 1
Introduction and Aim
of this Thesis
Chapter 1
Introduction
The word osteomyelitis is derived from the Greek "osteon" meaning bone and
"myelos" meaning marrow. Osteomyelitis is a refractory condition, potentially leading
to death or amputation. In practice it refers to an infection of the bone tissue. It is still
a major cause of morbidity and remains a difficult condition to treat by conventional
means. Clinically, the adagium is "once osteomyelitis, always osteomyelitis."
Throughout life, bone is constantly renewed and replaced, and small colonies of
bacteria can become sealed within the bone during longstanding infection. Here they
can survive in a dormant state for many years without producing any symptoms.
However, at times of lowered resistance and with increasing age, infection may break
out causing pain and discharge from sinuses surrounding the bone, and the use of
antibiotics often is a first option. Antibiotic misuse is, however, a worldwide problem
with the extent of the problem being greater in the developing countries through their
purchase (without prescription) in local pharmacists and drug stores, and through
inappropriate prescribing habits and an over-zealous desire to treat every infection.
The misuse involves both overuse and under-use, where both types of uses are
inappropriate. Growing misuse of antibiotics also has been reported in hospitals,
causing toxic effects and various infections due to resistant microorganisms that
increase the cost and duration of hospitalization. Increased cost of health care will
definitely jeopardize the capacity of the poor population to seek modern health care.
In developing countries, potential obstacles to the delivery of orthopedic care
include inadequate resources and difficulties in accessing health care services due to
geographic constraints. Large populations are concentrated in huge cities or the
capital city. In developing countries, motor vehicle accidents are now the third highest
cause of death 1. Factors include the large number of pedestrians and cyclists (many
2
Introduction
living below the poverty line) involved in these accidents; overcrowding of public
transport; poor maintenance of roads; few speed restrictions; and the failing medical
management and treatment of trauma. In these countries a large deficiency exists of
doctors and paramedical personnel, there are only a few orthopedic surgeons, and
hospital facilities are often very limited. Few patients can afford to pay for any
treatment at all, let alone for orthopedic appliances or hospital charges of around
USD 17.2 per day2. Moreover, in general there are no government funded health
insurances in developing countries. Therefore, most patients have to refuse medical
treatment for their traumatologic sequelae as for instance fractures and suffer from
them of an accident or illness until permanent deformities or death result.
Mismanagement of open fractures by traditional bonesetters is one of the
causes for osteomyelitis in developing countries. Interestingly, many of the patients
initially managed in a medical facility, leave this professional care voluntarily and
seek for these traditional forms of treatment. Although overall results of these would
be graded as poor in many cases by Western standards, the traditional bonesetters
have the respect of their local communities, and treat many patients who would
otherwise not have received care at all. From the view of orthopedics, it is not a good
idea to visit bonesetters since they can create a situation in which it may be very
difficult to manage the complications of their treatment.
In the first part of this chapter the pathophysiologic changes occurring in bone
infection, bacterial contamination and adhesion, biofilm development, non-implant
associated chronicity of the infection, the osteomyelitis staging system, as well as
diagnosis and the management of the infect defect will be dealt with. In the second
part, the health care situation in developing countries will be described and compared
with the Western world, both with emphasis on the treatment of osteomyelitis. The
3
Chapter 1
third part reviews the concept of antibiotics in bone cement as one of several
methods applied in the prevention and treatment of osteomyelitis. The final part of
this chapter describes the research objectives of this thesis.
Pathophysiologic changes in bone infection
The blood supply of bone is at risk when trauma and infection occur. Surgical and
traumatic wounds often disrupt the endosteal blood supply and strip the periosteal
vessels, which can lead to dead cortical bone. This dead cortical bone may be
revascularized and subsequently remodeled or be resorbed. When infection persists,
the local defense and repair mechanisms act to try to wall off, isolate and eradicate
the dead bone. The walled-off dead bone segment is a sequestrum, which harbors
bacteria with the potential to proliferate and attack surrounding tissue. The
granulation tissue that forms around the infected area is an attempt to isolate the
organisms while the inflammatory immunologic defense mechanisms eradicate the
infection. Reactive bone, or involucrum, subsequently is formed as a further attempt
to wall off the infection from the host. Once fibrous and bony encapsulation has
occurred, antibodies and inflammatory cells must cross this hypovascular fibrous
membrane and involucrum to eradicate the organisms. However, this protective
walling off which isolates the host from the infection also shields the microorganisms
from the host defense mechanisms, providing a host-generated bacterial line of
defense3•4.
In osteomyelitis, antibiotics do not readily penetrate bone, making infection
difficult to eradicate by conventional means. In addition, the biofilm mode of growth
protects the infecting bacteria against antibiotic therapy and the natural host defense
mechanisms. Biofilms were perceived (and pictured) as simple "slabs" of matrix
4
Introduction
material in which sessile bacteria are randomly embedded. Moreover, inadequate
treatment of the acute phase of osteomyelitis allows the local pathological process
either to persist and become chronic or to become relatively quiescent for a time,
only to recur at a later date.
Osteomyelitis can be caused by direct inoculation of bacteria as a result of
trauma, by spread of bacteria from adjacent infected areas such as ulcers, or by
seeding of bacteria from infections elsewhere in the body via bloodstream. The
causal organisms are usually Staphylococcus aureus, though in young children
Streptococcal infection is very common. Other references revealed around 50 to 70%
may be caused by Streptococcus pyogenes, Escherichia coli, Proteus and
Pseudomonas strains5•9•
Acute bacterial osteomyelitis carried 50% mortality in the pre-antibiotic era
because of overwhelming sepsis with metastatic abscesses 10• Although antimicrobial
drugs have dramatically changed the prognosis of the acute haematogenous form,
chronic bacterial osteomyelitis remains a challenging medical problem. Despite
advances in antibiotic development, it all too often remains refractory to
chemotherapeutic treatment alone 11•
Rodet produced the first experimental acute haematogenous osteomyelitis in
1884: injection into rabbits with "un micrococcus qu'il possede une couleur jaune
orange" (S. aureus) resulted in the occurrence of multiple bone abscesses 12• Since
then various animal models have been studied, including rodents, chicken, rabbits,
dogs and, more recently sheep 13. Some predisposing factors intervene at various
levels 14 (see Figure 1 ), such as the fact that healthy bone tissue is extremely
resistant to infection 14•15. The presence of bone necrosis, heavy contamination or
5
Chapter 1
foreign bodies, as well as general predisposing factors such as diabetes and
peripheral vascular disease tip the balance in favor of the bacterium.
Bacterial contamination (direct inoculus or haematogenous)
• Bacterial adhesion to bone or implant
f"'""'"""""'"'"'"'"' .....
Predisposing factors Acute Infection
L .................... ..,. •
Chronic infection
Figure 1. Pathogenesis of chronic bacterial osteomyelitis (adapted from Ciampolini and Harding 14).
Bacterial contamination, adhesion and biofilm development
S. aureus has been known to bind fibrinogen for some decades. This may well
provide an explanation for the ability of this microorganism to survive in body fluids.
Bacteria clump together and are covered in a layer of fibrinogen, thus protected from
host defense mechanisms and antibiotics 16. However, this alone does not explain
how bacteria adhere to bone matrix. Staphylococcus species express high affinity
receptors (adhesins) for fibronectin 17•18, collagen 19, and laminin20• Fibronectin, a
glycoprotein found in many body fluids and connective tissue matrices, appears to be
particularly relevant to the pathogenesis of chronic osteomyelitis: bacterial adherence
to polymers similar to the ones used in orthopedic surgery is mediated by
fibronectin21•22. Fibronectin, fibrinogen, and laminin have been demonstrated to be
responsible for adherence of S. aureus to the surface of a foreign body in an animal
6
Introduction
model23. The same glycoprotein has been shown to mediate bacterial adhesion to
metal plates and screws24• Sublethal doses of antibiotics have been shown to inhibit
mucosa! adhesion by group A streptococci and E. co/125•
Non-implant associated chronicity
Chronic osteomyelitis has been shown to result from persistence of the acute
haematogenous form in 4.4% of children in a recent Scottish study26• Waldvogel et
a/. in 1970 reported development of chronicity in 15% of adults 11• Chronic
osteomyelitis with one or two sequesters may present itself as a recurrent or
intermittent disease (see Figure 2), with periods of quiescence of variable duration.
Relapses of the disease several decades after the acute episode are well known.
Late reactivation of osteomyelitis up to 80 years after the primary illness had been
"cured", have been reported27•28• No definite predisposing factor was identified, and
both haematogenous and post-traumatic infections seemed equally to be involved14•
Figure 2. X-ray (AP and Lateral view) of a patient with chronic osteomyelitis of upper part of proximal right humerus. Large sequestered areas can be seen (see arrows).
7
Chapter 1
S. aureus, mainly non-encapsulated variants, can be internalized by chicken
osteoblasts29 and endothelial cells30 in vitro and survive intracellularly, protected from
host defense mechanisms and antibiotics. This might explain the known problem of a
flare up of osteomyelitis with no identifiable causative organism. Furthermore,
staphylococci can also acquire a very slow metabolic rate, in a phenotypic alteration
named small colony variant. Slowly growing bacteria have been known to be
resistant to antibiotics since 1942, active cell wall synthesis such as occurs in rapidly
dividing bacteria being necessary for penicillin to be bactericidal. Small colony
variants of S. aureus were described for the first time in 1932 by Hoffstadt and
Youmans as minuscule bacterial colonies (less than 1 mm) that grew very slowly and
often required magnification to be seen31 • Small colony variants were found to be
resistant to penicillin one year after its discovery by Fleming32• Small colony variants
may indeed account for the frequent failure to identify the causative microorganisms
in chronic osteomyelitis: these strains may be easily missed or overgrown in a busy
laboratory. They may also account for the frequent clinical presentation of chronic
osteomyelitis as a slow, indolent infection that causes little inflammatory response
and persists despite prolonged antimicrobial therapy.
The staging of osteomyelitis
The Cierny-Mader staging system is based on the status of the disease process, not
etiology, chronicity or other factors (Table 1 )33• The terms "acute" and "chronic" are
not used in the Cierny-Mader system. The stages in this system are dynamic and
may be altered by changes in the medical condition of the patients (host), successful
antibiotic therapy and other treatments. Although the classification systems for
osteomyelitis help describe the infection and determine the need for surgery, the
categories do not apply to special circumstances (i.e. infections involving prosthetic
8
Introduction
joints, implanted materials or smaller bones of the body) or special types of infection
(e.g. vertebral ostemyelitis).
Table 1. Ciemy-Mader staging system for osteomyelitis (adapted from Ciemy et al. 33)
Anatomic type
Stage 1 : medullary osteomyelitis
Stage 2: superficial osteomyelitis
Stage 3: localized osteomyelitis
Stage 4: diffuse osteomyelitis
Physiologic class
A host: healthy
B host:
Bs: systemic compromise
Bl: local compromise
Bis: local and systemic compromise
C host: treatment worse than the disease
Factors affecting immune surveillance, metabolism and local vascularity
Systemic factors (Bs): malnutrition, renal or hepatic failure, diabetes mellitus, chronic
hypoxia, immune disease, extremes of age, immunosuppression or immune deficiency
Local factors (Bl): chronic lymphedema, venous stasis, major vessel compromise,
arteritis, extensive scarring, radiation fibrosis, small-vessel disease, neuropathy, tobacco
abuse
The signs and symptoms as presented in diagnosis and management of
osteomyelitis
Patients with chronic osteomyelitis (local pain, persistent sinus tract drainage) are
suggestive, but often not diagnostic, of their condition. In Figure 3A, the picture of an
affected side is shown. This picture shows two large-size sinus tracts on the lateral
9
Chapter 1
side of the lower left femur in a patient with chronic osteomyelitis. Pus emerges from
these sinuses every day for already three months. From a lateral X-ray of the left
femur (see Figure 38), it can be seen that various sizes of bone defects with
sequester are in it.
Chronic osteomyelitis can seldom be completely eradicated until all the
infected dead bone has separated, or sequestrated, and has either been extruded
spontaneously through a sinus tract or been removed surgically (sequesterectomy).
Antibiotic therapy alone has led to dismal rates of eventual cure. Despite advances in
both antibiotics and surgical treatment, the long-term recurrence rate remains at
approximately 20-30%. As a result, chronic osteomyelitis has generally been
characterized as having variable periods of quiescence followed by flare-ups, which
may continue throughout the patient's lifetime34, even in this era of modern treatment
modalities.
Figure 3A. Two persistent sinus tracts on the lateral left upper leg of a chronic osteomyelitis case (see arrows) Figure 3B. Radiograph of the corresponding femur revealing osteolysis, periosteal reactions and sinus tracts ( see arrows)
10
Introduction
At surgery, multiple aerobic and anaerobic cultures are obtained from pus and
excised tissue from the persistent sinus tract. In developing countries, patients are
empirically put on fosfomycin (2 g twice a day for five days) or a more specific
antibiotic. For intravenous injection, fosfomycin is generally diluted in 100 ml of
sterile saline (0.9% NaCl) or 5% glucose in water and injected with infusion periods of
30 min. 2 g fosfomycin yields serum levels of around 259.3 µg/ml at 0 h, decreasing
after 8 h to around 6.8 µg/ml35. The site of osteomyelitis is extensively exteriorized
using curettes and guttering techniques to be sure that all infected and non-viable
bone and soft tissue are removed6• Using pulsatile lavage, the wound is cleansed
with between 4 to 6 L of sterile saline solution. Next, the wound is rinsed using full
strength Betadine and hydrogen peroxide while continuing pulsatile lavage. A final
washing using sterile saline solution follows. After the removal of all dead bone the
remaining defects are filled with fosfomycin-loaded beads. The wound is then packed
using only saline-soaked gauze with antibiotics or Betadine and the skin is loosely
closed to retain the gauze. In Figure 4, the X-ray (AP and Lateral views) of right
humerus was taken. It demonstrates the insertion of fosfomycin-loaded bone cement
beads in the bone after debridement. After 2 to 4 weeks in place, the beads are
removed and the space is filled with a cancellous bone graft from proximal tibia. If the
beads are inserted for a longer period of time, for say 4 to 6 weeks, beads become
surrounded by dense scar tissue and identification and removal of beads can
become extremely difficult.
The efficacy of the use of fosfomycin-loaded beads in combination with
surgical debridement and systemic administration in developing countries has never
been truly evaluated, although success rates of almost 40-50% are claimed.
Rigorous data are lacking however, due to lack of clinical follow-up (see also below).
11
Chapter 1
Figure 4. X-rays {AP and Lateral views) from a patient with chronic osteomyelitis of upper part of rightproximal humerus. Antibiotic-loaded beads can be observed in the middle of the bone defect (see arrows).
In Europe, commercially available gentamicin-polymethylmethacrylate (PMMA)
beads (see Figure 5) are an effective drug delivery system for local antibiotic therapy
in bone and soft-tissue infections36, and these are often used in combination with
systemically delivered antibiotics and surgical debridement. The gentamicin
concentrations at the site of infection are far higher than can be achieved after
systemic application of the same antibiotic and far above the minimal inhibitory
concentrations of most common pathogens. Because of the very low concentrations
in the serum and urine after implantation of the antibiotic bead chains, toxic side
effects are not to be feared. Radical debridement with removal of all sequestrated
bone fragments and removal of all alloplastic implants is mandatory before
implantation of gentamicin-PMMA chains into the infected bone cavity. Primary
wound closure is necessary to achieve high local concentrations. The chains are
12
Introduction
used for temporary filling of osteomyelitic cavities. However, since PMMA itself is
associated with a foreign body reaction, the beads should always be removed. There
are distinct advantages of this form of antibiotic therapy in chronic osteomyelitis, such
as increased patient comfort by primary wound closure, no need for prolonged
systemic antibiotic therapy with toxic side effects, no irrigation-suction-drainage, early
ambulation, shortening of hospitalization, and reduced cost.
Figure 5. Sample of commercially available gentamicin-loaded PMMA beads on an insertion device
The health care situation in developing countries
The health care situation in developing countries completely differs from the Western
world. Biomedical technology and biomaterials implants have become integral parts
of modern health care in the Western world. Organ replacement in kidney failure,
internal fixation for bone fractures or artificial hips, knees, shoulders and elbows for
the restoration of function after oncological surgery, trauma or due to advanced age
would be impossible and many diagnoses could not be made without the assets of
biomedical technology. The costs of these, however, are enormous. In developing
countries, the average life expectancy is low, and the types of diagnoses to be made
and the reasons why people need biomaterials implants differ from those in the
Western world. The devastating prospects of patients in need of a biomaterials
13
Chapter 1
implant while not being able to afford new ones, have led to the extensive re-use of
implants from one patient into another. Surgeons in developing countries do all they
can to bridge the gap between both worlds. In developing countries the orthopedic
work predominantly involves trauma and osteomyelitis. Treatment of most problems
is complicated by patient delays in obtaining medical care, usually related to
transportation difficulties in a steep mountainous land with few roads. Since the
people have difficulties in accessing health care, they often use the traditional
bonesetters to treat their illness. This occurs not only in rural areas, but it can also
occur in major cities. In referral hospitals, it is common for patients to present
themselves when the disease is in an advanced state, usually after having sought
help by a conventional native medicine or bonesetter. In treating open fractures, they
usually use herbs to put on the wound and pieces of woods for splinting on the
affected site to maintain bone alignment. It can also occur, that the patients come to
the orthopedic clinic with persistent sinuses on their legs with chronic osteomyelitis
after having had treatment by a bonesetter. In many developing countries, traditional
bonesetters currently continue to treat large numbers of patients. They can cure the
patients without considering possible deformities that may develop, such as
dislocation of the joint, malunion, non union of bone fracture, or even infection.
Generally they do not know how to manage open fractures or sterility of the
instruments. In developing countries, complex societal issues interfere with medical
care, such as the misuse of antibiotics by physicians, pharmacists, and the public;
the suboptimal quality of the drugs; and conditions such as crowding, lack of hygiene,
poor or nonexistent hospital infection control practices, or insufficient surveillance
(dissemination).
14
Introduction
There are many poor people in developing countries, for instance in Indonesia
that are unable to obtain good health care, in particular orthopedic cases. Chronic
infection of the bone is one example of a disease for which the orthopedic patient can
not get the appropriate treatment. This problem could be dealt with by the
implantation of antibiotic-loaded beads as made and sold in the Western world, but
the price of antibiotic-loaded beads is far too high for most patients in developing
countries. Therefore, the morbidity rate is quite high, leading to a high prevalence of
crippled people. New concepts should therefore be developed for the preparation of
antibiotic-loaded beads for curing infection, available for orthopedic application at
locally affordable costs.
As mentioned earlier, gentamicin-PMMA beads which were industrially
manufactured have been approved for their efficacy and effectiveness in treating
osteomyelitis. The antibiotic-loaded acrylic beads used in most of the Western world
are industrially made, highly porous, releasing 80% of their antibiotic content within
10 to 15 days37. In Indonesia, orthopedic surgeons use bone cement, mix it
themselves with antibiotic (mostly fosfomycin) in home-made molds and use them in
their patients. It can be doubted, however, whether such beads actually release
antibiotic due to their lack of porosity. When applied as cement for the fixation of
prostheses, it is known that this material generally does not release more than 15%
of its antibiotic content38.
In our experiences in Bandung, patients treated for osteomyelitis obey to do
follow-up regularly for the first 12 weeks after treatment and usually there are no
signs of flare-ups at this stage. After 24 weeks, however, osteomyelitis signs recurred
in 20% of all patients that continue to obey follow-up (an estimated 45% of all cases
do not come back for follow-up, see Figure 6).
15
Chapter 1
Patients showing recurrence are examined and receive extended
administration of oral antibiotics in addition to the routine osteomyelitis treatment. An
estimated 35% of these recurrency patients (7% of the initial patient group) are cured
while eventually the remainders of recurrency patients decide for whatever reason
not to re-present themselves at the hospital.
(45)
(55) 35 Cured
20 Sick
Repeat Treatment
13 stop treatment ( do not come back)
7 cured
Figure 6. Number of patients treated in Hasan Sadikin hospital together with the results
As mentioned, 45% of all cases do not come back for follow-up after 24
weeks, and we have no information about these patients. The reasons for this can
only be speculated upon. Some may be cured, but more likely ran out of money when
the disease recurs, or are disappointed in the hospital treatment due to the
recurrence and turn to a bonesetter (again). In this respect, it must also be realized
16
Introduction
that the distance people have to travel in order to reach a hospital can be quite large
and transportation is not always available, which makes it even more tempting to visit
a local bonesetter. This phenomenon may furthermore be influenced by the strong
belief of most people in Indonesia on their culture, traditional values, or even mystic.
The concept of antibiotics in bone cement
The use of local antibiotics for the treatment of musculoskeletal infection has become
increasingly popular for a variety of reasons. The basis for developing and using local
antibiotic delivery systems in the treatment of infection is either to supplement or to
replace the use of systemic antibiotics. High local levels of antibiotics facilitate
delivery of antibiotics by diffusion to avascular areas of wound infection that are
inaccessible by systemic antibiotics and in many circumstances the organisms that
are resistant to drug concentrations achieved by systemic antibiotics are susceptible
to the extremely high local drug concentrations provided by local antibiotic delivery.
The potential for antibiotic incorporation in bone cement was first proposed by
Buchholz and Engelbrecht39• By mixing gentamicin in acrylic bone cement, they
succeeded in reducing the infection rate. Many studies have now appeared on the
safety and efficacy of individual antibiotic and bone cement combinations. Fischer et
a/.40 and Greco et a/.41 have recorded the three criteria for a combination of antibiotic
with bone cement to be of clinical value:
(a) the antibacterial activity of the antibiotic must be conserved upon polymerization;
(b) the mechanical properties of the cement must not be affected by the antibiotic
additive (not for application in non-load bearing applications of course, as in beads);
(c) there must be no hypersensitivity reaction.
17
Chapter 1
Adverse chemical interaction between bone cement and antibiotic additive is
uncommon and theoretically most of the available antibiotics can be added in the dry
state. Mixing of the anhydrous polymer and antibiotic powder components should be
meticulous, and fluidization is advisable whenever possible. The mechanisms by
which antibiotics are released from bone cement is unclear. Diffusion through the
cement matrix has been postulated, although it was subsequently refuted42 • Release
of the drug through pores formed between the methacrylate matrix has been
suggested, although examination of the ultrastructure of cured cement does not
support this view43• In general, antibiotic is released from bone cement in a biphasic
manner, meaning high elution in the first hours to days post surgery and after a few
days it slows down considerably.
