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.

125

Ringkasan

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.

128

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

130

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

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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.

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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.

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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.

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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.

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