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103 Pages·2007·6 MB·English

Antimicrobial peptides
Experimental prevention of
osteomyelitis
We performed the experiments at the Oral Cell Biology Department
(OCB), Academic Centre for Dentistry (ACTA), VU. The work was
supported by:
AM-Pharma, Annafonds, Biomet, BV Cyclotron, Hereaus / Cavex, Johnson
& Johnson, Link, Livit Orthopedie, Nederlandse vereniging voor
Biomaterialen en Tissue Engineering, Nederlandse Orthopaedische
Vereniging, Ortho Biotech, Smithkline & Nephew, STEGA, Stichting Anna
Fonds, Stichting BIS, Stryker, Synthes Nederland, Wright Medical,
Zimmer
© Hein Stallmann, 2007. All rights reserved. No part of this book may be
reproduced, stored in retrievable form, or transmitted in any form or by
any means, without written permission from the author.
Cover: The Gross Clinic, (Thomas Eakins, 1875), surgical treatment of
osteomyelitis. With kind permission of Jefferson Medical College of
Thomas Jefferson University, Philadelphia.
Printed by Giethoorn ten Brink, Meppel. Fonts: Dutch823 and Verdana.
VRIJE UNIVERSITEIT
Antimicrobial peptides
Experimental prevention of osteomyelitis
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. L.M. Bouter,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de faculteit der Geneeskunde
op vrijdag 9 maart 2007 om 13.45 uur
in het auditorium van de universiteit,
de Boelelaan 1105
door
Hein Pieter Stallmann
geboren te Amsterdam
promotoren: prof.dr. P.I.J.M. Wuisman
prof.dr. A.C. van Nieuw Amerongen
copromotor: prof.dr. E.C.I. Veerman
Say it’s only a paper moon,

Contents
Chapter Page
Abbreviations 8
Outline and aims 9
1. Introduction.
AMPs: application in musculoskeletal infections 11
2. Continuous-release or burst-release of hLF1-11
from bone substitutes 23
3. In vitro gentamicin release from bone substitutes
Influence of carrier type on duration of release 31
4. Histatin and lactoferrin derived AMPs: activity and toxicity 41
5. In vivo release of hLF1-11 from calcium phosphate cement 51
6. Osteomyelitis prevention in rabbits using hLF1-11
or gentamicin containing cement 65
7. Summary and Discussion 77
8. Samenvatting NL 89
Dankwoord 101
Abbreviations
AMP antimicrobial peptide
AO Arbeitsgemeinschaft für Osteosynthesefragen
ATCC American Type Culture Collection
BHI brain heart infusion
CaP calcium phosphate
dH O demineralized water
2
DHVAR-5 synthetic histatin analogue
ELISA Enzyme Linked Immunosorbent Assay
ESR erythrocyte sedimentation rate
Fmoc 9-fluorenylmethoxycarbonyl
GS gentamicin sulphate
hLF1-11 human lactoferrin N-terminal amino acids 1-11
HPLC high performance liquid chromatography
KDL department of experimental animal surgery
LC-MS/MS liquid chromatography mass-spectrometry/mass-
spectrometry
LF lactoferrin
LPS lipopolysaccharide
MRSA methicillin resistant Staphylococcus aureus
PBS phosphate buffered saline
PMMA polymethylmethacrylate
Q-TOF MS quadrupole-time-of-flight mass-spectrometry
SCV small colony variant
STEGA Skeletal Tissue Engineering Group Amsterdam
VRSA vancomycin-resistant Staphylococcus aureus
VRSE vancomycin-resistant Staphylococcus epidermidis
WBC white blood cell count
8
Outline and aims
he first chapter introduces the main concepts of this manuscript:
osteomyelitis (bone infection), bacterial resistance and
antimicrobial peptides (AMPs). As part of a solution to the first
two problems, AMPs were investigated for the experimental
prevention of infection. The first chapter introduces several
AMPs which have been investigated in pre-clinical studies. In our
experiments hLF1-11 was used, it consists of amino acids one to eleven of
human lactoferrin. We investigated a biodegradable carrier-system for
local treatment or prevention of bone infection. The properties of several
biodegradable carriers were investigated in chapters two and three. The
chapters show the release profiles of hLF1-11 and gentamicin, a
conventional antibiotic, from a dozen different carrier materials. The
activity on bacterial strains was confirmed after the release process.
Chapter four describes the toxic properties and antimicrobial spectrum of
hLF1-11 and control peptides. No specific toxicity was observed on blood
and bone cells. The peptide rapidly killed all clinically problematic
bacterial strains. These results encouraged us to set up several animal
experiments. For these experiments a combination of biodegradable
cement (Bonesource) and hLF1-11 was selected. In the rabbit
experiments, the release of hLF1-11 from calcium phosphate cement was
used as a prophylactic agent against infection with Staphylococcus aureus,
the principal cause of osteomyelitis. Chapter five describes the release of
hLF1-11 from cement into bone, the AMP rapidly dissipated from the
carrier. This was comparable to the earlier in vitro experiments (chapters
two and three). Chapter six combines all results from earlier chapters in a
prevention study. The rabbits were surgically contaminated with S. aureus
and injected with cement and hLF1-11. After removal and culture of the
bones, a significant reduction of infection was measured by both hLF1-11
and (standard) gentamicin. This confirms the clinical potential in local
treatment of infection.
Study aims
• To develop a biodegradable carrier for release of hLF1-11.
• To evaluate the cytotoxicity and antimicrobial efficacy of hLF1-11.
• To compare the results to gentamicin and other antimicrobial peptides
for reference.
• To implant the carrier and the peptide in an animal model and study
its release end efficacy.
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