The effectiveness of gentamicin-PMMA beads is being hampered by the
emergence of gentamicin resistance44• New antimicrobial agents are therefore
needed. A class of antibiotics often used in developing countries is fosfomycin
sodium (Fosmicin). Fosfomycin sodium was first reported in 1977 by Woodruff et
a/.45• It is a nontoxic broad spectrum antibiotic, different in structure from all
previously described antibiotics, and acts selectively by inhibiting cell wall formation.
Fosfomycin sodium is an antibiotic with an extremely low molecular weight of 138 Da,
produced by strains of Streptomyces46, and is characterized by structural features of
an epoxide ring and a carbon-phosphorus bond. It is also characterized by its action,
which inhibits the first step of peptidoglycan biosynthesis, and is synergistic in
combination with many other antimicrobial agents45.47. Moreover, fosfomycin sodium
is an antibiotic that remains stable when exposed to the high temperatures generated
during the curing of the bone cement.
18
Introduction
New methods are needed to cure chronic osteomyelitis through considering
cost effectiveness and efficacy in killing the infecting bacteria. Currently, in Indonesia
handmade fosfomycin sodium-loaded beads with the artificial resin PMMA as a
matrix are commonly used as made according to local habits of the orthopedic
surgeons. No scientific reports exist on the actual efficacy of these beads. For the
purpose of this thesis, a number of orthopedic surgeons have been asked to prepare
antibiotic-loaded beads according to their own specific habits (see Figure 7). As can
be seen, there is already a great variety in size and shape. Note that the area to
volume ratio of the beads, preferably large for efficient release, varies greatly from 1
to 1.5 cm-1 , which is low compared to Septopal® beads, as used in the Western world
1 cm
4 cm
.... ..__.._��-------------� ---
Figure 7. Examples of handmade fosfomycin-loaded beads
1 9
Chapter 1
Aim of the thesis
The purpose of this thesis is to develop a new method for the preparation of
antibiotic-loaded beads for treating chronic osteomyelitis in developing countries, with
proven efficacy for orthopedic application at locally affordable costs.
20
Introduction
References
1 . Cumming, W.J. World Orthopedic Concern Newsletter 1 998;76:1-3.
2. Ministry of Health and Social Welfare Country Information: A Review of Health Development in Indonesia. A paper presented to the WHO DG. 2000, Jakarta.
3. Dougherty, S.H. Pathobiology of infection in prosthetic devices. Rev Infect Dis 1 988;1 0:1 102-1 1 1 7.
4. Evans, R.P., Nelson, C.L. and Lange, T.E. Pathophysiology of Osteomyelitis; in Mccollister Evarts C. editor. Surgery of the Musculoskeletal System. Ed 2. New York, Churchill Livingstone 1 990;4301-4312.
5. Dougherty, S.H. and Simmons, R.L. Infections in bionic man: the pathology of infections in prosthetic devices, part 2: Infections in implanted prosthetic devices. Current Problems in Surgery 1982;19:269-318.
6. Canale, S.T. Infection: in Campbell's Operative Orthopaedics, 9th ed, Vol. 1, Mosby Inc, St. LouisMissouri 1 998;551-559.
7. Apley, G.A. and Solomon, L. Infection: in System of Orthopaedic and Fractures, 8th ed, Oxford University Press Inc, New York 2001 ;31-43.
8. Spivak, J.M., Di Cesare, P .E., Feldman, D.S., Koval, K.J., Roklto, A.S. and Zuckerman, J.D. Post Traumatic Osteomyelitis; in Orthopaedics A Study Guide, 1 st International ed, McGraw Hills Co, New York 1 999:57:1-2.
9. Jones, M.E., Karlowsky, J.A., Draghi, D.C., Thornsberry, C., Sahm, D.F. and Nathwani, D. Antibiotic susceptibility of bacteria most commonly isolated from bone related infections: the role of cephalosporins in antimicrobial therapy. Int J Antimicrob Agents 2004;23:240-246.
1 0. Joyner, A.L. and Smith, D.T. Acute Staphylococcus osteomyelitis. Surg Gynecology Obstetry 1936;63:1-6.
1 1 . Waldvogel, F.A., Medoff, G. and Swartz, M.N. Osteomyelitis: A review of clinical features, therapeutic considerations and unusual aspects {first of three parts). N Engl J Med 1970;282: 1 98-206.
1 2. Rodet, A. Etude experimentale sur l'osteomyelite infectieuse. Comptes Rendus de l'Academie des Sciences 1 884;99:569-571 .
1 3. Kaarsemaker, S., Walenkamp, G.H. and Van De Bogaard, A.E. New model for chronic osteomyelitis with Staphylococcus aureus in sheep. Clin Orthop 1 997;339:246-52.
14. Ciampolini, J. and Harding, G.K. Pathophysiology of chronic bacterial osteomyelitis. Why do antibiotics fail so often? Postgrad Med J 2000;76:479-483.
1 5. Scheman, L., Janota, M. and Lewin P. The production of experimental osteomyelitis. JAMA 1 941 ; 11 7:1525-1529.
1 6. Duthie, E.S. The action of fibrinogen on certain pathogenic cocci. J Gen Microbial 1 955;13:383-393.
1 7. Kuusela, P. Fibronectin binds to Staphylococcus aureus. 1 978;276:71 8-720.
1 8. Estersen, F. and Clemmensen, I. Isolation of a fibronectin-binding protein from Staphylococcus aureus. Infect lmmun 1 982;37:526-531 .
1 9. Patti, J.M., Boles, J.O. and Hook, M. Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus. Biochemistry 1 993;32:1428-1435.
20. Lopes, J.D., Dos Reis, M. and Brentani, R.R. Presence of laminin receptors in Staphylococcus aureus. Science 1 985;299:275-277.
21 . Maxe, I., Ryden, C. and Wadstrom, T. Specific attachment of Staphylococcus aureus to immobilized fibronectin. Infect lmmun 1 986;54:695-704.
21
Chapter 1
22. Vaudaux, P.E., Waldvogel, F.A. and Morgenthaler, J.J. Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence. Infect lmmun 1984;45:768-774.
23. Herrmann, M., Vaudaux, P .E. and Plttet. D. Fibronectin, fibrinogen and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J Infect Dis 1988;158:693-700.
24. Fischer, B., Vadaux, P. and Magnin, M. Novel animal model for studying the molecular mechanisms of bacterial adhesion to bone-implanted metallic devices: role of fibronectin in Staphylococcus aureus adhesion. J Orthop Res 1996;14:914-920.
25. Alkan, M.L., and Beachey, E.H. Excretion of lipoteichoic acid by group A streptococci: influence of penicillin on excretion and loss of ability to adhere to human oral mucosa! cells. J Clin Invest 1978;61 :671-677.
26. Craigen, M.A.C., Waterra, J. and Hackett, J.S. The changing epidemiology of osteomyelitis in children. J Bone Joint Surg Br 1992;74:541-545.
27. Korovessis, P ., Fortis, A.P. and Spastris, P. Acute osteomyelitis of the patella 50 years after a knee fusion for septic arthritis: A case report. Clin Orthop 1991 ;272:205-207.
28. Gallie, W.E. First recurrence of osteomyelitis eighty years after infection. J Bone Joint Surg Br 1951 ;33:1 10-1 11 .
29. Hudson, M.C., Ramp, W.K. and Nicholson, N.C. Internalization o f Staphylococcus aureus by cultured osteoblasts. Microb Pathog 1995;19:409-419.
30. Balwit, J.M., Van Langevelde P., Vann, J.M. and Proctor, R.A. Gentamicin resistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells. J Infect Dis 1994;170:1033-1037.
31 . Hoffstadt, R.E. and Youmans, G.P. Staphylococcus aureus dissociation and its relation to infection and immunity. J Infect Dis 1932;51 :216-222.
32. Schnitzer, R.J., Camagnl L.J. and Buck, M. Resistance of small colony variants (G-forms) of a Staphylococcus towards the bacteriostatic activity of penicillin. Proc Soc Exp Biol Med 1943;53:75-78.
33. Cierny, G., Mader, J.T. and Pennick, J.J. A clinical staging system for adult osteomyelitis. Contemp Orthop 1985;1 0:17-37.
34. Bryson, A.F. and Mandell, B.B. Primary closure after operative treatment of gross chronic osteomyelitis. Lancet 1964;13:1 179-1182.
35. Goto, M., Sugiyama, M., Nakajima, S. and Yamashina, H. Fosfomycin kinetics after intravenous and oral administration to human volunteers. Antimicrob Agents Chemother 1981 ;20:393-397.
36. Klemm, K.W. Antibiotic bead chains. Clin Orthop 1993;295:63-76.
37. Wichelhaus, T.A., Dingeldein, E., Rauschmann, M., Kluge, S., Dieterich, R., Schafer, V. and Brade, V. Elution characteristics of vancomycin, teicoplanin, gentamicin and clindamycin from calcium sulphate beads. J Antimicrob Chemother 2001 ;48:117-1 19.
38. Van de Belt, H., Neut, D., Van Horn, J.R., Van der Mei, H.C., Schenk, W. and Busscher, H.J. Antibiotic resistance-to treat or not to treat ? Nature Medicine 1999;5:358-359.
39. Buchholz, H.W. and Engelbrecht, H. Depot effects of various antibiotics mixed with Palacos resins. Der Chirurg 1970;41 :51 1-515.
40. Fischer, L.P., Gonon, G.P., Carrett, J.P., Vulliez, Y. and De Mourgues, G. Association methacrylate de metyle (cimen acrylique) et antibiotiques. Etude bacteriologique et mecanique. Revue de Chirurgie Orthopedique 1977;63:361-372.
41 . Greco, F ., Rossi, A., De Palma, L. and Spagnolo, N. Studio sull'attivita antibatetterica in vitro di miscele cemento-antibiotici. Giornale Italiano di Ortopedia e Traumatologia 1981;7: 105-1 16.
42. Hill, J., Klenerman, L., Trustey, S. and Blowers, R. Diffusion of antibiotics from acrylic bone cement in vitro. J Bone Joint Surg Br 1977;59:197-199.
22
Introduction
43. Blenkharn, I.J. Antibacterial bone cement preparations. In: Coombs, R., and Fitzgerald, R.H, editors. Infection in the orthopaedic patient, London; Boston: Butterworths 1 989:1 1 6-1 1 9.
44. Neut, D., Van de Belt, H., Stokroos, I., Van Horn, J.R., Van der Mei, H.C., and Busscher, H.J. Biomaterial-associated infection of gentamicin-loaded PMMA beads in orthopaedic revision surgery. J Antimicrob Chemother 2001 ;47:885-891 .
45. Woodruff, H.B., Mata, J.M., Hernandez, S.O., Mochales, S., Rodriguez, A., Stapley, E.O., Wallick, H., Miller, A.K. and Hendlin, D. Fosfomycin: Laboratory studies. J Chemother (Suppl 1 ) 1977;23:1-22.
46. Kumon, H., Ono, N., Iida, M. and Nickel, J.S. Combination effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa growing in a biofilm. Antimicrobial Agents Chemother 1995;23:65-74.
47. Goto, S. Fosfomycin, antimicrobial activity in vitro and in vivo; in Fosmicin: New perspective therapy of infection. New perspective therapy of infection. J Chemother 1 997;23:65-74.
23
Chapter 2
Efficacy of Handmade
Fosfomycin-Loaded PMMA
Beads In Vitro
Chapter 2
Introduction
Chronic osteomyelitis is defined as a long-standing infection of bone characterized by
periods of remission interspersed with acute relapses, and in adults may result from
hematogenous spread of infection, open fractures, penetrating trauma, or surgical
intervention after inadequate management1 . Biofilm formation is the key in
development and persistence of these bone infections. A biofilm is an aggregation of
microbial colonies enclosed within an extracellular polysaccharide matrix (glycocalyx)
that adheres to devitalized bone1•2
• The biofilm mode of growth protects the organism
against antibiotics and host defense mechanisms, such as antibody adsorption and
phagocytosis and allows infections to exist in a sub-clinical state and recur1 •2.
The development of infection is facilitated by the virulence of the
microorganisms, (sub-)optimal conditions of the local environment, and possible
systemically compromised of the host3. Osteomyelitis is commonly polymicrobial, and
more than one organism is present in 32% to 70% of the patients in particular if
fistulae are or have been present3•4. The most common infecting organism is
Staphylococcus aureus, which can be identified either alone or in combination with
other pathogens in 65% to 70% of all patients4·5
. Pseudomonas aeruginosa, the
second most common infecting organism, is found in 20% to 30% of all patients3•4
.
Atypical mycobacteria or fungi may be the responsible pathogens in
immunocompromised patients.
In developing countries, such as Indonesia, there are many poor people that
make use of the services of traditional medicine, like bonesetters when dealing with
open bone fractures. Initially, the bonesetters reduce the fracture without cleaning the
wound, cover the wound by herb to kill infectious bacteria and fix the fracture using
wooden splints. Usually, these patients present themselves after several months to
26
Efficacy of handmade fosfomycin-beads
the orthopedic clinic with persistent sinus tract infection may produce pus in
abundant quantities, indicating chronic osteomyelitis has occurred (see Figure 1 ).
Chronic osteomyelitis in these countries is generally treated by clearing the cavity of
infected material, systemic administration of antibiotics and planting a chain of
handmade antibiotic-loaded beads, as shown in Figure 2. These beads are left in situ
for about fourteen days, after which the cavity is filled with bone graft.
Figure 1. Infected proximal tibia in a 36 year-old-male patient previously treated conservatively by a bonesetter. The intra-osseous cavity contains an avascular sequestrum and a quantity of pus leading to chronic discharging sinus (see arrows)
The application of polymethylmethacrylate (PMMA) beads for local delivery of
antibiotics in treating musculoskeletal infection is common 1•6-a. High local levels of
antibiotics facilitate delivery of antibiotics by diffusion to avascular areas of wounds
that are inaccessible by systemic antibiotics and in many circumstances organisms
that are resistant to drug concentrations achieved by systemic antibiotics, are
susceptible to the extremely high local concentrations provided by local antibiotic
delivery9• 10.
27
Chapter 2
Figure 2. X-ray (AP view) of lower leg with a chain of handmade antibiotic-loaded beads inserted down the tibial shaft after removal of infected material (see arrow)
Klemm and other investigators report good results with the use of gentamicin
loaded PMMA beads in the treatment of chronic osteomyelitis1 1 -13, although in vitro
studies have demonstrated bacterial adhesion and growth on antibiotic-loaded
cement, despite the release of antibiotics 13•1 4• The antibiotic is leached from the
PMMA beads into the postoperative wound hematoma and secretion, which acts as a
transport medium 12. Pharmacokinetic studies have shown that the local
concentrations of antibiotic achieved with these beads are up to 200 times higher
than levels achieved with systemic antibiotic administration9•1 1 , while maintaining low
serum levels and low systemic toxicity.
Antibiotic-loaded PMMA beads are commercially available under the name
Septopal® and widely use in the Western world at a price of approximately 148 euros
per chain, but the price of a chain of these beads is far higher in Indonesia (about
437 euros) due to shipping costs and taxes. This is much more that most patients in
28
Efficacy of handmade fosfomycin-beads
developing countries can afford. Therefore, orthopedic surgeons use PMMA bone
cement, mix it themselves with antibiotic in handmade molds, manually prepare
beads and apply them in their patients. Fosfomycin is the commonly used antibiotic
in these beads, because of its low costs, wide antibacterial spectrum, small molecular
weight (138 Da) and its ability to remain stable up to the high temperatures which are
reached during polymerization of the bone cement15. A chain of handmade beads
then only costs around 80 euros. Although the choice of fosfomycin as the antibiotic
to incorporate into the handmade PMMA beads seems disputable, as it is commonly
used for urinary tract infections and up till now has only16 limited applications in other
infections probably because of its limited effectiveness in vitro, despite the fact that
at least one author stated that fosfomycin may well be ineffective in vitro while being
effective in vivo17• Moreover, fosfomycin is seldom used as a mono-therapy, as it
rapidly stimulates antibiotic resistance.
The treatment of osteomyelitis in Indonesia generally yields satisfactory
clinical results although rigorous follow-up is lacking, but it is unknown up to what
extent the fosfomycin-loaded, handmade beads contribute to these results. The aim
of this study is to evaluate the in vitro antibacterial efficacy and kinetics of antibiotic
release of several currently used, handmade fosfomycin-loaded beads. Since it is
known, however, that fosfomycin in vivo works in concert with glucose-6-phosphate
(G-6-P)18, experiments will be done in the absence and presence of G-6-P. The in
vitro activity of fosfomycin can be greatly enhanced by inclusion of G-6-P in the test
medium. This substance acts as an inducer of the hexose phosphate transport
pathway, and fosfomycin can take advantage of this pathway to achieve elevated
intracellular concentrations 19•20• All results will be compared with those of
commercially available gentamicin-loaded PMMA beads (Septopal®).
29
Chapter 2
Materials and methods
Antibiotic-loaded beads preparation
A number of orthopedic surgeons in Indonesia were requested to describe the
personal method which they developed to prepare handmade antibiotic-loaded
beads. In general, PMMA bone cements were used, in combination with a variety of
different mixing techniques and sometimes templates. Fosfomycin-sodium was the
general antibiotic included. Based on this inventory, six methods were selected for
further research and the participating surgeons were asked to submit an extensive
protocol of their concept, as summarized in Table 1, together with samples of their
beads.
Antibiotic-loaded PMMA beads are commercially available under the name
Septopal® (Biomet Europe, Darmstadt, Germany). One bead (diameter, 7 mm,
area/volume ratio 8.6 cm"1) contains 7.5 mg of gentamicin sulphate (corresponding to
4.5 mg of gentamicin base) and 20 mg zirconium oxide (monoclinic) as an X-ray
contrast medium. One bead consists of methylmethacrylate-methyl acrylate
copolymer and glycine. One chain consists of 30 beads threaded on surgical wire21 .
Antibacterial efficacy
Beads no1 to no 6 and Septopal® beads were immersed in 10 ml of sterile phosphate
buffer saline, PBS (NaCl 8.76 g/L, K2HPO4 43.5 g/L, KH2PO4 34.5 g/L, pH 7.4) and
incubated at 37°C. After 24 h of incubation 1 5 µI samples were taken and these were
placed on bacterial streaked tryptone soya broth (TSB) agar plates (for composition
see Table 2). The zones of inhibition were established by measuring the diameters of
the clear areas around one drop of elution fluid. Absence of an inhibition zone was
30
Efficacy of handmade fosfomycin-beads
taken as a sign that the antibiotic concentration was too low to inhibit bacterial
growth.
Table 1. Six different local concepts developed by Indonesian orthopedic surgeons for preparing handmade antibiotic-loaded beads. All beads contained fosfomycin-sodium and mixing was done manually using a spatula. As a reference, the different properties of the Septopal® beads are added.
Cement base Fosfomycin Beads
[g] content per bead
[mg]
no 1 Zimmer, 40 1 88
no 2 Simplex P, 40 94
no 3 Simplex P, 40 94
no 4 Simplex P, 40 94
no s Simplex P, 40 94
no 6 Simplex P, 40 94
7.5 Septopal®
PMMA (gentamicin sulehate}
Table 2. Detailed composition of TSB
Formula
Pancreatic digest of casein
Papaic digest of soybean meal
Sodium chloride
Di-potassium hydrogen phosphate
Glucose
pH 7.3 ± 0.2
Diameter Shaping [cm]
Template 1 .7
Hand rolled 2.8
Hand rolled 1 .6
Handrolled 2
Handrolled 1 .6
Hand rolled 1 .6
Template 0.7
Area/volume ratio [cm·11
3.5
2 . 1
3.8
3.0
3.8
3.8
8.6
g/L
1 7.0
3.0
5.0
2.5
2 .5
31
Chapter 2
For this study, we used ten clinical strains isolated from patients with an
implant-related infection of the University Medical Center Groningen, The
Netherlands, as well as twenty clinical isolates from osteomyelitis patients from
Indonesia (see Table 3). Distribution of the different strains and species chosen are
in accordance with those reported to be involved in the occurrence of osteomyelitis3.4.
The bacterial strains were cultured on blood agar and each strain was suspended in
4 ml of 0.9% saline to a concentration of approximately 1 08 bacteria/ml. The
bacterial suspension was streaked on TSB agar plates and after drying for 20 min, 1 5
µL droplets of PBS with antibiotic were put on the bacterial streaked agar plates.
After overnight incubation at 37°C, the inhibition zone diameters (mm) were scored.
In addition, the minimal inhibitory concentration (MIC in µg/mL) of the isolated
bacteria and possible sub-populations against fosfomycin and gentamicin were
determined using an E-test (AB Biodisk, Dalvagen, Sweden). All gentamicin-resistant
S. aureus strains included were also tested for MRSA (methicillin resistant
Staphylococcus aureus) by Mee-A gene method22 •
Kinetics of antibiotic release
Six different handmade fosfomycin-loaded beads and Septopal® beads were
immersed in 1 0 ml sterile PBS and incubated at 37°C. At indicated time points (24 h,
48 h, 72 h, and 1 44 h) beads were transferred to fresh 10 ml PBS and again
incubated at 37°C. The kinetics of antibiotics release was established by taking 1 5 µL
elution fluid samples at the indicated time points and placing the droplets on the four
quadrants of a bacterial streaked TSB agar plate. Kinetics of antibiotic release was
only studied with strains toward which antibacterial efficacy could be established, as
32
Efficacy of handmade fosfomycin-beads
described above. After 24 h incubation at 37°c the inhibition zone diameters were
scored in all four quadrants.
In order to assess the efficacy of fosfomycin in the presence of G-6-P, beads
no 2 and no 5 were also evaluated in TSB medium, supplemented with G-6-P to a
final concentration of 25 µg/mL 23•24•
Scanning electron microscopy
For scanning electron microscopy (SEM), beads were sputter-coated with gold/
palladium (-3 nm). Examination was done at 2.0 kV in a JEOL field emission
scanning electron microscope type 6301 F on each of the different handmade beads
as mentioned in Table 1 and Septopal® beads on the surface as well as on the
fracture surface.
Results
Characteristics of the beads
The diameters of different handmade beads were large compared to Septopal®
beads, while furthermore they were not all uniform in size (see Table 1 ). The
antibiotic content per bead ranged from 94 to 188 mg, which may seem enormous
compared to the gentamicin sulphate content in Septopal® (7.5 mg), but this is
completely due to the large size of the handmade beads. Furthermore the area to
volume ratio of the handmade beads varies greatly from 2.1 to 3.8 cm·1 which is
much lower than the one of Septopal® beads (8.6 cm·1 ). Therefore, the use of a
proper template should be considered for bead preparation rather than the use of
oversized hand-rolled beads. We will address this problem in more detail in Chapter
4.
33
Chapter 2
Minimal inhibitory concentration (MIC values)
Dutch isolates. The effect of fosfomycin and gentamicin on the growth of ten bacterial
strains obtained from Dutch patients with an implant-related infection was evaluated
after incubation in vitro (see Table 3). The MIC values of fosfomycin for the Dutch
isolates ranged from 0. 19 to over 1024 µg/ml. Since the threshold for resistance
against fosfomycin is � 128 µg/ml 25, fosfomycin can be considered effective against
seven out of the ten strains, although for three of these strains resistant sub
populations could be observed within the inhibition zones (see Table 3). Efficacy of
gentamicin on the growth of these ten bacterial strains was also evaluated, and MIC
values ranged from 0.38 to over 256 µg/ml. Since the threshold for resistance
against gentamicin is � 4 µg/ml 26, gentamicin can be considered effective in seven
out of the ten strains.
Indonesian isolates. The efficacy of fosfomycin and gentamicin was also evaluated
on the growth of twenty bacterial strains from chronic osteomyelitis patients from
Indonesia after incubation in vitro. MIC values for these isolates ranged from 1.5 to
over 1024 µg/ml for fosfomycin.
Table 3. MIC values from different bacterial isolates used in this study for fosfomycin and gentamicin. The Dutch isolates were taken from implant-related infection, while Indonesian isolates were taken from osteomyelitis patients (P, B and F denoting Pus, Bone and Fistula respectively). Gentamicinresistant S. aureus strains were also tested for MRSA using the Mee-A gene method.
No. Bacterial strains
Dutch Isolates
34
2
3
4
Staphylococcus aureus 5298
Staphylococcus aureus 7323
Pseudomonas aeruginosa 5148
Pseudomonas aeruginosa 7348
MIC value (µg/mL)
Fosfomycin
96
0.1 9*
>1 024
32
Gentamicin
0.75
1 .5
2
4
Efficacy__ of handmade fosfomy__cin-beads
5 Coagulase negative staphylococcus 7368 48 0.38
6 Coagulase negative staphylococcus 7334 24 6
7 Coagulase negative staphylococcus 7391 0.50* >256
8 Klebsiella 333257 >1024 1 .0
9 Micrococcus 7397 192 0.5
10 Escherichia coli BS 6206 1 .5* 1 .0
Indonesian Isolates
Staphylococcus aureus In P1 (MRSA +) 2 >256
2 Staphylococcus aureus In B1 (MRSA +) 1 .5 >256
3 Staphylococcus aureus In P4 (MRSA -) 1 .5 1 .0
4 Staphylococcus aureus In B4 (MRSA -) 1 .5 1 .0
5 Staphylococcus aureus In B7 (MRSA -) 1 .5 1 .5
6 Staphylococcus aureus In F7 (MRSA -) 4 - 6 1 .0
7 Staphylococcus aureus In F1 0 (MRSA -) 4 1 .0
8 Staphylococcus hemoliticus In P3 128 >256
9 Staphylococcus hemoliticus In P5 96* 32
10 Staphylococcus saprophyticus In B5 >1024 >256
11 Pseudomonas aeruginosa In P4 >1024 2
12 Pseudomonas aeruginosa In P7 >1024 >256
13 Pseudomonas aeruginosa In B7 >1024 >256
14 Pseudomonas aeruginosa In F9 >1024 0.50
15 Klebsiella terrigena In B5 >1024 2
16 Klebsiella terrigena In P2 32* 1 .5
17 Klebsiella pneumonia In P7 32* 1 .0
18 Klebsiel/a pneumonia In F9 32* >256
19 Proteus mirabilis In P2 32* >256
20 Proteus In B2 32* >256
* resistant sub-populations could be observed within the inhibition zones and refer to the slow growth of certain variants on routine media, yielding unexpectedly small sub-populations in comparison to the normally growing parents strains; it does not imply actual genetic conversion27
"29
•
35
Chapter 2
Fosfomycin can be considered effective against fourteen out of twenty strains. Low
MIC values in the absence of resistant sub-populations were only seen in seven S.
aureus strains lnP1 ; lnB1 ; lnP4; In B4; lnB7; lnF7 and lnF1 0, taken from bone, pus
and fistulae. Six strains were resistant to fosfomycin (MIC over 1 28 µg/mL), and six
of the sensitive isolates showed resistant sub-populations.
The efficacy of gentamicin on the growth of the twenty Indonesian bacterial
strains was also examined, yielding MIC values from 0.50 to over 256 µg/ml, and
gentamicin can be considered effective in ten of the twenty isolates. Both gentamicin
resistant S. aureus strains were MRSA positive.
Table 4. The absence (-) or presence (+) of zones of inhibition around droP.lets of elution fluids from handmade fosfomycin-loaded PMMA beads and gentamicin-loaded Septopal® beads
No Bacterial Strains Bead Bead Bead Bead Bead Bead Septopai® no 1 no 2 no 3 no 4 no 5 no 6 S. aureus 5298 +
2 S. aureus 7323 + + + + +
3 P. aeruginosa 5148 +
4 P. aeruginosa 7348 +
5 CNS 7368 +
6 CNS 7334
7 CNS 7391 + + + + +
8 Klebsiella 333257 +
9 Micrococcus 7397 +
1 0 E. coli B S 6206 +
Antibacterial efficacy of the beads
The antibacterial efficacy of the beads was tested against Dutch isolates only, as
these were generally more susceptible to fosfomycin than the Indonesian isolates.
Table 4 shows that handmade fosfomycin-loaded beads were only effective against
36
Efficacy of handmade fosfomycin-beads
S. aureus 7323 and CNS 7391, which appeared resistant against Septopal® beads.
Bead no 1 shows the least antibacterial efficacy. Antibacterial efficacy of Septopal®
beads could be seen in all bacterial strains used, except for CNS 7334 and CNS
7391, which are both gentamicin-resistant.
Antibiotic release
Table 5 presents the diameter of the inhibition zones achieved with elution fluid from
the handmade fosfomycin-loaded beads after different elution periods. None of the
fosfomycin loaded beads showed antibacterial efficacy against S. aureus 7323
extending beyond 24 h, whereas beads no 1 and no 3 were not effective at all. In
contrast, all beads showed sustained antibacterial efficacy against CNS 7391,
although here resistant sub-populations were ubiquitously present. Therefore, we
conclude that at least in vitro fosfomycin is not effective against any of the bacteria
tested here. In contrast, Septopal® beads showed effectiveness against S. aureus
7323 for at least 144 h, while no inhibition zone could be measured against CNS
7391, due to its resistance against gentamicin.
Further evaluation was performed to find out whether the efficacy of
fosfomycin beads against S. aureus 7232 and CNS 7391 for beads no 2 and no 5
increased in the presence of G-6-P. Addition of G-6-P to TSB agar yielded only
improved efficacy for bead no 5 against S. aureus 7323 during the first 48 h, but no
major effects of the addition of G-6-P was seen for the other cases. These results
suggest that the presence of G-6-P in TSB media does not increase the efficacy of
fosfomycin during in vitro experiments as carried out here.
37
Chapter 2
Table 5. Diameters of inhibition zones (mm) around droplets of elution fluid after different elution times from fosfomycin-loaded PMMA beads for two strains susceptible to fosfomycin, as compared with commercially gentamicin-loaded beads (Septopal®). Beads no 2 and no 5 were also evaluated in the presence of G-6-P - = no zone; * resistant sub-populations could be observed within the inhibition zones
Bead S .aureus 7323 CNS 7391
Number 24h 48h 72h 144h 24h 48h 72h 144h
no 1 20 20 1 5 20
no 2 16 25* 20 20 20
no 2+G6P 22* 22*
no 3 1 5* 15 15
no 4 1 1 20* 20 20 20
no 5 7 1 1 25* 20* 20*
no 5+G6P 22* 6 1 1 23* 1 3*
no 6 1 3 25* 20 20 20
Septopal® 16 12 1 1 1 4
Scanning electron microscopy
Electron microscopy was done on the Septopal® beads and on all handmade beads.
Since there were differences in size between the handmade beads we selected one
of the smaller sized beads and the biggest bead, i.e. beads no 1 and no 2,
respectively to describe the surface texture and the fracture surface. The surface
texture of Septopal® beads is much more homogeneous than of the handmade beads
(compare Figure 3 A, B and C), and contains open structures on the bead surface
and inside, as can be seen from the fracture surfaces. Bead no1 appears to have a
highly dense surface, and although there are some openings visible, they do not
extend inside and the fracture surface does not reveal any porosity. The surface of
bead no 2 is dense and closed as well, but there are folds toward the inside. The
fracture surface demonstrates a clear porosity of the inside.
38
Efficacy of handmade fosfomycin-beads
39
Chapter 2
40
Efficacy of handmade fosfomycin-beads
Figure 3. Scanning electron micrographs of a Septopal® and handmade, fosfomycin-loaded beads under different magnifications. Series 1 indicate outersurfaces of the bead, while series 2 indicate fracture surfaces. A-1/A-2: Gentamicin-loaded Septopal® bead B-1/B-2: Indonesian bead no 1 C-1/C-2: Indonesian bead no 2 The bar equals 1 mm for low magnification micrograph, and 100 µm for the insert.
Discussion
Currently, especially in the Western world, antibiotic-loaded PMMA beads are often
used to sterilize and temporarily maintain dead space following debridement
surgery30-32• In developing countries, chronic osteomyelitis occurs relatively frequent
41
Chapter 2
and surgical debridement, systemic antibiotics and the use of handmade (fosfomycin
loaded) beads for 2 to 4 weeks is the therapy of choice. In general, this treatment
yields satisfactory clinical results, although hard evidence is lacking.
The characteristics of the beads
The size, area per volume ratio and the presence of interconnecting (or percolating)
pores extending to the bead surface are the most important characteristics of
effective antibiotic releasing beads33. With respect the current characterization of
Indonesian beads, it should be noted that due to practical reasons all bead-related
results were obtained for single beads and the use of multiple beads was impossible.
In general, the antibiotic release from Indonesian handmade beads is inadequate
and strategies need to be developed in order to improve the release kinetics. As will
be discussed in Chapter 4, we proceeded to design a template to make beads of
uniform size, thus ensuring a uniform antibiotic content of all beads, although the use
of a template yields the risk that surface pores, required for proper release are
closed.
Antibacterial efficacy of the beads
The choice of fosfomycin as an antibiotic to incorporate into the beads can be
questioned based on the current results. Fosfomycin was only effective against eight
out of the twenty Indonesian isolates involved, while five of the isolates showed
resistant sub-populations existing within the inhibition zones in the Indonesian
isolates. This may be due to the fact that fosfomycin easily induces one-point
mutations causing resistance34, although there are some authors who state that it
does not imply actual genetic conversion2 7-2 9. In view of the potential risk of one point
42
Efficacy of handmade fosfomycin-beads
mutations, fosfomycin is seldom used in the Western world as a monotherapy and
mostly in combination with vancomycin or teicoplanin23•35. Furthermore, it can be
seen that relatively high concentrations of fosfomycin are needed to achieve any
efficacy, regardless of the absence or presence of G-6-P. Such high concentration
can under normal circumstances only be achieved in urine, and indeed in Germany
fosfomycin in combination with J3-lactams, ofloxacin, ciprofloxacin, aminoglycoside is
sometimes used to control urinary tract infections36•37. Concludingly, fosfomycin is not
the ideal antibiotic for inclusion in antibiotic-loaded beads.
Conclusions
1. The handmade beads were not uniform in size, lacked sufficient porosity at the
surface, had a sub-optimal area-per-volume ratio and may contain non-uniform
antibiotic-loading.
2. Fosfomycin is not the ideal antibiotic for inclusion in antibiotic-loaded beads.
Acknowledgements
The author would like to thank to letse Stokroos, Department of Cell Biology and
Electron Microscopy for his help with the electron microscopy. This work is funded by
Eric Bleumink Fund, University of Groningen, the Netherlands, the Research Institute
"Biomedical engineering, Materials Science and Application" (BMSA).
We also like to thank Mrs. G. Kampinga, medical microbiologist for her pertinent
advice.
43
Chapter 2
References
1. Patzakls, M.J. and Zalavras, C.G. Chronic posttraumatic osteomyelitis and infected nonunion of the tibia: Current management concepts. J Am Acad 2005;13:41 7-427.
2. Costerton, J.W. Biofilm theory can guide the treatment of device-related orthopaedic infections in: Biofilms in orthopaedic infections. Clin Orthop 2005;437:7-1 1 .
3. Schmidt, A.T. and Swiontkowskl, M.F. Pathophysiology of infections after internal fixation of fractures. J Am Acad Orthop Surg 2000;8:285-291 .
4. Patzakis, M.J., Wilkins, J., Kumar, J., Holtom, P., Greenbaum, B. and Ressler, R. Comparison of the results of bacterial cultures from multiple sites in chronic osteomyelitis of long bones: A prospective study. J Bone Joint Surg Am 1994;76:664-666.
5. Perry, C.R., Pearson, R.L. and Miller. Accuracy of cultures of material from swabbing of the superficial aspect of the wound and needle biopsy in the preoperative assessment of osteomyelitis. J Bone Joint Surg Am 1 991 ;17:90-95.
6. Wahlig, H. Gentamicin-PMMA beads: A drug delivery system in the treatment of chronic bone and soft tissue infections. J Antimicrob Chemother 1982;10:463-465.
7. Mclaren, A.C., Nelson, C.L., Mclaren, S.G. and Declerk, G.R. The effect of glycine filler on the elution rate of gentamicin from acrylic bone cement. A pilot study. Clin Orthop 2004;427:25-27.
8. Hanssen, A.O. Local antibiotic delivery vehicle in the treatment of musculoskeletal infection in: Local antibiotic delivery systems. Clin Orthop 2005;437:91-96.
9. Buccholz, H.W., Elson, R.A. and Engelbrecht, E. Management of deep infection of total hip replacement. J Bone Joint Surg 1 981 ;63:342-353.
10. Canale, S.T. Infection: in Campbell's operative orthopedics, 9th ed, Volume I, Mosby Inc, St-LouisMissouri 1 998;561-91 3.
1 1 . Seligson, D. and Henry, S.L. Newest knowledge of treatment for bone infection: antibioticimpregnated beads. Clin Orthop 1 993;295:2-18.
12. Klemm, K.W. Antibiotic bead chains. Clin Orthop 1 993;295:63-76.
1 3. Mclaughlin, R.E., Reger, S.I., Barkalow, J.A., Allen, M.S. and Difazio, C.A. Methylmethacrylate: A study of teratogenicity and fetal toxicity of the vapor in the mouse. J Bone Joint Surg 1978;3:355-358.
14. Neut, D., Van de Belt, H., Stokroos, I., Van Horn, J.R., Van der Mei, H.C. and Busscher, H.J. Biomaterial-associated infection of gentamicin-loaded PMMA beads in orthopaedic revision surgery. J Antimicrob Chemother 2001 ;47:885-891.
15. Woodruff, H.B., Mata, J.M., Ndez, S.H., Mochales, S., Rodrigues, S., Stapley, E.O., Wallick, H., Miller, A.K. and Hendlin, D. Fosfomycin: Laboratory studies. Chemotherapy 1977;23:1 -22.
16. Goto, S. Fosfomycin, antimicrobial activity in vitro and in vivo in: Chemotherapy 1 977;23:65-74.
17. Scorttl, M., Lora, L.L., Wagner, M., Calero, I.C., Losito, P. and Boland, J.A. Coexpression of virulence and fosfomycin susceptibility in Listeria: molecular basis of an antimicrobial in vitro-in vivo paradox. Nature Medicine 2006;12:515-517.
1 8. Winkler, H.H.. Distribution of an inducible Hexose-Phosphate Transport System among various bacteria. J Bacterio/ 1973;116:1 079-1 081.
19. Greenwood, D., and Whitley, R. Fosfomycin Trometamol: Activity in vitro against urinary tract pathogens. Infection 1990;18:60-64.
20. Kahan, F.M., Kahan, J.S., Cassidy, P.J. and Kropp, H. The mechanism of action of fosfomycin (phosphonomycin). Ann N Y Acad Sci 1974;235:364-386.
21. Septopal®. The time-tested http://www.biometbiomaterials.com
44
local antibiotic therapy. Available from:
Efficacy of handmade fosfomycin-beads
22. Wielders, C.L.C., Fluit, A.C., Brisse, S., Verhoef, J. and Schmitz, F.J. Mee-A gene is widely disseminated in Staphylococcus aureus population. J Clin Microbiology 2002;40:3970-3975.
23. De Cueto, M., Lopez, L., Hernandez, J.R., Morillo, C. and Pascual, A. In vitro activity of fosfomycin against extended-spectrum-�-lactamase-producing Escherichia coli and Klebsiella pneumoniae: Comparison of susceptibility testing procedures. Antimicrob Agents Chemother 2006;50:368-370.
24. Lopez, L., De Cueto, M., Di'az, M.A., Morillo, C. and Pascual, A. Evaluation of the E-test method for fosfomycin susceptibility of ESBL-producing Klebsiella pneumoniae. J Antimicrob Chemother 2007;59:810-812.
25. Andrews, J.M., Baquero, F., Beltrau, J.M., Canton, E., Crokaert, F., Gobernado, M., GoomezLuz, R., Loza, E., Navarr, M., Olay, T., Rodriguez, A., Vicente, M.V., Wise, R. and Yourassowsky, E. International collaborative study on standardization of bacterial sensitivity to fosfomycin. J Antimicrob Chemother 1 983;12:357-361 .
26. National Committee for Clinical Laboratory Standards (1 998). MIC Interpretive Standards (µg/mL) for Enterobacteriaceae, Pseudomonas aeruginosa and other non-Enterobacteriaceae-Approved Standard M7-A4 (M1 00-S8; Table 2A-2B), NCCLS, Villanova, PA.
27. Neut, D. Biomaterial-associated infections in orthopaedics, prevention and detection [dissertation]. Rijksuniversiteit Groningen; 2003.
28. Roggenkamp, A., Sing, A., Hornef, M., Brunner, U., Autenrieth, B. and Heesemann, J. Chronic prosthetic hip infection caused by a small-colony variant of Escherichia coli. J Clin Microbial 1 998;36:2530-2534.
29. Proctor, R.A. Microbial pathogenic factors: small colony variants. In: Bisno AL, Waldvogel FA, editors. Infections associated with indwelling medical devices. Washington, D.C: American Society for Microbiology 1 994;79-90.
30. Mader, J.T., Calhoun, J. and Cobos, J. In vitro evaluation of antibiotic diffusion from antibiotic-impregnated biodegradable beads and polymethylmethacrylate beads. Antimicrob Agents Chemother 1 997;41 :41 5-41 8.
31 . Cierny, G. and Mader, J.T. Adult chronic osteomyelitis. Orthopedics 1984;7: 1 557-64.
32. Calhoun, J.H. and Mader, J.T. Antibiotic beads in the management of surgical infection. Am J Surg 1 977;157:443-449.
33. Van de Belt, H., Neut, D., Uges, D.R., Schenk, W., Van Hom, J.R. and Van der Mel, H.C., Busscher, H.J. Surface roughness, porosity and wettability of gentamicin-loaded bone cement and their antibiotic release. Biomaterials 2000;21: 1981-1 987.
34. Ellington, M.J., Livermore, D.M., Pitt, T.L., Hall, L.M.C. and Woodford, N. Mutators among CTX-M b-lactamase-producing Escherichia coli and risk for the emergence of fosfomycin resistance. J Antimicrob Chemother 2006;58:848-852.
35. Plstella, E., Falcone, M., Balocchi, P ., Pompeo, M.E., Pierclaccante, A., Penni, A. and Venditti, M. In vitro activity of fosfomycin in combination with vancomycin or teicoplanin against Staphylococcus aureus isolated from device-associated infections unresponsive to glycopeptide therapy. lnfez Med 2005;13:97-1 02.
36. Mirakhur, A., Gallagher, M.J., Ledson, M.J., Hart, C.A. and Walshaw, M.J. Fosfomycin therapy for multiresistant Pseudomonas aeruginosa in cystic fibrosis. J Cystic Fibrosis 2003;2:1 9-24.
37. Mazzei, T., Cassetta, M.I., Fallani, S., Arrigucci, S. and Novelli, A. Pharmacokinetic and pharmacodynamic aspects of antimicrobial agents for the treatment of uncomplicated urinary tract infections. J Antimicrob Agents Chemother 2006;28:35-41 .
45
Chapter 3A
Concepts for Increasing the
Gentamicin Release from Bone
Cement Beads
Chapter 3A
Introduction
In Europe, commercially available gentamicin-loaded polymethylmethacrylate
(PMMA) beads constitute a proven effective drug delivery system for local antibiotic
therapy in bone and soft-tissue infections 1 •2 , in combination with systemically
delivered antibiotics and surgical debridement. The gentamicin concentrations
reached at the site of infection are far higher using antibiotic-loaded acrylic beads
than the concentrations achieved by systemic administration of the same antibiotic 1
and far above the minimal inhibitory concentrations of most common pathogens3•
The use of antibiotic-releasing acrylic beads furthermore yields very low antibiotic
concentrations in serum and urine to prevent toxic side effects4•
Gentamicin-loaded acrylic beads, however, are not commercially available in
some parts of the world, like in the USA, or they are too expensive for common use
in other areas of the world. Therefore, orthopedic surgeons worldwide make
antibiotic-loaded beads themselves, sometimes using a template system, but most
often by hand-rolling. The antibiotic release kinetics from PMMA bone cements
depends on the penetration of dissolution fluids into the polymer matrix and the
subsequent diffusion of the dissolved drug from the beads. Both steps require a
certain porosity of the cement. Commercially prepared gentamicin-loaded acrylic
beads are porous and they show much higher release rates than hand-rolled,
nonporous antibiotic-loaded acrylic beads5• Unfortunately, the exact way of pore
production in these beads has not been disclosed.
In order to increase antibiotic release from hand-rolled acrylic beads, McLaren
et al. proposed to add soluble fillers, like glycin, xylitol, sucrose or erythritol as to
increase their porosity5•7 and consequently the penetration of the dissolution fluids.
These soluble fillers all increased the gentamicin release from acrylic beads5•7.
48
Concepts for increasing the gentamicin release
Moreover, gentamicin release from an acrylic-glycine mixture increased with
increasing amounts of glycine5• Furthermore, xylitol appeared more effective in
increasing the antibiotic release than glycine: for example, on day one xylitol
increased the daptomycin release by a factor of 2. 7, whereas glycine increased it 1.8
times when compared with beads without fillers6•
Although addition of soluble fillers as described above yielded a considerable
and significant increase in antibiotic release from hand-rolled beads (total release
after 7 days amounted approximately to 1 0% in the presence of soluble fillers,
whereas beads in the absence of fillers released only 5% of their gentamicin
content), the release properties were still inferior to those of commercially available
beads (total release after 7 days amounts around 60% of the total antibiotic content)8.
Therefore, the aim of this study was to develop a simple, cheap and effective
formulation and process to prepare acrylic beads with gentamicin release properties
similar to those observed for commercially available beads. To this end, acrylic beads
were first prepared with variable monomer contents to yield increased gentamicin
release through the creation of a less dense polymer matrix. Subsequently, after an
optimal monomer content had been defined, different gel-forming polymeric fillers
such as polyvinylpyrrolidone (PVP) (at two different molecular weights) and,
hydroxypropylmethylcellulose (HPMC) were added to enhance the permeability by
dissolution fluids and gentamicin release. After selection of the most favorable
biodegradable filler, its concentration was varied and the antibiotic release of the final
beads was compared with the gentamicin release from antibiotic-loaded Septopal®
beads.
49
Chapter 3A
Materials and methods
Commercially antibiotic beads
Commercially available antibiotic-loaded bone cement beads Septopal® (Biomet
Merck, Darmstadt, Germany) containing 2.25 w/wo/o gentamicin base in each bead (7
mm in diameter) was used in this study.
Beads preparation with different concentration of monomer
Simplex-P bone cement powder (Stryker Howmedica OSTEONICS, Howmedica
International S, Limerick, Ireland) was mixed with powdered gentamicin sulphate
(Gracia Pharmaceutical, Indonesia) for 2 min in a ceramic bowl employing a spatula.
1 g of gentamicin sulphate was added to 40 g of polymer powder. The resulting
mixture was subsequently combined with 20 ml of monomer in a ceramic bowl and
mixed for 2 min with a spatula according to the manufacturer instructions. Thus
prepared beads (500 µIlg polymer) will be denoted "100% monomer''. In addition,
beads were prepared with 75% (375 µUg polymer) and 50% (250 µUg polymer) of
the prescribed amount of monomer. The material is mixed until a doughy phase is
obtained, and the gentamicin-PMMA-MMA mixture was hand-rolled into beads.
Beads preparation with different polymeric fillers
Powdered PMMA and gentamicin sulphate (1 g gentamicin sulphate and 40 g PMMA
powder) were mixed. Subsequently one of the polymeric fillers was added to this
powder mixture. Three different gel-forming polymeric fillers were used: Polyvinyl
pyrrolidone of a molecular weight of 28000-34000 Da (PVP 90K) (Genfarma,
Zaandam, The Netherlands) and Polyvinylpyrrolidone of a molecular weight of 7000-
11000 Da (PVP17) (Kollidon®-17PF) (BASF, Germany), the third polymer
50
Concepts for increasing the gentamicin release
investigated was (Hydroxypropyl)methyl cellulose (HPMC) (Sigma-Aldrich Chemie
GmbH, Steinham, Germany). Polymeric fillers were mixed at a concentration of
1 0w/w% with respect to the amount of polymer powder. The resulting mixtures were
finally combined with 50% (250 µUg polymer) of the prescribed amount of monomer
and the beads were prepared as described above.
Beads preparation with different concentration of PVP 1 7
Powdered PMMA and gentamicin sulphate ( 1 g gentamicin sulphate and 40 g PMMA
powder) were subsequently mixed with different amounts of PVP17 (5w/w%,
1 0w/w%, and 15w/w% with respect to the amount of polymer powder) and beads
were prepared with 50% (250 µUg polymer) monomer as described above.
Analysis of the release kinetics of gentamicin from the beads
A gentamicin-loaded acrylic bead was immersed in 10 ml of sterile phosphate buffer
saline, PBS (NaCl 8.76 g/L, K2HPO4 43.5 g/L, KH2PO4 34.5 g/L, pH 7.4) and
incubated at 37°C. At designated time intervals (6, 24, 48, 72, 168, 336 h), 500 µL
aliquots of the gentamicin-PBS solution were taken and the amount of buffer restored
to 10 ml.
Gentamicin concentrations were measured using a procedure described by
Sampath et al.9• Briefly, an o-phtaldialdehyde reagent was made and stored for 24 h
in a dark environment. The gentamicin aliquot, o-phtaldialdehyde reagent and
isopropanol were mixed in equal proportions and stored for 30 min at room
temperature. The o-phtaldialdehyde reacted with the gentamicin amino groups and
chromophoric products were obtained, whose absorbances were measured at 332
nm using a Spectronic® 20 GenesysTM spectrophotometer (Spectronic Instruments,
51
Chapter 3A
Inc. Rochester, NY 14625, USA). A calibration curve was used to calculate the
gentamicin concentrations in the samples. The gentamicin percentages released of
the total amount incorporated were calculated for all acrylic beads used and
gentamicin release was compared with the gentamicin release from Septopal®
beads.
Scanning electron microscopy
To compare the polymer matrix of our home made beads with the one of Septopal®
beads, scanning electron microscopy (SEM) was performed. Examination was done
at 2.0 kV in a JEOL field emission scanning electron microscope type 6301 F. Beads
were sputter-coated with a 3 nm thick conductive layer of gold/ palladium (80/20).
Statistical analysis
The release experiments with beads prepared with different concentrations of PVP17
(see section 2.4) were performed in triplicate and a statistical analysis was done in
order to compare the gentamicin release rates of the hand-rolled beads with those
from commercial Septopal® beads. To this end, the Student's t-test for independent
samples was used. A 95% (p<0.05, two-tailed) confidence interval was applied for
statistical significance.
Results
Characteristics of the beads
The different handmade beads had an average diameter of 13. 7 mm and their
average weight varied from 1.45 g, 1.34 g, to 1.23 g for beads prepared with 100%,
75%, and 50% monomer, respectively.
52
cu n, cu
C
:Q n,
C cu (!J
Concepts for increasing the gentamicin release
25 .----------------------------�
20
15
r � �
10
.,A • • •
5
0 -+----�------�-------�---------<
0 so 100 150 200 250 300 350
Time ( h )
Figure 1 . Cumulative percentage of gentamicin release from PMMA beads made with different dosages of monomer (+, 1 00%; o, 75%; .&. , 50%), as a function of time during exposure to phosphate buffered saline.
Gentamicin re/ease
Figure 1 shows the gentamicin release from acrylic beads prepared with different
amounts of monomer. Gentamicin release from beads prepared with 100% monomer
leveled off within the time interval of the experiment and was confined to about 8% of
the total amount of gentamicin included. Reduction of the amount of monomer
caused incomplete polymerization and melting of the polymer beads, which resulted
in an almost twofold increased gentamicin release when 50% of monomer was
employed compared to a bead with 100% monomer.
53
Chapter 3A
100 --,-----------------------------,
cu u, ns cu
75
'ii so -C ·u ·e ns -
C cu (!J
25
0
0
'--------·----•-------------------------•---------------------------------------------•
so 100 1 50 200 250 300 350
Time ( h )
Figure 2. Cumulative percentage of gentamicin release from PMMA beads made with 1 0 w/wo/o of different polymeric fillers (•, control; o, PVP17; •, PVP 90K; A, HPMC) and 50% of the advised monomer amount, as a function of time during exposure to phosphate buffer saline.
Figure 2 presents the gentamicin release from acrylic beads made with 50% of
monomer prescribed and different biodegradable fillers. Inclusion of a biodegradable
filler almost tripled the gentamicin-release with respect to its release in the absence
of fillers. There was little difference between the different fillers.
Based on the latter observation and the fact that PVP17 has the highest purity,
it was decided to vary the amount of PVP17, as presented in Figure 3, together with
the release kinetics of gentamicin from Septopal® beads. The gentamicin release
from beads prepared with 50% monomer increases upon increasing of the amount of
PVP17 in the beads. Beads containing 15% PVP17 released 71 % of their antibiotic
content in 336 h. Importantly, this is significantly (p<0.05, two-tailed,) more than the
54
Concepts for increasing the gentamicin release
gentamicin-release from Septopal® beads, their release is confined to only 56% after
336 h.
-
� 0
- so QJ U) n, QJ "ii � C
:� 25 E n, ., C QJ �
0
0 50 100 150 200
Time ( h )
250 300 350
Figure 3. Cumulative percentage of gentamicin release from PMMA beads made with different dosages of PVP17 (0, 5 w/w%; o, 1 0 w/w%; A , 1 5 w/w%} and 50% of the advised monomer amount, as a function of time during exposure to phosphate buffer saline, in comparison with the gentamicin release from commercial Septopal® beads (•}. Error bars denote the SD over 3 different beads of hand-rolled and Septopal® beads.
55
Chapter 3A
Figure 4a
Figure 4b
56
Figure 4c
Figure 4d
Concepts for increasing the gentamicin release
57
Chapter 3A
Figure 4e
Figure 4 . SEM micrographs of gentamicin-loaded, fractured acrylic beads: (a) prepared with the prescribed monomer amount. (b) beads prepared with 50% of the prescribed monomer amount. (c) beads prepared with 50% of the prescribed amount of monomer and 15w/w% PVP 1 7 (before release). (d) beads prepared with 50% of the prescribed amount of monomer and 1 5w/w% PVP 1 7 (after release). ( e) commercial Septopal®. SEM was taken from fracture side of the beads. Scale bars equals 100 µm for low and high magnifications micrograph and for the insert.
SEM evaluation of bead porosities
Figure 4 visualizes the porosities of differently prepared beads. In Figure 4a, it can be
seen that beads prepared with 100% monomer form a dense and massive material
with little porosity. Reduction of the amount of monomer with respect to the
prescribed amount causes a major increase in porosity and it can be seen that
polymer particles are fussed less well together and appear "sintered" together (Figure
4b ). Septopal® beads clearly present a porous structure (Figure 4e) that is more
58
Concepts for increasing the gentamicin release
open than of sintered beads with 15% PVP17 added (Figures 4c). Small particles of
around 10 µm can be discerned which disappeared after antibiotic release, leaving
pores of equal size (see arrows, Figures 4d).
Discussion
In this paper we describe a simple, cheap and effective formulation and process to
prepare gentamicin-releasing acrylic beads, with release kinetics better than that of
commercially available Septopal® beads. The improved release kinetics is first of all
the result of an increased porosity of the beads, which is the result of using only 50%
of the prescribed amount of monomer, which causes sintering rather than
polymerization fusion of polymer particles, leaving a porous matrix. Furthermore, the
addition of a gel-forming polymeric filler, PVP17, ensures penetration of fluids into all
parts of the matrix thereby increasing the total amount of drug that is released.
With the reduction of the amount of monomer, the hardening time of the
acrylic reduces considerably and it requires some dexterity to prepare beads within
the time available. Yet, it is possible to produce beads although especially the last
beads prepared out of a batch appear brittle. In general however, manual
examination of the 50% monomer beads yielded the conclusion that the beads had
sufficient strength for this non-load bearing application of bone cement. Reduction of
the monomer content to below 50% was impossible as no integrity between polymer
beads could be obtained at lower concentration 10. Considering the short time
available to prepare beads, a template system should be considered.
The use of gel-forming polymeric filler turned out indispensable to increase the
gentamicin release to levels comparable or higher than the release of gentamicin by
commercially available Septopal® beads. Although we chose to use PVP17 for this
59
Chapter 3A
purpose, other biodegradable fillers may also have served the purpose. PVP17 filled
beads showed excellent release profiles of the gentamycin and the Kollidon 1 7PF
grade is considered safe for parenteral use in humans 11. Moreover, the PVP17 could
easily be combined in the production process with the acrylic polymer.
Also McLaren et al. 7 investigated the use of biodegradable fillers, but did not
combine this with the use of less monomer, and therewith no additional intrinsic
porosity was created, i.e. a porosity achieved without dissolution of any filler material.
In line with the present findings, McLaren et al., showed that it is not the filler material
that is crucial but rather the particle size7 •1 2 and fillers with a larger particle size lead
to larger pores, less pore interconnectivity, and faster fluid penetration 1 2. Smaller size
particles lead to smaller pores, greater pore interconnectivity and smaller areas
between the pores with no fluid penetration 12•
Conclusion
The release of gentamicin from acrylic beads can be increased by decreasing the
amount of monomer used for polymerization. This increases the porosity of the
beads. A further increase in the extent of antibiotic release could be achieved by
increasing the permeability of the matrix by adding gel-forming polymeric fillers. The
beads developed in this study have an improved gentamicin release compared to the
release from commercially available Septopal® beads.
The formulation and process described meets the requirements set in the
introduction of being cheap, simple and effective.
60
Concepts for increasing the gentamicin release
References
1 . Buccholz H.W. and Engelbrecht, H. Depot effects of various antibiotics mixed with Palacos resins. Chirurg 1 970;41 :51 1 -51 5.
2. Klemm, K.W. Antibiotic bead chains. C/in Orthop 1 993;295:63-76.
3. Wahlig, H., Dingeldein, E. and Bergman, R. The release of gentamicin from polymethylmethacrylate beads. An experimental and pharmacokinetic study. J Bone Joint Surg Br 1 978;60-B:270-275.
4. Diefenbeck, M., Muckley T. and Hofmann, G.O. Prophylaxis and treatment of implant-related infections by local application of antibiotics. J Care Injured 2006;37:95-104.
5. McLaren, A.C, Nelson, C.L., McLaren, S.G. and DeCLerk, G.R. The effect of glycine filler on the elution rate of gentamicin from acrylic bone cement: a pilot study. Clin Orthop Re/at Res 2004;427:25-27.
6. McLaren, A,C,, McLaren, S.G. and Smeltzer, M. Xylitol and glycine fillers increase permeability of PMMA to enhance elution of daptomycin. Clin Orthop Re/at Res 2006;451:25-28.
7. McLaren, A.C., McLaren, S.G. and Hickmon, M.K. Sucrose, xylitol, and erythritol increase PMMA permeability for depot antibiotics. Clin Orthop Re/at Res 2007;461 :60-63.
8. Walenkamp, G.H.I.M., Vree, T.B. and Van Rens, T.J. Gentamicin-PMMA beads. Pharmakokinetic and nephrotoxicological study. Clin Orthop re/at Res 1 986;205: 1 71-183.
9. Sampath, S.S. and Robinson, D.H. Comparison of new and existing spectrophotometric methods for the analysis of tobramycin and other aminoglycosides. J Pham1 Sci 1 990;79:428-431 .
10 . Willert, H.G., Mueller, K. and Semlitsch, M. The morphology of polymethylmethacrylate (PMMA) bone cement. Surface structure and causes of their origin. Arch Orthop Traumat Surg 1979;94 :265-292.
1 1 . Buhler, V. Kollidon® Polyvinylpyrrolidone for the pharmaceutical industry, BASF, 4th ed., 1 999.
12. McLaren, A.C., McLaren, S.G., Mclemore, R. and Vernon, B.L. Particle size of fillers affects permeability of polymethylmethacrylate. Clin Orthop Re/at Res 2007;461:64-67.
61
Chapter 38
Addition of Soluble Fillers to
Achieve Percolation in Antibiotic
Loaded PMMA Beads
Chapter 3B
Introduction
Post-operative and post-traumatic infections of bone, soft tissue and joints continue
to be a major challenge in modern surgery, and are among the most serious
complications occurring. Substantial progress in treating these diseases has been
made by the invention of antibiotic-loaded PMMA beads providing high local
antibiotic delivery1 •4•
The antibiotic-loaded bead system commercially available is Septopal®, but
Septopal® beads are not readily available worldwide for different reasons. In
developing countries, for instance, the price of these beads is far too high for most
patients. Therefore, orthopedic surgeons manually add and mix antibiotics to bone
cement, and prepare these beads themselves. Previous results have shown that
effects of fosfomycin release from such beads are not comparable with the effects of
gentamicin release from commercially available bead systems (see Chapter 2 of this
thesis).
Antibiotic release from bone cement is a complex process and important
variables include: type of antibiotic and powder size5•6
, type of bone cement7 and the
mixing conditions8•9
•1 0
• Due to the controlled pharmacokinetics, the antibiotic is
released from PMMA beads by way of diffusion, which is dependent on the material
properties of the beads. The PMMA matrix is structured to provide for optimum
interaction between the carrier matrix and the antibiotic11• Initially, however,
antibiotics adhering to the surface of the PMMA beads will dissolve rapidly to create
a so-called burst-release, which is followed by the prolonged release of antibiotic
from the PMMA matrix by diffusion. Clearly, prolonged release depends on the
porosity of bone cement12. The effects of porosity and pore size distribution on water
permeability and drug release 13 are included in so-called percolation models 14-11. The
64
Addition of soluble fillers
application of the principles of percolation theory is useful to design bead systems
with an optimal antibiotic release. In essence a "percolating cluster'' can be described
as a function of relative volume ratios of one or more components in a matrix1 5•1 6 and
when their concentration exceeds a certain threshold, a system with interconnecting
pores is obtained yielding optimal antibiotic release.
In order to enhance the porosity of PMMA bone cements, various fillers, such
as dextran 18, glycine 19•20 , sodium chloride (NaCl), or a second antibiotic have been
added21• Here we propose to improve the porosity and consequently increase the
antibiotic release of PMMA beads by adding glycine, a biologically and chemically
inert, water soluble and inexpensive material22 in combination with NaCl to
gentamicin-loaded cement systems in order to produce effective antibiotic-releasing
beads. Results will be qualitatively interpreted in terms of percolation theory.
Theory: The concept of percolation
Figure 1 schematically presents an antibiotic-loaded PMMA bead containing various
types of porosities. In Figure 1A, only three of the antibiotic particles are connected
with the bead surface through these pores, yielding the remaining beads inaccessible
for dissolution and release. Hypothetically, such a system may be able to ultimately
release, say 50% of its antibiotic content. The system depicted in Figure 1 B is
percolating, because all antibiotic particle are connected through pores with the bead
surface and ultimately 100% release of its antibiotic content can be expected.
Whereas the same is true for Figure 1 C, pores connect in this percolating from one
side of the bead to another, yielding faster release than expected for the system in
Figure 18.
65
Chapter 3B
Antibiotic release
© 1 00%
50% non-percolating system
time A
Antibiotic release
® 1 00%
percolating system 50%
B time
Antibiotic release
1 00%
flow through percolating bead
50%
C time
Figure 1. Percolation and hypothetical antibiotic release from a porous polymer matrix, expressed in terms of the percentage of the total amount of antibiotic incorporated. e antibiotic particle; / pore.
Soluble fillers, like for instance glycine, NaCl, polyvinylpyrrolidone (PVP) or
hydroxypropylmethylcellulose (HPMC) can be added to increase the porosity of bead
systems over time and aid to create a percolation system (see Figure 2B and C).
Soluble fillers incorporated in a bone cement matrix will slowly dissolve, leaving
behind a porous structure and assisting percolation.
66
non-percolating system
non-percolating
i dissolution
of filler
non-percolating
' dissolution of filler
Antibiotic 1 00
50%
Antibiotic
1 00%
Antibiotic 100%
50%
Addition of soluble fillers
fimA
timA
fimA
Figure 2. Percolation and hypothetical antibiotic release from a porous polymer matrix with increasing amount of soluble fillers from A (no filler) to C, expressed in terms of the percentage of the total amount of antibiotic incorporated • antibiotic particle; / pore; 0 soluble filler particle.
Materials and methods
Cement preparation with g/ycin and different concentration of sodium chloride
8 g PMMA powder (Simplex P®, Stryker Howmedica OSTEONICS, Howmedica,
Ireland) is first mixed with 0.2 g of gentamicin sulphate powder (Gracia
Pharmaceutical, Indonesia), 0.6 g crystalline glycine (Merck, Darmstadt, Germany)
and different amounts (0 g, 12 g, 16 g and 20 g) of NaCl (Merck, Darmstadt,
Germany) in a ceramic bowl for 2 min. Once the antibiotic powder and soluble fillers
are fully blended, the monomer (methacrylate/MMA) is poured over the powder,
allowed to wet it and again blended according to the manufacturer instructions using 67
Chapter 3B
a spatula for 2 min to yield a free flowing paste. Subsequently, beads with an
approximate diameter of 1 2 mm are hand-rolled, for which 30 min are available until
complete polymerization.
Particle size distributions of gentamicin sulphate, PMMA powder Simplex-P
bone cement, crystalline glycine, NaCl were measured by laser diffraction
spectrometry (Helos H0503, Sympatec GmbH, Germany) using a 200 mm lens. The
powders were dispersed with a Sympatec Rados dry disperser.
Determination of gentamicin release
The gentamicin-loaded beads were placed in 10 ml of phosphate buffer saline (PBS)
and incubated at 37°C. At designated time intervals (6, 24, 48, 72, 168, 336 h), 0.5
ml aliquots of the gentamicin-PBS solution were taken and their gentamicin
concentrations were measured using an o-phtaldialdehyde reagent, made and stored
for 24 h in a dark environment23•24
. The gentamicin sample, o-phtaldialdehyde
reagent and 2-mercaptoethanol were mixed in equal proportions and stored for 30
min at room temperature. The o-phtaldialdehyde reacted with the gentamicin and a
chromophoric product was obtained. The absorbance was measured at 332 nm25
using a Spectronic 20 Genesys spectrophotometer. Gentamicin release was
expressed as a percentage of the total amount incorporated.
Results
Particle size measurements
Table 1 summarizes the size of the gentamicin particles size which are commonly
used in Indonesia and also used in this in vitro study. As can be seen, the 50%
distribution of the gentamicin particles is 18 µm and of the PMMA powder it is 15 µm.
68
Addition of soluble fillers
The soluble filler particles of NaCl and glycine are larger and having a mean size of
304 µm and 272 µm respectively.
Table 1. Particle size distribution of bead components as measured by laser diffraction. Diameters represent the cumulative powder volume up to 1 0, 50, and 90% respectively.
Substance d10 [µm] d50 [µm] d90 [µm]
Gentamicin sulphate 5.9 18. 1 42. 1
Simplex-P PMMA powder 1.8 15.5 51.6
Crystalline glycine 43.7 271.8 493. 1
NaCl 117 304.0 487.2
Gentamicin release after addition of soluble fillers
Addition of only glycine to gentamicin-loaded PMMA bone cement yielded only a
minor increase in gentamicin release (see Figure 3), as compared with the effects of
adding NaCl. The effects of adding NaCl to the gentamicin-glycine-PMMA mixture
are shown in Figure 3. The addition of NaCl clearly enhances gentamicin release
stronger than the enhancement achieved by the addition of glycine only. Gentamicin
release increases when the NaCl content is increased up to 16 g per 8 g PMMA but
in the range between 16 and 20 g NaCl there is no further increase in gentamicin
release anymore, demonstrating that the percolation threshold has probably been
reached.
69
Chapter 3B
100
75
j 50
. . . . . .. . . . .. . . . . - - - - · · · · · · · · · · · · ·
� ,,. ... -- -+--- • -- - ------- - ------+-- - - -------- - - - ----------- ---•"
25
0 +-----�---�--�---�-----.-----..----i
48 96 144 1 92 240 288 336
Time (hours)
Figure 3. Cumulative gentamicin release expressed as a percentage of the total amount of gentamicin incorporated of gentamicin-loaded PMMA bone cement in combination with glycine and different concentration of NaCl as a function of time during exposure to phosphate buffer saline. A, 0 g NaCl; +, 12 g NaCl; o, 1 6 g NaCl; A , 20 g NaCl
Discussion
In this study we examined whether addition of NaCl in combination with glycine could
increase gentamicin release from antibiotic-loaded PMMA beads. Gentamicin release
increased with an increasing amount of NaCl and percolation was achieved when 0.6
g glycine and more than 1 6 g of NaCl was added to 8 g of PMMA. Addition of 0.6 g
glycine alone did not achieve percolation, which could be partly due to the large
particle size of glycine, as generally higher amounts of filler are needed to achieve
percolation when the particle size increases 16. For developing countries, where
antibiotic-releasing beads are hand-made and release of antibiotics out of these
beads is low if not absent, the concepts forwarded here are extremely important as
70
Addition of soluble fillers
they show that with cheap and simple means, i.e. the addition of glycine and NaCl, a
percolating system can be achieved with enhanced antibiotic release.
Conclusion
The gentamicin release from PMMA bone cements increases by adding glycine and
NaCl, with the percolation threshold occurring between 1 2 g and 1 6 g NaCl added to
8 g PMMA with 0.6 g glycine.
71
Chapter 3B
References
1 . Buranapanitkit, B., Wongsiri, S., lngviya, N., Chamniprasas, K. and Kalnauwakul, S. In vitro inhibitive effect of antibiotic beads to common orthopaedic pathogens: Home-made vs commercial beads. The Thai J Orthop Surg 2000;25:48-52.
2. Winiger, D.A. and Fass, R,J. Antibiotic-impragnated cement and beads for orthopedic infections. Antimicrob Agents Chemother 1 996;40:2675-2679.
3. Kent, M.E., Rapp, R.P. and Smith, K.M. Antibiotic beads and osteomyelitis: Here today, What's coming tomorrow? Orthopedics 2006;29:599.
4. Brian, W.W., Salvati, E.A., Klein, R., Brause, B. and Stern, S. Antibiotic impregnated bone cement in total hip arthroplasty: an in vivo comparison of the elution properties of tobramycin and vancomycin. Clin Orthop 1 993;296:242-248.
5. Penner, M.J., Masri., B.A. and Duncan, C.P. Elution characteristics of vancomycin and tobramycin combined in acrylic bone cement. J Arthroplasty 1 996;11 :939-944.
6. Lawson, K.J., Marks, K.E., Brems, J. and Rehm, S. Vancomycin vs tobramycin elution from polymethylmethacrylate: an in vitro study. Orthopedics 1 990;71:625-629.
7. Penner, M.J., Duncan, C.P. and Masri, B.A. The in vitro elution characteristics of antibioticloaded CMW and Palacos-R bone cements. J Arthroplasty 1 999;14:209-214.
8. Deluise, M. and Scott, C.P. Addition of hand-blended generic tobramycin in bone cement: effect on mechanical strength. Orthopedics 2004;27:1 289-1291 .
9. Lewis, G., Janna, S. and Bhattaram, A. Influence of the method of blending an antibiotic powder with an acrylic bone cement powder on physical, mechanical, and thermal properties of the current cement. Biomaterials 2005;26:431 7-4325.
1 0. Neut, D., Van de Belt, E., Van Horn, J.R., Van der Mei, H.C. and Busscher, H.J. The effect of mixing on gentamicin release from polymethylmethacrylate bone cements. Acta Orthop Scand 2003;74:670-676.
1 1 . Wahlig, H. Gentamicin-PMMA beads, a drug delivery system; basic results (1 980). In: van Rens Th JG, Kayser FH. Local antibiotic treatmentin osteomyelitis and soft-tissue infections, International Congress Series 556, S. 9, Excerpta Medica.
1 2. Baker. A.S. and Greenham, L.W. Release of gentamicin from acrylic bone cement. Elution and diffusion studies. J Bone Joint Surg Am 1 988;70: 1 551 -1 557.
1 3. Van Veen, B. Compaction of powder blends. Effects of pores, particles and percolation on tablets strength. (dissertation). Groningen University; 2003.
1 4. Stauffer, D. Introduction to percolation theory. 1 985, London and Philadelphia: Taylor & Francis. 1 -120.
1 5. Sahimi, M. Applications of Percolation Theory. Taylor & Francis, Bristol, 1 994, PA.
1 6. Caraballo, I., Millan, M., Fini, A., Rodrigues, L. and Cavalarri, C. Percolation thresholds in ultrasound compacted tablets. J Controlled Release. 2000;69:345-355.
1 7. Sahimi, M. Flow and transport in porous media and fractured rock: From classical to modern approaches. VCH, 1 995, New York, NY.
1 8. Kuechle, D.K., Landon, G.C., Musher, D.M. and Noble, P.C. Elution of vancomycin, daptomycin, and amikalin from acrylic bone cement. Clin Orthop 1991 ;264 :301-308.
1 9. Mc Laren, A.C., Mc Laren, S.G. and Smeltzer, M. Xylitol and glycin fillers increase permeability of PMMA to enhance elution of Daptomycin. Clin Orthop 2006;451 :25-28.
20. Mc Laren, A.C., Nelson, C.L., Mc Laren, S.G. and DeClerk, G.R. The effect of glycin filler on the elution rate of gentamicin from acrylic bone cement. Clin Orthop Rel Res. 2004;427:25-27.
21 . Penner, M.J., Masri, B.A. and Duncan, C.P. Elution characteristics of vancomycin and tobramycin combined in acrylic bone cement. J Arthroplasty 1 996;11:939-944.
72
Addition of soluble fillers
22. DiCicco, M., Duong, T., Chu, A. and Jansen, S.A. Tobramycin and gentamicin elution analysis between two in situ polymerizable orthopedic composites. J Biomed Mater Res B Appl Biomater 2003;65:1 37-149.
23. Sampath, S.S. and Robinson, D.H. Comparison of new and existing spectrophotometric methods for the analysis of tobramycin and other aminoglycosides. J Pharm Sci 1 990;79:428-431 .
24. Zhang, X., Wyss, U.P., Pichora, D . and Goosen, M.F. Biodegradable controlled antibiotic release devices for osteomyelitis: optimization of release properties. J Pharm Pharmacol 1994;46:71 8-724.
25. Frutos, C.P ., Diez, P .E., Baralles-Rienda, J.M. and Frutos, G. Validation and in vitro characterization of antibiotic-loaded bone cement release. Int J Pharm 2000;209:1 5-26.
73
Chapter 4
A Template to Produce
Antibiotic-Loaded PMMA-Beads
in the Operating Room
Chapter 4
Introduction
The concept of local antibiotic therapy using antibiotic-loaded bone cement and
antibiotic loaded PMMA beads to treat infected arthroplasties was introduced in the
1970s 1. Due to its success in effectively treating arthroplasty infections, interest
developed in applying antibiotic-loaded cement as a therapy for chronic
osteomyelitis2 ·3.4. Chronic osteomyelitis is an infection of the bone which is difficult to
cure. For the clinical picture of chronic osteomyelitis we refer to Chapter 1. In 1979,
gentamicin-loaded cement beads were first used to fill the dead space created by
debridement of infected bone5 • Since then it is used routinely in clinical practice for
the treatment of osteomyelitis.
One of the properties of the commercially available antibiotic containing beads
is their biphasic antibiotic release kinetics, occurring primarily during the first hours to
days after implantation (the so-called "burst-release"), during which very high local
concentrations of the antibiotic can be attained which never can be reached by any
other way of administering the drug ; the remaining elution persists for weeks and
sometimes years5• The prolonged elution of the antibiotic out of a bead depends
mainly on the porosity of the cement of which the bead is composed. (see Chapter
3).
In a developing country, like Indonesia, commercially available gentamicin
loaded PMMA beads (Septopal® beads, Biomet, Germany) are not readily available
throughout the country and application in an average patient is impeded due to its
high prize.
As a solution, up to now orthopedic surgeons make these beads themselves
by mixing PMMA-components with antibiotics, hand-rolling them during surgery and
trying to make beads with a spherical shape and diameter of around 7 mm, similar to
76
A template to produce antibiotic loaded PMMA-beads
commercial beads. In Chapter 2, we have shown that this generally yields beads with
a highly variable diameter and little antibiotic release.
In order to aid orthopedic surgeons in developing countries with the
preparation of more effective homemade antibiotic-loaded beads of a uniform size,
we first developed a method to increase the efficacy of antibiotic release of hand
rolled beads through the inclusion of biodegradable fillers as has been mentioned in
Chapter 3, but that solution has not yet been tested in beads manufactured with an
operating room procedure as is described in this chapter. Also we have not studied
the release kinetics of the antibiotics from the beads which have been manufactured
with the template described in this chapter neither have we compared these release
kinetics with those from the hand rolled beads. We will address these two subjects in
a separate chapter.
Two options for the design of a template system were considered: either
producing the beads elsewhere in large series and storing them under sterile
conditions or producing them during or immediately prior to surgery. The latter
method was preferred, because it is more cost effective and obviates storage. On the
other hand this implies that the surgeon or staff has to deal with the complete
manufacturing in the operating room prior to or during the operation, perhaps thereby
prolonging the operating time.
It is the aim of this chapter to describe the design of a suitable template
system.
Requirements for the beads to be produced by the system
• The system should be made of non-corrosive materials, and withstand high
temperatures such as are reached during sterilization in an autoclave and the
77
Chapter 4
curing of PMMA cement, i.e. a temperature of more than 120 degrees
Centigrade. The materials that have been chosen are stainless steel and
PTFE.
• The system should be durable against wear such as will occur by repeated
handling.
• The system should be easy to handle, allowing even minimally schooled
people to assemble and disassemble the construct, in other words the
template should be user friendly in daily use.
• To obtain one chain of hand-made antibiotic loaded beads, the beads should
be connected by a smooth stainless steel wire (0.8 mm in diameter). The
number of beads per wire should be 30, the ideal length for one chain of
beads, and must fit to the length of the adult long bone and for the
manufacturing process need 10 min to finish after pouring into the template.
• To create one chain of beads with an approximate diameter of 8 mm using the
template, about 20 min will be allowed for hardening. The template system
must be reproducible. The manufacturing of the beads should be done under
sterile conditions by the orthopedic surgeon during the operative procedure.
Material and methods
The system that was developed consists of a template in which 30 beads can be
produced in a single process. The template was designed in a ring mode to facilitate
easy manufacturing. The template materials chosen are stainless steel and PTFE6•
78
A template to produce antibiotic loaded PMMA-beads
Description of the template
In general, the template is designed in such a way as to create a uniform spherical
shape of all 30 beads on one chain. It consists of two parts i.e. two PTFE plates and
a set of three stainless steel rings, which have to be assembled and fitted together in
order to create a uniform size and spherical shape of the beads. These five
components will be described in the next paragraphs
PTFE plates
The main part consists of two PTFE plates (upper plate 1 and lower plate 2) with an
outside diameter of 145 mm, 28 mm wide and 5 mm thick. In each plate 30 half holes
with an inner diameter of 8 mm are prepared and also a groove across and in the
middle of the holes and in the PTFE connecting the holes. The distance between
each hole is 4 mm. The two plates are connected together on top of each other to
create 30 PMMA beads with 8 mm in diameter. The groove in the PTFE allows to
place an 0.8 mm stainless steel wire. Thereby, the beads can be connected to each
other. The schematic design of this PTFE part is depicted in detail in Figures 1 and 2.
PTFE plates 1 and 2 serve to create spherically shaped beads. Each plate contains
30 half spherical holes. When the two plates are assembled in the right way the half
spheres of the upper plate match those of the lower one and thus form a full sphere
with a funnel in each sphere in the upper plate that can be filled with PMMA (see
Figure 2). In the bottom part of each sphere there is a small hole. The function of this
small hole is to evacuate the air that will be entrapped when the PMMA in its doughy
phase is pushed into the spheres.
79
Chapter 4
Figure 1. Schematic design of PTFE plate-1 , an outside diameter of 145 mm, 28 mm wide, and 5 mm
thick.
I WIXNQQ 21-s QSXX&MI I
Figure 2. Schematic design of PTFE plate-2, an outside diameter of 145 mm, 28 mm wide, and 5 mm
thick, showing small holes indicated as dots in the holes on the bottom of the spherical shape
template.
PTFE plates 1 and 2 serve to create spherically shaped beads. Each plate contains
30 half spherical holes. When the two plates are assembled in the right way the half
spheres of the upper plate match those of the lower one and thus form a full sphere
with a funnel in each sphere in the upper plate that can be filled with PMMA (see
Figure 2). In the bottom part of each sphere there is a small hole. The function of this
80
A template to produce antibiotic loaded PMMA-beads
small hole is to evacuate the air that will be entrapped when the PMMA in its doughy
phase is pushed into the spheres.
Stainless steel rings
The additional part of the system consists of three stainless steel rings which will
reinforce the PTFE rings and are able to withstand compression to the plates.
1. An upper ring with an outside diameter of 166 mm. This ring has a screw thread
inside (Figure 3).
2. A center ring with an outside diameter of 112 mm. It has a screw thread on the
outside (Figure 5).
3. A base ring with an outside diameter of 145 mm. It has a screw thread on the
outside corresponding to the one on the inside of the upper ring and a screw
thread on the inside corresponding to the one on the outside of the center ring
(Figure 4).
This system is made of a non corrosive material that is heat stable during
autoclaving.
D 1 . i I o I 1
Figure 3. Schematic design of the upper ring.
81
Chapter 4
II J:l D
Figure 4. Schematic design of base ring.
o
I I 1 I I j I I
Figure 5. Schematic design of center ring.
The schematic design of the beads mould in detail
The mould of the beads is made of PTFE that is heat stable so as to allow
sterilization. In cross section, the bead mould is divided into three parts namely neck,
upper half hole and bottom half hole. In the middle of the spherical shape part, a 0.8
mm diameter groove is located. The function of this groove is to admit a smooth
stainless steel wire which has to hold the beads together once they are formed within
82
A template to produce antibiotic loaded PMMA-beads
the holes (see Figures 6 and 7). The neck part which is situated in the upper plate 1
resembles a funnel, which should be the entrance for PMMA mixture into the hole.
The size of this neck is 6 mm in diameter and 1 mm in height.
0 6 mm
PTFE plate-1
0.8 mm in diameter
PTFE plate-2 4 mm
0 8 mm
Figure 6. Cross section of the mould of one bead with the 0.8 mm diameter stainless steel wire in the middle of this bead.
Assembly of the beads template system
All parts of the system must be attached to each other in the sequence, depicted in
Figure 8. Under sterile conditions, the two PTFE plates are coupled after a stainless
steel flexible wire of 0.8 mm diameter is inserted into the groove on and across the
holes of the PTFE plate-2 which is placed on the base-ring, eventually, the wire will
be inserted through the holes of this ring and the wire is tightened from the outside,
after which plate one is put on top of plate two. Then, the center ring is put on top of
the coupled PTFE plates fixing them tightly to the base ring by means of a screw
thread on the outside of the centre ring which corresponds to a similar screw thread
on the inside of the base ring. At the end of the assembly the upper steel ring has to
be attached to the base ring by screw thread fixation thereby strengthening the whole
construct. With this the beads template system is completely assembled.
83
Chapter 4
e (A)
L Small hole for air removal
Figure 7. Schematic design demonstrating {A) a small hole for air removal, which is located in the bottom of the PTFE plate-2; {B) an example of a chain of completely assembled moulds connected to each other by a flexible stainless steel wire.
The realization of beads template system
The pair of PTFE plates form the primary part of the system and has an important
role in the formation of the spherical shape of the beads. It is divided in the neck and
a half hole in plate one and a half hole in plate two. In Figure 9 the completely
assembled beads template system is shown. The bead mould PTFE component and
strengthener component (stainless steel) together form the complete template
system that is needed to manufacture the PMMA beads.
The upper and the centre ring on top of the assembly form a gutter in which all the
funnels that give access to the bead moulds are visible. This gutter can be used to
divide the doughy cement over all the openings of the funnels in order to fill the
moulds.
84
o I r
o I I
o I I
o ! I
). I
I I I 5Y&6NQYY:pYY9519Yjl i
Id! lj
; i
A template to produce antibiotic loaded PMMA-beads
(E)
(D)
(C)
(B)
(A)
Figure 8. Assembly of a semi manual beads template system consisting of the stainless steel part (A=base ring, D=centre ring and E=upper ring) and the PTFE plates (B=plate two and C=plate one).
Figure 9. The completely assembled template system for the preparation of PMMA beads.
85
Chapter 4
Preparation of the beads after the template is completely assembled
Under sterile conditions, cement powder is mixed with powdered gentamicin sulphate
in a ceramic bowl using a spatula. 1 g of gentamicin sulphate is added to 40 g of
polymer powder. The resulting mixture is subsequently combined with monomer 20
ml in a ceramic bowl and mixed for 2 min with a spatula according to the
manufacturer's instructions, which will result in the formation of doughy cement. This
is then spread over all the holes in the gutter on top of the assembly between the
upper and centre stainless steel rings. Eventually doughy cement fills up all the holes
by manual pressure distribution using a PTFE stick as a tool for pushing all the
doughy mixture obtaining a homogeneous distribution in all the holes. At 20 min as
judged by the temperature of the cement the rings are pulled off the PTFE plates and
the chain of beads can be harvested.
Results
Characteristics of the PMMA beads
The filled template system can be seen in Figure 1 0. The entire process of filling the
mould with PMMA cement and creating beads requires approximately 20 min,
including the time required for the curing of the cement which seems to be about
twice faster than by hand rolling.
We compared the characteristics of two strings of beads prepared using the template
system. The diameter of the beads prepared appeared approximately uniform in size
with equal distance from each other. Table 1 summarizes the characteristics of both
strings of beads prepared. The area to volume ratio of the template beads is 7 cm·1
which is slightly lower than of Septopal® beads (8.6 cm·1).
86
A template to produce antibiotic loaded PMMA-beads
Figure 10. Photographs of chain of antibiotic-loaded beads. (A): PMMA beads prepared using the template system on the surface of the PTFE plate-2. (B) Chain of beads attached to a stainless steel wire. The beads are more or less uniform in spherical shape and have a diameter of 8 mm with a distance of 3-4 mm in between.
Table 1. Characteristics of two different strings of beads prepared using the template system described. All beads were prepared from Simplex-P bone cement, mixing was done manually using a spatula.
Strains Beads distance (mm) Diameter (mm) AN ratio (cm·1)
Average Min Max Average
Strain-1 3.5 6.3 9.3 8.8 6.6
Strain-2 3.2 8.4 9.2 8.7 6.8
AN ratio: area per volume ratio
87
Chapter 4
Discussion
The study of Chapter 4 is focused on the engineering part. Production of a chain of
antibiotic-loaded PMMA beads during or immediately prior to surgery was considered
preferable because it is more cost effective.
The area per volume ratio in our experiment is 7 cm·1 which is only slightly less than
of commercial ones (8.6 cm·1). This ratio is important to stimulate elution of
antibiotics: the smaller size of the bead will allow more antibiotic to be released from
the bead.
In Figure 10 the beads do not exhibit a perfectly spherical shape, likely due to
the fact that during pouring the part of the doughy mass, which is poured in first will
immediately polymerized and the remaining mixture which is located in the upper
PTFE will be delayed in hardening. Since the mould beads have a neck part,
assuming that doughy mass will first fill in all the holes until the neck is filled. These
assumptions clarify the non-uniform spherical shape of some beads produced by the
template system.
This template system seems to be resistant to wear such as will occur by
repeated handling. It is easy to handle allowing even minimally schooled people to
assemble and disassemble the construct, in other words this bead template is user
friendly in daily use.
Conclusion
In conclusion, the template system of a relatively simple design as discussed here
can be used to produce a chain of 30 antibiotic-loaded PMMA beads under operating
room conditions in approximately 20 min. Beads produced have a more or less
88
A template to produce antibiotic loaded PMMA-beads
spherical shape of 8.5 mm in diameter, with a distance between the beads of around
4 mm.
89
Chapter 4
References
1 . Buchholz, H.W. and Engelbrecht, H. Depot effects of various antibiotics mixed with Palacos resins. Chirurg 1970;41 :51 1 -515
2. Mader, J.T., Calhoun, J. and Cobos, J. In vitro evaluation of antibiotic diffusion from antibioticimpregnated biodegradable beads and polymethylmethacrylate beads. Antimicrob. Agents Chemother 1997;41 :41 5-418.
3. Cierny, G. and Mader, J.T. Adult chronic osteomyelitis. Orthopedics 1984;7: 1 557-1 564.
4. Calhoun, J.H. and Mader, J.T. Antibiotic beads in the management of surgical infection. Am. J. Surg 1977;1 57:443-449.
5. Ziran, B.H. and Rao, N. Infections, in Orthopaedic Knowledge Update; Trauma 3, American Academy of Orthopaedic Surgeon, MOS, 3rd ed, 2005; 1 31 -1 39.
6. Lee, H.B., Kim, S.S. and Khang, G. Polymeric Biomaterials. In: Bronzino JD, editor. The Biomedical Engineering Handbook. CRC Press, Inc, IEEE Press, USA, 1 995; 581 -597.
90
Chapter 4-append ix
Gentamicin-Release from
Template Made Beads
Including PVP1 7
Chapter 4 - appendix
Gentamicin-release from PMMA beads is partly a surface phenomenon, and partly
controlled by diffusion out of the bulk. Since it is possible that template-made beads
have different surface porosity than hand-rolled beads, we verified using the methods
described in Chapter 3 whether gentamicin was still released from template-made
beads. Figure 1 demonstrates the effect of adding different concentration of PVP17 in
half doses of monomer using the template system. The results clearly show that
template-beads with a biodegrable filler release antibiotics like handmade beads with
a biodegrable filler (see Chapter 3).
100
80
60
:� 40
20
0 0 48 96 144 192 240 288 336
Time, hours
Figure 1. The cumulative gentamicin release from PMMA beads made with different dosages of PVP1 7 (�. 5 w/w%; •, 10 w/w%; A , 1 5 w/w%) and 50% of monomer amount, expressed as a percentage of the total amount of gentamicin incorporated as a function of time during exposure to phosphate buffer saline. Error bars denote the SD over 3 different beads made by template system.
92
Chapter 5
In Vitro Evaluation of Hand-Made
Gentamicin-Loaded PMMA Beads
to Prevent Biofilm Formation
Chapter s
Introduction
Biofilms have been identified on sequestra of dead bone and on bone grafts, from
which they can incite an invasive infection called chronic osteomyelitis. Chronic
osteomyelitis remains an important and daunting orthopedic and clinical problem.
Staphylococcus aureus is an organisms identified as causative to osteomyelitis in
many patients and biofilms of S. aureus show a characteristically higher degree of
resistance to host immune responses and antimicrobial treatments than planktonic
cells 1 • Systemic antibiotic administration is therefore often unsuccessful.
High systemic levels of gentamicin imply the risk of toxicity and organ failure,
such as hearing or kidney damages. In 1970 Buchholz and Engelbrecht2 introduced
the addition of gentamicin to polymethylmethacrylate (PMMA) bone cement,
delivering an effective antibiotic at a sufficiently high concentration to the area of
infection3-5 but with very low systemic concentrations, suggesting advantages such as
infection control and lack of side-effects6• Nowadays, gentamicin-loaded PMMA
beads are used for the treatment of osteomyelitis7•8
. Implantation of these beads (in
the form of chains) after debridement of a focus of infection fills dead space with
antibiotic releasing beads. Gentamicin is mostly the antibiotic of choice because it is
active against Gram-positive and Gram-negative organisms in low minimal inhibitory
and bactericidal concentrations, it is highly soluble in water and stable at relatively
high temperatures.
Gentamicin-loaded beads are commercially available in many countries under
the name Septopal®, but in some parts of the world (in particular developing
countries) these beads can not be applied as their price is far too high. Therefore,
antibiotic-loaded PMMA beads are hand-rolled by the orthopedic surgeons.
Unfortunately, these hand-rolled beads are largely ineffective in their release
94
Efficacy of handmade gentamicin-beads
of antibiotic. In the previous chapters we demonstrated that this could be solved by
using half the amount of prescribed monomer and adding a biodegradable filler to the
bead system. However, hand-rolled beads are not uniform in size and usually larger
than commercially available beads, with a more favorable area-to-volume ratio. To
solve this problem, a template system was designed (Chapter 4 ), in which beads of
uniform size and small diameter could be produced on a chain.
The aim of this study is to compare the antibacterial efficacy of the gentamicin
loaded beads (hand-rolled and template-made) with the one of commercially
available beads.
Materials and methods
Gentamicin-loaded bone cement beads
Preparation hand-rolled beads. Simplex-P bone cement powder (Stryker
Howmedica OSTEONICS, Howmedica International S, Limerick, Ireland) was mixed
with powdered gentamicin sulphate (Gracia Pharmaceutical, Indonesia) and PVP17
(BASF, Germany) for 2 min in a ceramic bowl employing a spatula. Powdered PMMA
and gentamicin sulphate (1 g gentamicin sulphate and 40 g PMMA powder) were
mixed with different amounts of PVP17 (15w/w% with respect to the amount of
polymer powder) and beads were prepared with 50% of the prescribed amount of
monomer. Powder and liquid were combined in a ceramic bowl and mixed for 2 min
with a spatula according to the manufacturer instructions. The material is eventually
mixed until a doughy phase is obtained, and the gentamicin-PMMA-MMA mixture
was hand-rolled into beads with an approximate diameter of 12.3 mm. One bead
contains 16 mg of gentamicin sulphate.
95
Chapter s
Preparation beads using a template system. The gentamicin-PMMA-MMA mixture
was prepared as described above and poured into the funnel surface of the template
system {see Chapter 4), yielding beads with an approximate diameter of 8.6 mm.
One bead contains 5 mg of gentamicin sulphate.
Commercially available beads. Gentamicin-loaded PMMA beads are commercially
available under the name Septopal® {Biomet Deutschland, Darmstadt, Germany).
One bead {diameter of 7.0 mm) contains 7.5 mg of gentamicin sulphate.
Preparation of elution media
One template-made, hand-rolled or Septopal® bead was immersed in 5 ml Trypton
Soya Broth {TSB, CM 01 29, OXOID) and incubated at 37°C. Each bead was
transferred daily to 5 ml fresh TSB and again incubated at 37°C yielding broth
containing antibiotic released over a 24 h time span for biofilm growth studies. Only
broth collected after 1 , 2, 3, 7 and 1 4 days was used for further evaluation. Elution
media were stored in a refrigerator at 4°C until further use. Experiments were
performed in triplicate.
Growth of biofilm
Biofilms were grown using a clinical strain, S. aureus 5298, isolated from a patient
with an implant-related osteomyelitis of the University Medical Center Groningen, The
Netherlands. This strain was gentamicin sensitive with a MIC value of 0. 75 µg/ml. A
preculture of the strain was used to fill 96-wells plates with 200 µL bacterial
suspension {2 µL preculture + 1 98 µL fresh TSB, or TSB collected as described
above after antibiotic release from an immersed bead). Biofilms were grown for 24 h
at 37°C. Subsequently, the wells were flushed with 200 µL PBS to remove free-
96
Efficacy of handmade gentamicin-beads
floating bacteria. Then, the wells were stained with 200 µL 1 % crystal violet for 30
min, washed with 200 µL demineralized water to remove the excess stain, and the
crystal violet was solubilized in 200 µL of ethanol-aceton (80:20, vol/vol). The
absorbance at 575 nm was determined by using microtiter plate reader (Fuostar
Optima, BMG, Labtech) and expressed as a percentage with respect to the
absorbance of control biofilms, grown in the absence of released antibiotics. All
experiments were repeated three times with separately grown bacteria.
Statistical analysis
Statistical analysis was done in order to compare the efficacy of gentamicin release
from hand-rolled and template-made beads with the one of commercial Septopal®
beads. Differences were pair-wise analyzed for significance by the Student's t-test,
defining significance at ps0.05.
Results
Figure 1 shows the reduction in biofilm growth achieved by the various amounts of
antibiotics released from the different bead systems evaluated. All bead systems
showed high reductions for up to at least 14 days after immersion.
There is no significant difference in biofilm growth reduction between
template-made beads Septopal® beads (p > 0.4, two-tailed), but hand-rolled beads
were less effective than Septopal® beads at day-14 (p < 0.003).
97
Chapter s
C: 0
1 20
1 00
� 80 ::::,
"C Cl)
.c 60
e (!)
.§ 40
20 I
® • Septopal
2
I
3 Time, days
Template-made
I
7 14
• Hand-rolled
Figure 1. Biofilm growth reduction (%) as a function of time by different gentamicin-releasing beads, calculated as a percentage UV absorbance with respect to a control, i .e. the absorbance of a biofilm grown in broth without antibiotics released from beads. Bars represent the mean ± SD over triplicate results with separately grown bacteria.
Discussion
The novel gentamicin-loaded bead system based on the use of half the prescribed
amount of monomer and the addition of a biodegradable filler and including a
template has been found to reduce biofilm formation equally well as commercially
available Septopal® beads. Hand-rolled beads were slightly less effective than
template-made beads, presumably due to a less favorable area-to-volume ratio: 4.9
cm-1 and 7.0 cm-1 for hand-rolled and template-made beads, respectively. Moreover,
hand-rolled beads yielded much larger standard deviations than obtained with
template-made beads, attesting to the larger reproducibility of preparation of
template-made beads.
98
Efficacy of handmade gentamicin-beads
These novel formula and beads template system can be proposed to
orthopedic surgeons in developing country to apply this system in creating a chain of
antibiotic-PMMA beads. Apart for the costs of the template system, the price of these
template beads is approximately 3x cheaper than of commercial ones.
Conclusion
In conclusion, gentamicin is effectively released from beads prepared with half the
amount of prescribed monomer and biodegradable filler added, regardless whether
the beads were hand-rolled or template-made. Biofilm reduction achieved by these
beads were high and comparable with the one achieved by a commercial bead
system, Septopal®. The system proposed is simple and cheap and constitutes only
minor modifications with respect to the routines used by orthopedic surgeons in
developing countries to prepare antibiotic releasing beads themselves. However, its
efficacy is far superior.
99
Chapter 5
References
1 . Costerton, J.W., Stewart, P.S. and Greemberg, E.P. Bacterial biofilms: a common cause of persistent infections. Science 1999;284 : 1318-1 322.
2. Buchholz, H,W. and Engelbrecht, H. Depot effects of various antibiotics mixed with Palaces resins. Chirurg 1 970;41 :51 1 -515.
3. Buchholz, H.W., Elson, R.A., Engelbrecht, E., Lodenkamper, H., Rottger, J. and Siegel, A. Management of deep infection of total hip replacement. J Bone Joint Surg (Br) 1981 ;63:342-353.
4. Cierny, G., Mader, J.T. and Pennick, J.J. A clinical staging system for adult osteomyelitis. Contemp Orthop 1 985;10:1 7-37.
5. Calhoun, J.H. and Mader, J.T. Antibiotic beads in the management of surgical infections. Am J Surg 1 989;157:443-449.
6. Grieben, A. Treatment of bone and soft tissue infections with gentamicin-polymethyl-methacrylate chains. A review of clinical trials involving 1 500 cases. S Afr Med J 1 981 ;10:395-397.
7. Blaha, J.D., Nelson, C.L., Frevert, L.F., Henry, S.L., Seligson, David, Esterhal, J.L. Jr., Heppenstal, R.B., Calhoun, J., Cobos, J. and Mader, J. The use of Septopal (polymethylmethacrylate beads with gentamicin) in the treatment of chronic osteomyelilis. In Instructional Course Lectures, The American Academy of Orthopaedic Surgeons 1990;39:509-514.
8. Calhoun, J.H. and Mader, J.T. Antibiotic beads in the management of surgical infections. Am J Surg 1 989;157:443-449.
100
Append ix
Appendix
Appendix
The following tables describe: • A comparison between the price of commercial beads (Septopal®) and the
material cost of gentamicin/fosfomycin-loaded beads (30 beads chain). • Unit cost for recurrent treatment and revision surgery. • Approximate cost of bone setter.
Table 1. A comparison between the price of commercial beads (Septopal®) and material cost for local handmade antibiotic beads in Indonesia.
No. Item
1 . Septopal® (30 gentamicin-loaded beads per chain)
2. Material cost of handmade antibiotic-loaded beads (30 beads per chain):
• Simplex-P® bone cement
• Fosfomycin antibiotic powder
• Stainless steel wire (0: 0.8 mm)
Total:
3. Material cost of handmade antibiotic-loaded gentamicin beads
102
(30 beads per chain):
• Simplex-P® bone cement
• Gentamicin antibiotic powder
• Stainless steel wire (0: 0.8 mm)
Total:
Price (USD)
350
90
1 8.82
1 5
123.82
90
7
1 5
1 12
Table 2. Unit cost for recurrent treatment
No. Activities
1 . Surgery for implant related osteomyelitis
• Plate-screws removal & debridement under anesthesia
• Implantation of handmade fosfomycin beads
• Intravenous antibiotic {one week) o 2 g Fosfomycin in 1 00 ml Dextrose 5% {two
times daily for a week)
• Oral antibiotic { one week), two times daily
Total:
2. Revision surgery
• Open reduction and internal fixation under anesthesia {re-plating and re-screwing)
• Intravenous antibiotic {one week}
• Oral antibiotic { one week)
Total:
Appendix
Cost (USO)
1200
123.82
200
21
1 544.82
1400
200
21
1621
Unit cost of bone setters is around USO 5 to USO 10 for one time consultation, including the treatment.
103
General d iscuss ion
General discussion
Introduction
Osteomyelitis is an infection of the bone 1 . The pathogenesis of osteomyelitis has
been explored clinically and different types of osteomyelitis can be classified
according to the source of the infection (i.e. heamatogenous or contiguous focus)
and the vascular capability of the host (i.e. with or without generalized vascular
insufficiency)2. Clinically, the old adage "once osteomyelitis always osteomyelitis"
has not lost its relevance. The multi-factorial nature of these infections demands an
approach that addresses all aspects related to the patient, wound, implant, and
pathogen.
Throughout life, bone is constantly renewed and replaced, and small colonies
of bacteria can become sealed within the bone during longstanding infection. Here
they can survive in a dormant state for many years without producing any symptoms.
Chronic osteomyelitis often requires surgical debridement and local antibiotic
treatment. Disadvantages of currently used non-biodegradable polymethyl
methacrylate (PMMA) carriers include incomplete, low antibiotic release by the
cements and the requirement of surgical removal3 • Moreover, resistant bacteria may
appear on the carrier-surface during later stages of low-level antibiotic release4 •
Although application of biomaterials has been one of the major assets in
modern medicine to improve the quality of life of patients, occurrence of chronic
osteomyelitis is still a serious health threat to the individual patient especially when
treated late in the disease process as often the case in developing countries.
Chronic osteomyelitis can result in morbidity affecting the viability of a diseased limb.
Biofilm formation on a-vascularized bone protects pathogens and leads to
persistence of infection.
106
General discussion
Evaluation of the existing antibiotic-loaded PMMA beads as made
and used in developing country
In developing countries such as Indonesia, the problem is characterized by
accelerating rates of resistance driven by antibiotic misuse and shortfalls in infection
control and public health. The World Health Organization targets factors such as
regulated drug availability, drug quality control, and surveillance in its containment
strategies5•
In Indonesia, fosfomycin-loaded PMMA beads are handmade by orthopedic
surgeons in order to treat osteomyelitis. The efficacy of these beads as compared to
the commercially available ones has hitherto remained unreported, and therefore the
clinical result is still in doubt: Improvement can either come from systemic antibiotics,
the beads themselves or surgical intervention. However, to have better antibiotic
release out of bone cement beads, some requirements should be considered such
as uniform size in diameter, spherical shape, sufficiently high porosity, and antibiotic
concentration. In empirical studies described in Chapter 1, current methods used in
Indonesia are compared with those in the Western-world for the treatment of chronic
osteomyelitis and reasonable clinical results are claimed for hand-made fosfomycin
loaded beads. The in vitro study described in Chapter 2 has been performed in order
to clarify the efficacy of the currently used handmade-fosfomycin PMMA loaded
beads. The study,includes determination of the minimal inhibitory concentrations
(MIC) of fosfomycin against a variety of Indonesian and Dutch bacterial strains,
isolated from orthopedic implants and osteomyelitis patients and measurement of the
release of fosfomycin in relation to the cement porosity and other properties of
fosfomycin-beads as compared with those of Septopal® beads ("the golden
standard" in the treatment of infected bone). Fosfomycin shows efficacy against 70% 107
General discussion
of the bacterial strains from osteomyelitis patients, out of which 40% are
Staphylococcus aureus. However, hand-made fosfomycin beads were able to kill
only two bacterial strains namely S. aureus 7323 and CNS 7391. In this experiment,
fosfomicin-loaded PMMA beads do not yield good release because beads are not
uniform in size, lack sufficient porosity at the surface, and also have a sub-optimal
area-per-volume ratio and may contain non-uniform loading. It means that
fosfomycin is not the ideal antibiotic for inclusion in PMMA beads. To improve this
performance, porosity should be introduced (see Chapter 3), and a bead template
system should be designed (see Chapter 4). Final analysis of a new, cheap and
effective bead system for use in developing countries is required (see Chapter 5),
preferably using in vivo methods.
Methods to establish the antimicrobial efficacy of antibiotic-loaded
bone cements
Since the pioneering work of Bucholz and Engelbrecht10 who proposed incorporating
antibiotic powder directly into the bone cement for prophylaxis of infection, the
question of whether this approach could be used to treat osteomyelitis has been
raised. Klemm answered it in 1979 by producing antibiotic-impregnated PMMA
beads and investigating their use in the treatment of acute and chronic
osteomyelitis 11 . The combination of debridement, gentamicin-PMMA beads, and
intravenous antibiotics resulted in an infection control rate up to 100% 12. In
developing countries such as Indonesia, up to now, gentamicin-loaded PMMA beads
are the only commercially available beads under the name of Septopal®, and are
used in chronic bone infections, without knowing the infecting bacteria and their
sensitivity for gentamicin. However, in many cases Septopal® beads are too 108
General discussion
expensive in the absence of a proper health-care insurance system. The existing,
alternative hand-made fosfomycin-loaded beads do not yield sufficiently effective
release for killing bacterial strains from Dutch and Indonesian patients Gentamicin
was considered as an alternative antibiotic as it has been used successfully as a
locally applied antibiotic in orthopedic surgery6-8 Its broad antimicrobial spectrum,
covering most bacteria commonly involved in osteomyelitis, and its bactericidal
effect, even on non-proliferating microorganisms7•9 makes it favorable for local
application.
Chapter 3 describes in detail the release kinetics of gentamicin from hand
made beads after adding soluble fillers such as glycine or sodium chloride, or
biodegradable polymers such as PVP17 in different concentrations. Moreover,
polymerization of the cement with a half dose of monomer is attempted. The
combination of gentamicin-15% PVP17-PMMA with a half dose of monomer
demonstrates good antibiotics release.
The study presented in Chapter 4 of this thesis was focused on developing a
template system, that can be used to prepare a chain of hand-made antibiotic-loaded
beads with a uniform size. The newly designed system allows to produce a chain of
antibiotic-loaded PMMA beads during or immediately prior to surgery, which is most
cost-effective and obviates storage, while moreover all beads are uniform in size.
By using this novel concept, beads produced with 15% PVP17 and half the
required dose of monomer showed prolonged antibiotic release, comparable to the
one of Septopal® beads. More importantly, biofilm formation by S. aureus 5298 was
reduced, as presented in Chapter 5.
109
General discussion
Therewith, this concept provides a good alternative for the hand-made
fosfomycin-loaded beads, currently hand-made in developing countries. Its costs are
similar as currently used hand-made beads and antibiotic-release and efficacy with
respect to biofilm control proven.
Future research in this field
Although the novel concept, including the template system, fulfills the general goal of
this thesis, a number of further studies might be explored:
• The identification of "safety zones" of local antibiotic concentrations, based on
specific antibiotic classes, will need to be developed because it seems that with
high antibiotic concentrations that can be achieved with local delivery vehicles,
there will be trade-off between decreased systemic toxicity and a potential
increase in local toxicity.
• Gentamicin is slowly released from impregnated beads for prolonged periods of
time. In this study, up to 71 % of the incorporated gentamicin is released from
mixture of gentamicin-15% PVP17, prepared with half the required dose of
monomer, and has provided high gentamicin release. Although the release is
comparable to the one of commercially available Septopal® beads, in vivo studies
should be carried out, including precise assessment of kidney function and
function of the middle ear.
• Low antibiotic release from gentamicin-loaded PMMA beads has not been
considered to be therapeutically effective, and has been associated with the
development of gentamicin-resistant bacteria 13· 14, which has been recognized as
an emerging clinical problem 15. However, a clear causative relationship between
110
General discussion
low antibiotic release and the occurrence of gentamicin resistance has not yet
been shown, and should be further explored.
• Biodegradable beads will avoid an extra surgery necessary to remove non
biodegradable beads and allow all antibiotic to be released from the bead.
Recent research at other laboratories has explored the use of antibiotic-loaded
biodegradable beads for potential use in the local delivery of debrided
osteomyelitis bone, such as, gentamicin-high-molecular-weight (high-MW)
biodegradable poly(D,L-lactide), gentamicin-loaded calcium hydroxyapatite
biodegradable beads. Biodegradable bead systems should therefore be also
further exploited for their potential use in developing countries, both in terms of
clinical as well as in terms of costs-effectiveness and feasibility.
1 1 1
General discussion
References
1 . Mader, J.T., Mohan, D., and Calhoun, J. A practical guide to the diagnosis and management of bone and joint infections. Drugs 1 997:54:253-264.
2. Lew, D.P., and Waldvogel, F.A. Osteomyelitis. Lancet 2004;364:369-379.
3. Henry, S.L., and Galloway, K.P. Local antibacterial therapy for the management of orthopaedic infections. Pharmacokinetic considerations. Clin Pharmacokinet 1 995;29:36-45.
4. Neut, D., Van de Belt, H., Stokroos, I., Van Horn, J.R., Van der Mei, H.C., and Busscher, H.J . Biomaterial-associated infection of gentamicin-loaded PMMA beads in orthopaedic revision surgery. J Antimicrob Chemother 2001 ;47:885-891 .
5 . World Health Organization. WHO Global Strategy for Containment of Antimicrobial Resistance. WHO Report 2002.
6. Bauer, T.W., and Schills, J. The pathology of total joint arthroplasty. Mechanisms of implant fixation. Skeletal Radiology 1 999;28:423-432.
7. Southorn, P.A., Plevak, D.J., and Wright, A.J. Adverse effects of vancomycin administered in the perioperative period. Mayo Clin Proc 1 986;61 :721-724.
8. Taylor, G.J., Bannister, G.C., and Calder, S. Perioperative wound infection in elective orthopaedic surgery. J Hosp Infect 1 990;16:241 -247.
9. Erron, L.J. Prevention of infection following orthopedic surgery. J Antibiot Chemother 1985;33:140-164.
1 0. Bucholz, H.W., and Engelbrecht, H. Depots effects of various antibiotics mixed with Palaces resins. Chirurg 1 970;41 :51 1 -515.
1 1 . Klemm, K. Gentamicin-PMMA beads in treating bone and soft tissue infection. Zentralbl Chir 1979;104:934-942.
1 2. Evans, R.P ., and Nelson, C.L. Gentamicin-impregnated polymethylmethacrylate beads compared with systemic antibiotic therapy in the treatment of chronic osteomyelitis. Clin Orthop Relat Res 1 993;295:37-42.
13. Van de Belt, H., Neut, D., Van Horn, J.R., Van der Mei, H.C., Schenk, W., and Busscher, H.J . . . . or not to treat?. Nat Med 1 999;5:358-359.
14. Neut, D., Hendriks, J.G.E., Van Horn, J.R., Van der Mei, H.C. and Busscher, H.J. Pseudomonas aeruginosa biofilm formation and slime excretion on antibiotic-loaded bone cement. Acta Orthopaedica 2005;76: 109-1 14.
15. Weber, F.A., and Lautenbach, E.E. Revision of infected total hip arthroplasty. Clin Orthop 1986;21 1 :108-1 15.
1 1 2
Summary
Summary
Although application of biomaterials has been one of the major assets in modern
medicine to improve the quality of life of patients, occurrence of a chronic
osteomyelitis is still a serious health threat to the individual patient. Chronic
osteomyelitis can result in morbidity affecting the viability of an affected limb. Biofilm
formation on avascular bone protects pathogens and leads to the persistence of
infection. Fosfomycin-loaded polymethylmethacrylate (PMMA) beads are handmade
by orthopedic surgeon in Indonesia to treat the disease. Up to now, the
efficaciousness of these beads as compared to the commercially available ones
(such as Septopal®) has never been studied, therefore, the clinical result is still in
doubt, whether it comes from the systemic antibiotics, the beads itself or surgical
intervention. To have better antibiotic release from the bone cement beads some
requirements should be considered such as uniform size in diameter, spherical
shape, porosity, and antibiotic concentration.
Chapter 1 reviews the background of osteomyelitis management in
developing countries, including the use of handmade antibiotic-loaded PMMA beads.
The health care situation in developing countries completely differs from the Western
world and surgeons in developing countries do all they can to bridge the gap
between both worlds. Often, however, finances do not allow them to apply biomedical
technologies common in the Western world. The antibiotic-loaded beads used in the
Western world are industrially made, highly porous acrylic beads, releasing 80% of
their antibiotic content within 10 to 15 days. In Indonesia, orthopedic surgeons use
bone cement, mix it themselves with antibiotic in handmade molds and apply them in
their patients. It can be doubted, however, whether such beads actually release
antibiotic due to their lack of porosity. The purpose of this thesis is (1) to clarify the
characteristics of current handmade-fosfomycin loaded PMMA beads in relation to
114
Summary
the bone cement porosity, antibiotic concentration and duration of releasing
antibiotics from bone cement; (2) to propose novel concepts in preparing handmade
antibiotic-loaded PMMA beads; design and realization of a bead template system to
produce antibiotic-loaded PMMA beads (uniform in size, shape and porosity).
Analysis of the existing handmade antibiotic-loaded PMMA beads is done in
Chapter 2. Release of antibiotic from six locally made beads based on Zimmer® and
Simplex® P bone cements and fosfomycin were studied and compared with
Septopal® beads. The area per volume ratio was measured and compared with
Septopal® beads and their efficacy was determined against various clinical, Dutch
and Indonesian bacterial isolates on agar plates. Minimal inhibitory concentrations for
fosfomycin and gentamicin of the different bacterial strains were determined with an
E-test. Bone cement samples were put in phosphate buffered saline up to 144 h and
in vitro release kinetics were monitored by measuring bacterial inhibition zones on
agar plates at different points in time. Scanning electron microscopy was done on
both handmade and Septopal® beads to determine porosity on the outer as well as
on fracture surfaces. Fosfomycin efficacy could only be shown in 70% from the
tested bacterial strains, out of which 40% of them are S. aureus. Fosfomycin-loaded
PMMA beads are able to kill only 20% (2110) of the tested bacterial strains S. aureus
7323 and CNS 7391, whereas Septopal® beads were effective in 80% (8110).
Concepts for increasing gentamicin release from bone cement beads are
proposed. Chapter 3A describes the effect of soluble fillers in enhancing the
antibiotic release. Acrylic beads were first prepared with variable monomer contents:
500 µIlg polymer (100%), 375 µIlg polymer (75%), and 250 µIlg polymer (50%) to
increase gentamicin release through the creation of a less dense polymer matrix.
After an optimal monomer content had been defined, different gel-forming polymeric
1 15
Summary
fillers were added to enhance the permeation of fluids into the beads.
Polyvinylpyrrolidone 17PF (PVP17) was selected as a suitable filler, its concentration
was varied and the antibiotic release of the final beads was compared with the
gentamicin release from Septopal® beads. Gentamicin release rate and extend of
release from beads prepared with 50% monomer increased upon increasing the
PVP17 content in the beads. Beads with 15% PVP17 released 71 % of their antibiotic
content after 336 h. Importantly, this is significantly (two-tailed, p < 0.05) more than
the gentamicin-release from Septopal® beads, that is confined to only 56% after 336
h. Electron microscopy shows that the use of 50% of the prescribed amount of
monomer causes sintering of the polymer beads rather than polymerizing into a solid
mass and therewith create a certain porosity needed for antibiotic release.
Dissolution of the soluble PVP17 subsequently ensured penetration of the dissolution
fluids and prolonged release levels above those of Septopal®.
In Chapter 3B, commercially available antibiotic-loaded beads are shown to
provide an efficient vehicle for antibiotics in local delivery systems, but are not readily
available in all parts of the world. In the absence of commercially available bead
systems, orthopedic surgeons have designed various ways to prepare an efficient
bead system themselves. The efficacy of any antibiotic-releasing bead system
depends on whether a percolating system is obtained or not. The purpose of this
study is to optimize the release kinetics of gentamicin from hand-made beads by
adding soluble fillers, such as glycin and sodium chloride (NaCl), and to qualitatively
analyze the data in terms of percolation theory. Gentamicin-loaded
polymethylmethacrylate (PMMA) bone cement beads (0.2 g gentamicin added to 8 g
of PMMA powder) were prepared with the inclusion of glycin (0.6 g) and different
amounts of NaCl (12 g to 20 g). In order to determine the gentamicin release, beads
116
Summary
were placed in 10 ml of phosphate buffered saline and aliquots were taken at
designated times to measure the released amounts of gentamicin. The antibiotic
release increases by adding glycin and NaCl, with the percolation threshold occurring
between 12 g and 16 g of NaCl.
Chapter 4 describes the design and realization of a simple template system to
manufacture under operating room (OR) conditions antibiotic-loaded
polymethylmethacrylate (PMMA) beads, which can be used for local antibiotic
therapy. The template system has been designed to produce 30 PMMA beads with
an almost spherical shape and regular diameter of 8 mm which are connected with a
stainless steel wire. The system consists of two polytetrafluoroethylene (PTFE)
plates each of which contain 30 half holes with a spherical shape and which when
combined serve as bead templates and a set of stainless steel rings, which serve as
an additional structure to strengthen the construct. The system can withstand high
temperatures such as are generated during polymerization and autoclaving, and can
be manufactured in the OR within half an hour using loaded PMMA bone cement with
or without antibiotics. With the template system presented here one is able to
produce antibiotic-loaded beads in operating theatre conditions, which beads can be
used in the same way as the commercially available antibiotic-loaded beads.
Eventually, in vitro evaluation of handmade gentamicin-loaded PMMA beads
to prevent biofilm formation has been described in Chapter 5. S. aureus is an
opportunistic human pathogen capable of forming a biofilm under physiological
conditions that can persist despite long-term antibiotic treatment. PMMA beads
releasing antibiotics are frequently used to treat osteomyel itis. In the Western-world,
these beads are commercially available, but in developing countries hand-made
beads are used, generally proven to be ineffective. With some minor modifications,
117
Summary
hand-made beads could be prepared that released high amounts of antibiotics. The
modification consisted of using half the amount of prescribed monomer for bead
preparation and adding a biodegradable filler (PVP17). Here we determine the
efficacy of these beads against S. aureus 5298 biofilms, grown in 96-wells plates.
Effects of hand-rolled and template-made beads were compared with the effects of
commercial Septopal® beads. After 1, 2, 3, 7 and 14 days, biofilms were stained with
crystal violet (CV), and the absorbance at 575 nm served as an index for biofilm
formation. Hand-rolled and template-made beads reduced bacterial growth up to
98.7%, which is a similar reduction as achieved Septopal®.
In conclusion, with some minor and cheap modifications of existing
methodologies to hand-made antibiotic releasing bead systems in developing
countries, a bead system can be obtained with an efficacy similar to the one of
commercially available Septopal® beads. In order to accomodate in creating a chain
of antibiotic-PMMA beads, therefore, a relatively simple design of semi-manual
beads template system has been finally developed that produces gentamicin
releasing beads at costs affordable in developing countries.
118
Samenvatti ng
Samenvatting
Ofschoon de toepassing van biomaterialen een van de belangrijke aanwinsten in de
moderne geneeskunde is geweest om de kwaliteit van leven van patienten te
verbeteren is het v66rkomen van chronische osteomyelitis nog steeds een ernstige
bedreiging voor de individuele patient. Chronische osteomyelitis kan resulteren in
afwijkingen die het behoud van een aangedane ledemaat in gevaar kunnen brengen.
Kralen gemaakt van polymethylmethacrylaat (PMMA) waarin fosfomycine is
vermengd worden ter behandeling van de osteomyelitis in Indonesia met de hand
gemaakt door de orthopedisch chirurg. Tot op heden is de effectiviteit van deze
kralen in vergelijking met de commercieel beschikbare kralen (zoals bv Septopal®)
nooit bestudeerd, zodat het nog steeds niet zeker is of het klinisch resultaat van de
behandeling het gevolg is van de systemisch toegediende antibiotica, de kralen zelf
of de chirurgische interventie. Om het vrijkomen van het antibioticum uit de kralen te
verbeteren moeten enkele voorwaarden hiertoe nader beschouwd worden zoals
bijvoorbeeld de uniforme diameter, de sferische vorm, de porositeit en de
concentratie antibioticum in elke kraal.
Hoofdstuk 1 geeft een overzicht van de literatuur betreffende de achtergrond
van het behandelingsprotocol van osteomyelitis in ontwikkelingslanden, inclusief het
gebruik van met de hand gemaakte antibioticumhoudende PMMA kralen. De
gezondheidszorg in ontwikkelingslanden verschilt volledig van die in de westerse
wereld en chirurgen in ontwikkelingslanden doen al het mogelijke om op dit terrein
de afstand die er bestaat tussen beide werelden te overbruggen. Vaak echter laat de
financiele situatie het hun niet toe om biomedische technologieen toe te passen die
gemeengoed zijn in de westerse wereld. De antibioticahoudende PMMA kralen die in
de westerse wereld worden gebruikt worden industrieel gemaakt en bestaan uit
acrylaten en zijn in hoge mate poreus, waardoor 80% van hun antibiotische lading
120
Samenvatting
binnen 1 O tot 1 5 dagen in het omringende milieu vrijkomt. In Indonesia gebruiken de
orthopedisch chirurgen botcement dat zij zelf met antibiotica mengen, met de hand
vormen tot kralen en bij hun patienten inbrengen. Er is echter twijfel mogelijk over
het feit of uit zulke kralen ook werkelijk antibioticum vrijkomt in verband met het
gebrek aan porositeit van het cement waaruit deze kralen zijn vervaardigd.
Het doel van de studie die aan dit proefschrift ten grondslag ligt is (1)
opheldering te verschaffen omtrent de karakteristieke eigenschappen van de huidige
met de hand gemaakte fosfomycine bevattende PMMA kralen in verband met de
porositeit van het cement, de concentratie van het vrijgekomen antibioticum en de
duur van het vrijkomen van het antibioticum uit het botcement; (2) om nieuwe
concepten betreffende de productie van handgemaakte antibioticumhoudende
PMMA kralen voor te stellen evenals het ontwerp en de realisatie van een set mallen
om antibioticumhoudende kralen te maken die uniform zijn in afmeting, vorm en
porositeit.
Een analyse van de bestaande handgemaakte antibioticumhoudende PMMA
kralen wordt gepresenteerd in Hoofdstuk 2. Het vrijkomen van antibioticum uit 6
verschillende lokaal, van Zimmer en SimplexP cement en fosfomycine, vervaardig
de kralen werd bestudeerd en vergeleken met Septopal® kralen. De oppervlak per
volume ratio werd gemeten en vergeleken met Septopal® kralen and hun effectiviteit
werd op agarplaten bepaald tegen diverse klinisch relevante Nederlandse en
lndonesische bacteriele isolaten. De minimale inhibitoire concentratie (MIC) waarden
voor fosfomycine en gentamycine van de diverse bacterie stammen werd bepaald
met de E-test. Samples botcement werden tot 144 uur gebracht in fosfaat gebufferde
zoutoplossing en de kinetica van het in vitro vrijkomen van het antibioticum werd
bestudeerd door de bacteriele groeiremmingszones op agar platen op verschillende
121
Samenvatting
tijdstippen te meten. Scanning electronen microscopie werd uitgevoerd op zowel de
met de handvervaardigde als de Septopal® kralen om de porositeit zowel aan het
oppervlak als op breukvlakken vast te stellen. Fosfomycine effectiviteit kon slechts
warden vastgesteld bij 70% van de geteste bacteria stammen, van deze behoorde
40% tot de stam Stafylococcus aureus. Fosfomycin bevattende PMMA kralen waren
in staat slechts 20% (2/10) van de bacteriestammen de S. aureus 7323 en CNS
7391 te doden, terwijl Septopal® kralen effectief waren in 80% (8/10).
Methoden om het vrijkomen van gentamycine uit botcement kralen te doen
toenemen warden in de volgende hoofdstukken voorgesteld. Hoofdstuk 3A
beschrijft het effect van oplosbare vulstoffen om het vrijkomen van antibioticum te
vergroten. Acrylkralen warden eerst vervaardigd met variabele monomeer
hoeveelheden: 500 µ1/g polymeer (100%), 375 µ1/g polymeer (75%) en 250 µ1/g
polymeer (50%) om het vrijkomen van gentamycine te doen toenemen door het
creeren van een minder dichte polymeer matrix. Nadat een optimale
monomeerconcentratie was bepaald werden verschillende gel-vormende polymere
vulstoffen toegevoegd om het binnendringen van vloeistoffen in de kralen te doen
toenemen. Polyvinylpyrrolidone 17PF (PVP17) werd geselecteerd om te dienen als
een geschikte vulstof, de concentratie ervan werd gevarieerd en het vrijkomen van
antibioticum uit de uiteindelijke kralen werd vergeleken met het vrijkomen van
gentamycine uit Septopal® kralen. De snelheid waarmede gentamycine vrijkwam
evenals de hoeveelheid ervan uit kralen die waren gemaakt met 50% monomeer
namen toe met de toename van de PVP17 concentratie in de kralen. Uit kralen met
15% PVP17 kwam 71 % van hun antibiotische inhoud vrij na 336 uur. Van belang is
dat dit significant meer is dan het vrijkomen van gentamycine uit Septopal® kralen,
welke slechts beperkt is tot 56% na 336 uur. Electronenmicroscopie toont aan dat
122
Samenvatting
het gebruik van 50% van de voorgeschreven hoeveelheid monomeer sinteren van
de polymeer korrels tot gevolg heeft en niet polymerisatie tot een solide massa en
dat daardoor een zekere mate van porositeit wordt gecreeerd welke nodig is voor het
vrijkomen van het antibioticum. Door het PVP17 op te lossen werd verdere
penetratie van oplosmiddelen mogelijk gemaakt hetgeen leidde tot niveaus van
vrijkomen van het antibioticum boven die van Septopal®.
In Hoofdstuk 3B wordt voor commercieel beschikbare antibioticumhoudende
kralen aangetoond dat zij een efficient vehiculum zijn voor het lokaal toedienen van
antibiotica, maar zij zijn niet overal in de wereld gemakkelijk verkrijgbaar. Waar deze
kralen niet commercieel beschikbaar zijn hebben orthopedisch chirurgen diverse
methoden ontworpen om zelf efficiente kralensystemen te maken. De
doeltreffendheid van elk antibioticumhoudend kralensysteem hangt af van het feit of
er sprake is van een percolerend systeem. Het doel van deze studie is om de
kinetica van het vrijkomen van gentamycine uit met de hand vervaardigde kralen te
optimaliseren door oplosbare vulmiddelen als glycine en natrium chloride (NaCl) toe
te voegen en om kwalitatief de gegevens te analyseren met betrekking tot de
percolatie theorie. Gentamycine houdende polymethylmethacrylaat (PMMA) bot
cement kralen (0,2 g gentamycine toegevoegd aan 8 g PMMA poeder) werden
vervaardigd en hieraan werd toegevoegd glycine (0,6 g) en verschillende
hoeveelheden NaCl (12-20 g). Teneinde het vrijkomen van gentamycine te bepalen
werden de kralen geplaatst in 10 ml fosfaat gebufferde zoutoplossing en monsters
werden op vastgestelde tijden genomen om de vrijgekomen hoeveelheden
gentamycine te meten. Het vrijkomen van het antibioticum neemt toe door glycine
en NaCl toe te voegen waarbij de drempel voor de percolatie optreedt tussen 12 g
en 16 g NaCl.
1 23
Samenvatting
Hoofdstuk 4 beschrijft het ontwerp en de verwezenlijking van een eenvoudig
systeem van mallen om onder operatiekamer condities antibioticumhoudende PMMA
kralen te maken die gebruikt kunnen worden voor lokale antibiotische therapie. De
mal is ontworpen om 30 PMMA kralen te vervaardigen met een vrijwel sferische
vorm en een uniforme diameter van 8 mm en die verbonden zijn door middel van
een roestvrije stalen draad. Het systeem bestaat uit twee polytetrafluoroethylene
(PTFE) platen die elk 30 halve gaten bevatten met een sferische vorm en die
wanneer ze gecombineerd worden dienen als een mal voor de kralen en een stel
roestvrij stalen ringen die dienen om de constructie te versterken. Het systeem kan
hoge temperaturen verdragen zoals die gegenereerd worden tijdens het
polymerisatie proces en het autoclaveren en kan binnen een half uur in de
operatiekamer in elkaar gezet worden om kralen te maken waarbij gebruik gemaakt
wordt van PMMA botcement met of zonder antibiotica. Met het mallen systeem zoals
dat hier gepresenteerd wordt is men in staat om antibioticumhoudende kralen onder
operatiekamer condities te maken, welke kralen op dezelfde manier gebruikt kunnen
worden als de commercieel beschikbare antibioticumhoudende kralen.
Tenslotte wordt een in vitro evaluatie beschreven in Hoofdstuk 5 omtrent de
preventie van biofilm vorming. S. aureus is een opportunistische humane
pathogene bacteria die in staat is biofilm te maken onder fysiologische
omstandigheden welke kan blijven bestaan ondanks langdurige antibiotische
behandeling. PMMA kralen die antibiotica afgeven worden frequent gebruikt om
osteomyelitis te behandelen. In de westerse wereld zijn deze kralen commercieel
verkrijgbaar maar in ontwikkelingslanden worden met de hand gemaakte kralen
gebruikt welke in het algemeen bewezen hebben ineffectief te zijn. Met enkele kleine
modificaties konden met de hand gemaakte kralen worden vervaardigd die grote
124
Samenvatting
hoeveelheden antibioticum afgaven. De modificatie bestond uit het gebruik van de
helft van de voorgeschreven hoeveelheid monomeer voor het vervaardigen van de
kralen en het toevoegen van een bio- afbreekbare vulstof (PVP17). In deze studie
hebben wij de werkzaamheid van deze kralen getest tegen S. aureus 5298 biofilms,
welke waren gegroeid in 96-wells platen. De effecten van het gebruik van met de
hand gerolde en met de mal gemaakte kralen werden vergeleken met die van de
commerciele Septopal® kralen. Na 1, 2, 3, 7 en 14 dagen warden de biofilms
gekleurd met crystal violet, en de absorptie op 575 nm diende als een maat voor
biofilm vorming. Met de hand gerolde en met de mal gemaakte kralen reduceerden
de bacteriele groei met 98. 7%, hetgeen een vergelijkbare reductie is als die wordt
bereikt met Septopal® kralen.
Concluderend kan worden gesteld dat met enkele kleine en goedkope
wijzigingen van bestaande methoden om antibioticumhoudende kralen te maken in
ontwikkelingslanden een kralen systeem kan worden verkregen met een
doelmatigheid die vergelijkbaar is met die welke bereikt kan worden met een van de
commercieel beschikbare (Septopal®) kralen. Teneinde het vervaardigen van
antibioticumhoudende PMMA kralensnoeren mogelijk te maken is een relatief
eenvoudig ontwerp gemaakt om semi-manueel antibioticumhoudende PMMA
kralensnoeren bestaande uit antibioticum vrijmakende kralen middels een mallen
systeem te vervaardigen tegen kosten die ontwikkelingslanden zich kunnen
veroorloven.
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Ringkasan
Walaupun aplikasi biomaterial merupakan modal utama di dalam dunia kedokteran
modern untuk memperbaiki kualitas hidup pasien, namun timbulnya osteomielitis
kronis masih tetap merupakan masalah kesehatan yang perlu mendapat perhatian
khusus. Osteomielitis kronis merupakan suatu penyakit pada jaringan tulang yang
dapat mempengaruhi kelangsungan hidup bagi anggota gerak tubuh. Biofilm yang
berkembang di sekitar tulang mati (sekuester) akan melindunginya terhadap bakteri
patogen, sehingga dapat menimbulkan infeksi yang persisten. Apabila hal ini terjadi
maka perlu dilakukan tindakan pembedahan untuk membuang sekuester dan
membuat seuntai antibiotik lokal yang berbentuk tasbih (beads). Untuk melakukan ini
maka para ahli orthopaedi di Indonesia secara manual membuat sendiri campuran
antibiotik dengan semen tulang/bone cement (polymethylmethacrylate/PMMA) yang
akan dibentuk menyerupai untaian beads, untuk selanjutnya akan ditanam dalam
defek tulang melalui tindakan pembedahan selama dua sampai empat minggu. Saat
ini antibiotik yang sering digunakan adalah golongan fosfomycin sodium, namun
efektifitas pemakaian metode ini masih belum pernah dipublikasikan dalam
kepustakaan untuk mengetahui seberapa banyak pelepasan antibiotik dari
campuran ini. Keberhasilan campuran antibiotik dengan polimer PMMA yang
berbentuk beads ini masih meragukan. Terdapat beberapa faktor yang dapat
mempengaruhi keberhasilan pengobatan, apakah dari pemberian antibiotik
parenteral, beads itu sendiri, atau keberhasilan tindakan pembedahan.
Untuk memperoleh pelepasan antibiotik yang optimal, maka beads harus
mempunyai keseragaman bentuk dan ukuran, mempunyai porositas yang cukup,
serta konsentrasi antibiotik yang tepat, hal ini dapat di produksi dengan
menggunakan semi-manual beads template system.
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Ringkasan
Bab 1 memberikan gambaran mengenai penanganan osteomielitis di negara
berkembang. Kesehatan masyarakat di dunia ketiga khususnya di Indonesia sangat
berbeda dengan dunia barat. Para ahli bedah di dunia ketiga mengerjakan seluruh
yang dapat mereka kerjakan untuk menjembatani celah diantara keduanya.
Seringkali hambatan keuangan tidak memungkinkan mereka untuk dapat
mengaplikasikan teknologi biomedika seperti apa yang dikerjakan dunia barat.
Sebagai contoh pada infeksi pasca pemasangan protese sendi panggul, cara
mengatasi infeksi ini adalah dengan mengangkat protese yang terinfeksi untuk
selanjutnya dilakukan debridement, kemudian ditanamkannya antibiotika yang
dicampur dalam semen tulang (antibiotic-loaded bone cement) pada daerah infeksi,
kemudian akan dilepaskan kembali pada tindakan pembedahan berikutnya sekaligus
melakukan pemasangan kembali prostesis baru. Tidak dapat dipungkiri bahwa
tindakan ini merupakan hal yang sangat mahal dan merupakan trauma utama bagi
seorang pasien.
Di dunia barat teknik pemakaian antibiotic-loaded bone cement telah banyak
digunakan, telah di produksi dan dipasarkan secara luas, berbentuk tasbih (beads)
terbuat dari acrylic dan berpori, campuran ini akan melepaskan sekitar 80%
kandungan antibiotika selama 10 sampai 15 hari. Di Indonesia, para spesialis
orthopaedi menggunakan semen tulang untuk dicampur dengan antibiotika, dibentuk
menyerupai tasbih dan dirangkaikan dengan menggunakan kawat baja halus serta
tahan karat, dan menamkannya pada tulang pasien yang mengalami infeksi. Melihat
keadaan seperti ini pelepasan antibiotika dari semen tulang ini masih belum optimal
karena bentuk tasbih tersebut mempunyai ukuran yang tidak seragam.
Oleh karena itu tujuan dari penelitian ini adalah:
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Ringkasan
1. Mempelajari karakteristik beads yang mengandung campuran antibiotik dan
PMMA terhadap porositas beads, konsentrasi antibiotik dan durasi pelepasan
antibiotik dari PMMA beads.
2. Mengembangkan dan mengusulkan konsep baru dalam pembuatan beads
mengandung campuran antibiotik dengan PMMA.
3. Merancang dan merealisasikan semi-manual beads template system untuk
memproduksi antibiotic-loaded PMMA beads yang mempunyai bentuk dan ukuran
yang seragam serta porositas yang cukup baik.
Evaluasi dari handmade antibiotic-loaded PMMA beads yang biasa digunakan
dijelaskan Bab 2. Untuk mengetahui sejauh mana efektifitas antibiotik yang keluar
dari beads PMMA yang biasa digunakan (existing fosfomycin-loaded PMMA beads),
maka dilakukan berbagai percobaan in vitro, untuk mempelajari pelepasan antibiotik
yang berkesinambungan dengan konsentrasi yang berada dalam koridor optimum
dalam durasi waktu tertentu. Penelitian dilakukan terhadap berbagai strain bakteri
dari pasien yang menderita infeksi akibat osteomielitis kronis dan pemasangan
implan. Dilakukan berbagai percobaan untuk menilai efektifitas antibiotik dengan
menentukan kadar hambat minimal (KHM), selanjutnya dilakukan evaluasi terhadap
kinetika antibiotik melalui zona hambat (inhibition-zones) serta melakukan penilaian
terhadap porositas beads dengan menggunakan scanning electron micrsocopy
(SEM). Hasil yang diperoleh akan dibandingkan dengan beads antibiotik dari produk
impor, yang merupakan standar baku untuk terapi infeksi tulang.
Hasil evaluasi tersebut ternyata handmade fosfomycin PMMA beads tidak
memperlihatkan pelepasan antibiotik yang memuaskan, hal ini disebabkan
karakteristik beads yang tidak ideal, dimana beads berukuran besar serta tidak
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Ringkasan
seragam dan mempunyai porositas yang sangat kurang. Ukuran beads yang
berukuran kecil perlu dipertimbangkan perannya dalam pelepasan antibiotik.
Permukaan beads merupakan karakteristik yang sangat penting dalam efektifitas
pelepasan antibiotik. Existing beads yang dievaluasi mempunyai ukuran yang sangat
tidak seragam dengan kisaran 1.6 sampai 2.8 cm bila dibandingkan dengan produk
impor Septopal® (0.7 cm), sehingga menunjukkan surface area per-volume ratio
yang tidak optimal (2. 1 sampai 3.8 cm-1 ) dibanding dengan Septopal® (8.6 cm-1 ),
sehingga mempunyai beban antibiotik yang berbeda.
Pilihan fosfomycin sebagai antibiotik yang dicampur dengan PMMA dalam
bentuk beads dapat dipertanyakan apakah antibiotik ini dapat berfungsi dengan
baik?. Karena dari penelitian in vitro diperoleh bahwa fosfomycin memperlihatkan
efikasinya terhadap 60% (12/20) terhadap strain bakteri osteomielitis, dan 58%
(7/12) adalah bakteri Staphylococcus aureus. Disamping itu fosfomycin-PMMA
beads hanya mampu membunuh 20% (2/10) yaitu Staphylococcus aureus 7323 dan
CNS 7391. Sementara itu Septopal® efektif pada 80% (8/10).
Untuk meningkatkan pelepasan gentamisin beads dijelaskan pada Bab-3A,
dengan mempelajari kinetika pelepasan gentamicin melalui penambahan polimer
biodegradable. PMMA beads pertama-tama dipesiapkan dengan mencampurkan
berbagai konsentrasi polimer yaitu: 500 µIlg polimer (100%), 375 µIlg polimer (75%)
dan 250 µIlg polimer (50% ). Pada beads dengan polyvinylpyrrolidone 17PF (PVP17)
menunjukkan hasil yang baik, berada di dalam koridor "dosis optimal" dan
memperlihatkan sustained release. Pelepasan gentamisin menunjukkan
peningkatannya pada beads dengan campuran 50% monomer dan konsentrasi
cukup tinggi PVP17. Kecepatan dan kuantitas pelepasan gentamisin yang sangat
bermakna terlihat pada beads berisi 15%PVP17 yaitu 71% pada hari ke-14 (two-
1 31
Ringkasan
tailed, p < 0.05), bila dibandingkan dengan gentamisin yang keluar dari Septopal®
pada waktu yang bersamaan, pelepasan gentamisin menunjukkan 56%. Tampak
pelepasan gentamisin yang berasal dari template system memperlihatkan
peningkatan 1 .3 kali dibanding Septopal® dengan perbedaan yang bermakna (p <
0.05). Mikroskop elektron memperlihatkan bahwa komposisi beads dengan 50%
monomer dapat menyebabkan perlengketan (sintering) diantara polimer di dalam
beads itu sendiri dari pada proses polimerisasi yang terjadi di dalam masa padat, hal
ini akan meningkatkan porositas beads yang diperlukan untuk pelepasan antibiotik.
Dengan larutnya PVP1 7 akan mempermudah penetrasi cairan ke dalam beads,
kemudian akan menyebabkan pelepasan antibiotik secara berkesinambungan,
sehingga dapat melebihi tingkat pelepasan gentamisin dari Septopal®.
Bab-3B mempelajari pengaruh soluble fillers seperti glycine, sodium chloride
(NaCL) terhadap kinetika gentamicin agar diperoleh pelepasan antibiotik secara
optimal. Analisis data dilakukan secara kualitatif melalui teori perkolasi. Terlihat
bahwa campuran gentamicin-glycine dengan penambahan konsentrasi tinggi NaCL
dapat meningkatkan pelepasan gentamicin yang keluar dari handmade beads. Hal
ini disebabkan tercapainya sistim perkolasi pada konsentrasi tersebut. Terlihat
bahwa ambang perkolasi terlihat pada 1 6 g dan 24 g NaCl.
Melihat kepentingan rasio beads yang demikian berpengaruh maka telah di
laksanakan perancangan dan realisasi dari semi-manual beads template system
yang dapat membuat beads berbentuk bola dengan diameter yang seragam dan
berjarak sama satu sama lain. Perancangan dan realisasi dari semi-manual beads
template system ini dapat dilihat pada Bab 4. Template ini sangat diperlukan untuk
memperoleh surface area per-volume ratio yang besar, dengan mengacu dari
produk impor Septopal® yaitu 7 mm. Template ini dapat membentuk 30 buah PMMA
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Ringkasan
beads dengan bentuk dan ukuran yang seragam yaitu sekitar 8 mm dengan jarak 4
mm satu sama lain. Dapat dilaksanakan di kamar bedah dengan cepat (sekitar 20
menit). Dengan menggunakan semi-manual beads template system diharapkan
dapat memproduksi beads yang mempunyai kualitas yang setara dengan kualitas
produk impor, dengan harga yang terjangkau bagi masyarakat di negara
berkembang.
Akhirnya analisis in vitro untuk melakukan evaluasi terhadap efikasi
handmade gentamicin-loaded beads dalam mencegah terbentuknya biofilm telah
dijelaskan pada Bab 5. S. aureus merupakan bakteri patogen yang oportunistik dan
mempunyai kemampuan untuk membentuk biofilm dalam keadaan fisiologis, dapat
bertahan dalam pengobatan yang cukup lama dengan antibiotik. Di negara barat
beads ini tersedia tapi di negara berkembang digunakan handmade beads yang
kadangkala tidak menunjukkan efektifitasnya. Bab ini menjelaskan efikasi beads
terhadap terbentuknya biofilm S. aureus 5298 yang berkembang di dalam 96
lempengan sumur. Selanjutnya efikasi antibiotik secara handrolled dan
menggunakan template telah dibandingkan dengan beads Septopal®. Dalam waktu
yang telah ditentukan yaitu hari ke-1, ke-2, ke-3, ke-7 dan hari ke-14, biofilm di beri
pewarnaan dengan crystal violet (CV), dengan nilai absorbans pada 575 nm sebagai
indeks dari formasi biofilm. Hasil uji laboratorium menunjukkan bahwa gentamicin
dapat menghambat terbentuknya biofilm sampai 98. 7%, setara dengan Septopal®.
Akhimya dapat disimpulkan, bahwa dengan beberapa perubahan minor serta
modifikasi dari metodologi yang telah ada terhadap sistem pelepasan antibiotik dari
handmade antibiotik di negara berkembang, maka suatu sistem beads antibiotik
telah dikembangkan dengan memperlihatkan efikasi yang sama dengan produk
133
Ringkasan
impor yaitu Septopal®. Untuk membantu dalam membuat satu rangkaian beads
antibiotik-PMMA, telah dikembangkan semi-manual beads template system yang
cukup sederhana dengan harga yang terjangkau.
134
Acknowledgements
Acknowledgements
Initially, I could not imagine conducting research at the Rijksuniversity Groningen
(RUG), The Netherlands. But, this opportunity has been realized through the
dedication of many to share knowledge and to increase the public health orthopedic
status for developing countries. These activities could not be completed without the
help of many individual non-author contributors. These are the true "unsung heroes"
of this thesis, and deserve my acknowledgements and sincere thanks.
The author is grateful to the Eric Bleumink Fund (EBF), University of Groningen, The
Netherlands, and the Research Institute BioMedical Engineering/Materials Science
and Application (BMSA) for the financial assistance and support. I am also pleased
to record my gratitude to the Departtment of Biomedical Engineering, School of
Electrical Engineering and Informatics (SEEi), lnstitut Teknologi Bandung, and the
Department of Orthopedic and Traumatology Hasan Sadikin Hospital/Faculty of
Medicine Universitas Padjadjaran, Bandung.
I owe many individuals for the realization and production of this thesis over the past
three years. Here is my sincere "thank you so much" to these special individuals.
Professor Soegijardjo Soegijoko, thank you for introducing me to Professor Henk
and Professor Jim for the first time at the BME seminar at 1TB, Bandung. After
several meetings with them, I finally got the change to conduct research in
Groningen. You are a great teacher for me since you always gave me insights in a
new world that is called "the scientific environment". I want to thank you for all your
encouragement and support during my study.
Professor Henk Busscher is gratefully acknowledged for offering me the opportunity
to work at his department in this interesting field of research and for the continuous
136
Acknowledgements
interest in the project. I have nothing to say other than that you are a perfect
supervisor, promoter, great teacher and friend. Your patience has changed part of
my personality. Dear Professor Henk, this thesis would not have been possible
without your expert guidance and high level of scrutiny and many constructive
discussions. I enjoy our working relationship which is based on trust and sound
scientific knowledge and on which I could always fallback when a problem arose.
You taught me the art of writing scientific papers, of critically appraising my own
work, and of thinking laterally in science. You also give me a chance to explore my
research ability which will undoubtedly impact the way I practice orthopedics in future
years. You were never too busy to give me the ideas that I needed. For all good
things and quality time I spent with you, I would like to express my gratitude. Thank
you so much for everything.
Professor J.R van Hom, thank you first of all for encouraging and enabling me to
undertake this Ph.D. research in Groningen. Without that, I would not have such a
wide choice of paths for my future. I am not only greatly inspired by your enthusiasm
and dedication to orthopedic education in various parts of the world, but also
benefited tremendously from your generous encouragement, assistance, and care at
both professional and personal levels. Thank you for giving me the guided freedom
throughout the course of the research and the promotion of this thesis. I treasure you
as a motivator, a mentor, and a dear friend indeed.
Professor Henny van der Mei, again, I have nothing to say other than that you are a
perfect supervisor, promoter, great teacher and friend: it has been very nice working
with you. I thank you so much for your patience and understanding in dealing with an
inexperienced laboratory researcher like myself. You have truly helped me with the
learning and execution of this laboratory-based research. You also provided
137
Acknowledgements
consistently strong support and reinforcement throughout the course of this
research, in view of my clinical background with limited knowledge of microbiology. I
learned a great deal of scientific writing from you. The words "thank you" may not be
enough for what you have been given to me. You always tried to give your best to
support this research. I would never have completed it without your help. Once again
"thank you very much".
Danielle Neut PhD, the words "thank you" may be not enough for what you have
provided to me. You showed me how to work in the laboratory, and how to calculate
the percentage of antibiotic release and most above all: how interesting it is.
Observing experiments could sometimes be boring, but working with you Danielle,
was never boring. It was a pleasure to work with you. You are very patience in
correcting and explaining how things went in my experiments. Thank for your
cooperation and the friendly atmosphere at the department.
Professor Dr. H.W. Frijlink, I like to say thank for introducing me to the percolation
theory.
I would like to thank Professor Bart Verkerke, for your suggestion about the beads
template.
Professor Tati Mengko, I sometimes got frustrated and excited facing this program,
but it was you who could always ease me nerves. Thank you.
I also like to thank Mrs. G. Kampinga, medical microbiologist for your pertinent
advice.
letse Stokroos, thank you for your assistance with SEM analysis.
Joop de Vries, thanks for your expertise and help with the XPS experiments.
138
Acknowledgements
Professor Gerhard Rakhorst, thank you so much for DEC application, that
unfortunately was never approved.
I would also like to thank Wya Kloppenburg for your patience regarding the financial
issues during my study.
Ellen, you have done a great job in helping me with the lay-out of this thesis and
looking for the best company to print my thesis. Thank you for your good work. I
appreciate it.
Ina Heidema, I thank you so much for your hospitality during and after working
hours.
I would like to thank the reading committee: Professor. dr. J.M.M. Hooymans,
Professor dr. W.M. Molenaar and Professor. dr. ir. G.J. Verkerke, for providing your
time to read my thesis.
Dear Marten and Shanti, I have so many things to thank you for. Thank you much for
participating and spending your time in my Ph.D. defense as a paranymphs. Above
all, thank you for being my good colleagues and friends. I wish you all every success
in each of your careers.
I would like to thank Prof. Darmadji lsmono, as the head of Department of
Orthopedic and Traumatology Hasan Sadikin Hospital / Faculty of Medicine
Universitas Padjadjaran, Bandung for allowing me to minimize my duties during my
work in the laboratory and on this thesis. Thank you for your understanding and the
wisdom you showed me.
My special thanks and appreciation to my dear friends Dr. Ir. Johanes Tjandra
Pramudito, MT., Donny Danudirdjo MT., and Agung Wahyu Setiawan MT, and my
139
Acknowledgements
colleagues at the Robovis laboratory STEI 1TB, thank you for being my good
colleagues and friends.
Thank you also for my Indonesian colleagues: Pak Ketut, Pak Tri, Pak Punto, Titik,
and Tita. I hope we are able to continue our communication to create a research
environment in Indonesia.
I am grateful to my family who live in Holland. I want to thank Tante Tati, Djanti, Rika,
and Retty for your encouragement and continuous support during all the years of my
study.
Above all, to my beloved wife Nita, thank you for your time to accompany me during
my study in Groningen, and for your pray as well. The words "thank you" may be not
enough for what you have been doing for me. And also, I would like to thank to my
sons Adam and Aldi for your patience, encouragement and support.
Finally, I'd like to acknowledge the support of my late mom and dad, who gave me a
love of learning and much support over the years. Last but not least, my mother in
law, thank you for your prays and continuous support.
140
Curricu lum Vitae
Curriculum Vitae
The author of this thesis was born in Jakarta, Indonesia, on December 22nd 1957.
After graduating his study from the Faculty of Medicine Universitas Padjadjaran
Bandung in 1985, he worked as a general physician in rural health centre in
Pandeglang, West Java. Since 1988 he commenced to work as a resident at the
Department of Orthopedic Surgery at the same University. He completed his study in
January 1993, and started to serve the communities as an orthopedic surgeon in the
Dr. Soedarso General Hospital at Pontianak, West Kalimantan. He joined the Faculty
of Medicine Universitas Padjadjaran / Dr. Hasan Sadikin Hospital Bandung, as a
teaching staff since early 1997, where he is now a senior lecturer. In 1998, he
completed a ten month internship training on shoulder surgery in the Takai University
Hospital, lsehara, Japan. His Master Degree on Biomedical Engineering has been
obtained from the Department of Electrical Engineering, lnstitut Teknologi Bandung,
in 2002. Since 2003, he has been entirely engaged in his doctorate research
program for the project on "osteomyelitis therapy by antibiotic-loaded beads" at the
same department at 1TB. Moreover, the author has started his activities as a
researcher in a collaborative project with the Department of BioMedical Engineering,
Materials Science and Application (SMSA), University Medical Center Groningen
(UMCG), University of Groningen, the Netherlands, since the winter of 2005.
Selected Publications
• Hermawan N. Rasyid, Henny C. van der Mei, Soegijardjo Soegijoko, Henk J.
142
Busscher and Danielle Neut. Inhibitive effect of antibiotic-loaded beads to cure
chronic osteomyelitis in developing country: hand-made vs. commercial beads.
IFMBE book chapter. Springerlink, Berlin Heidelberg, 2007;1 5: 113-117.
Curriculum Vitae
• Hermawan N. Rasyid, Jim R. van Horn, Henny C. van der Mei, Soegijardjo
Soegijoko, Henk J. Busscher and Danielle Neut. Inhibitive effect of antibiotic
loaded beads to cure chronic osteomyelitis in developing country: hand-made vs.
commercial beads. Kuala Lumpur International Conference on Biomedical
Engineering (Biomed), 2006.
• Hermawan N. Rasyid, Kuspriyanto, Tati R. Mengko, Soegijardjo Soegijoko, Jim
R. van Horn, Henny C. van der Mei, Henk J. Busscher and Danielle Neut. In vitro
inhibitive effect of antibiotic-loaded beads to chronic osteomyelitis: hand-made vs
commercial beads. Bandung Medical Journal-MKS, Volume XXVIII, No. 2, 2006.
• Hermawan Nagar Rasyid, Kuspriyanto, Tati L Mengko, Soegijardjo Soegijoko.
The development of PC-based BME-ITB beads to evaluate antibiotic release
kinetics from bone cement: a preliminary report. Proc. of the 7th Asean Science
Congress and Sub Committee Conferences, August 5-7, 2005, Jakarta,
Indonesia.
143
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