Pathogenesis and prevention of biomaterial centered infection

Abstract

One of the major drawbacks in the use of biomedical materials is the occurrence of biomaterials centered infections. After implantation, the host interacts with a biomaterial by forming a conditioning film on its surface and an immune reaction towards the foreign material. When microorganisms can reach the biomaterials surface they can adhere to it. Adhesion of microorganisms to an implant is mediated by their physico-chemical surface properties and the properties of the biomaterials surface itself. Subsequent surface growth of the microorganisms will lead to a mature biofilm and infection, which is difficult to eradicate by antibiotics. The purpose of this review is to give an overview of the mechanisms involved in biomaterials centered infection and the possible methods to prevent these infections.

Full text

JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE 13 (2002) 717±722
Pathogenesis and prevention of biomaterial
centered infections
B. GOTTENBOS, H. J. BUSSCHER*, H. C. VAN DER MEI
Department of Biomedical Engineering, University of Groningen, Faculty of Medical
Sciences, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
P. NIEUWENHUIS
Department of Histology and Cell Biology, University of Groningen, Faculty of Medical
Sciences, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
E-mail: h.j.busscher@med.rug.nl
One of the major drawbacks in the use of biomedical materials is the occurrence of
biomaterials centered infections. After implantation, the host interacts with a biomaterial by
forming a conditioning ®lm on its surface and an immune reaction towards the foreign
material. When microorganisms can reach the biomaterials surface they can adhere to it.
Adhesion of microorganisms to an implant is mediated by their physico-chemical surface
properties and the properties of the biomaterials surface itself. Subsequent surface growth of
the microorganisms will lead to a mature bio®lm and infection, which is dif®cult to eradicate
by antibiotics. The purpose of this review is to give an overview of the mechanisms involved
in biomaterials centered infection and the possible methods to prevent these infections.
#2002 Kluwer Academic Publishers
1. Introduction
Foreign materials are used more and more in modern
medicine after trauma, oncological surgery or wear to
replace, support or restore human body function, for
example in hip prostheses, prosthetic heart valves or
catheters. The extended use of these materials, usually
referred to as biomaterials, has some major drawbacks.
One of these is the possible occurrence of biomaterials
centered infections (BCI) [1]. The incidence of this type
of infections varies from 4% for hip prostheses [2] to
100% for urinary tract catheters after 3 weeks use [3] (see
Table I). In BCI microorganisms are present in close
association with the biomaterials surface forming a so-
called bio®lm. BCI can cause severe problems, from
disfunctioning of the implanted device to lethal sepsis of
the patient. Furthermore, treatment of BCI is compli-
cated, as microorganisms in a bio®lm are more resistant
to antibiotics [4] than their planktonic counterparts [5].
As a consequence and if possible, the only remedy is
removal of the infected implant at the expense of
considerable costs and patient's suffering. A more
convenient way to deal with this problem is to prevent
the development of an infectious bio®lm on the
biomaterials surface. To achieve this, a thorough under-
standing of how these bio®lms develop is necessary.
2. Host±biomaterials interactions
2.1. Conditioning ®lms
The interaction of biomaterials with the body of the host
depends on the place where the implant or device is
situated, for example in the oropharyngeal cavity, the
urinary tract, different body tissues or in the circulatory
system. When a biomaterial is inserted, ®rst a so-called
conditioning ®lm from organic matter present in the
surrounding ¯uid is deposited on the biomaterials
surface. Depending on the body site the surrounding
¯uid can be saliva, urine, tear ¯uid, tissue ¯uid, serum, or
blood, and the conditioning ®lm will mostly consist of
adsorbed proteins, which for serum are mainly albumin,
immunoglobulin, ®brinogen, and ®bronectin. The
composition of the conditioning ®lm depends on the
physico-chemical properties, i.e. chemical composition,
hydrophobicity and charge, of the biomaterials surface.
For example, on polyethylene hydrophobicity gradients
exposed to blood serum it was found that at the
hydrophobic end less proteins were adsorbed with
relatively more ®brinogen, while on the hydrophilic
end more albumin was present [6]. In time, smaller
proteins like albumin are usually replaced by higher
molecular weight proteins, like ®brinogen and ®bro-
nectin. With blood contacting biomaterials, also blood
cells will adhere to the biomaterials surface. Especially
adhesion of blood platelets to arti®cial vascular grafts
can initiate the blood coagulase cascade, causing
thrombosis, a frequent complication in these applica-
tions. Another unwanted phenomenon at the biomaterials
surface is calci®cation of the implant, which can
decrease the necessary ¯exibility of, for example,
prosthetic heart valves. Ideally host derived cells will
colonize the implanted biomaterials, forming a thin
capsule around the implant.
*Author to whom correspondence should be addressed.
0957±4530 #2002 Kluwer Academic Publishers Review Paper 717

2.2. Immune reactions
The host will also actively interact with the biomaterials
surface if it is invasively implanted, as this is a normal
reaction to any foreign body that enters the host. The
coagulation cascade and complement system are acti-
vated, leading to formation of a ®brin network and
opsonization of the biomaterial [7, 8]. These processes
will attract and activate the innate immune system, i.e.
macrophages and polymorphonuclear cells, leading to
in¯ammation [9, 10]. This immune response can dis-
appear when the wound is healed and the biomaterial is
encapsulated. However, in many cases the host±
biomaterials interface remains in a state of chronic
in¯ammation, as few metals and plastics are chemically
inert in the warm, wet, oxygenated environment of living
tissues, causing the release of in¯ammatory compounds
from the biomaterial, like corrosion products, plastici-
zers, and monomers [1, 11]. Chronic in¯ammation
impairs host cell growth on the implant [12] and can
cause chronic pain.
3. Pathogenesis of biomaterials centered
infections
The presence of a foreign material signi®cantly
compromises the host to cope with microorganisms. In
a classical study in man it was shown that the presence of
a subcutaneous suture reduced the required inoculum to
produce infection with Staphylococcus aureus,an
infamous virulent pathogen, from 10
6
to only 200
bacteria [13]. Furthermore, the relatively avirulent
Staphylococcus epidermidis, normally not capable of
establishing infection, is the most common infecting
organism in BCI [14].
3.1. Inoculation
Probably the most important factor determining the
occurrence of BCI is the chance that microorganisms will
reach the biomaterials surface. Biomaterials in contact
with the outer part of the body, for example intravenous
catheters, peritoneal dialysis catheters, or urinary tract
catheters are readily reached by microorganisms and
consequently have a higher incidence of BCI than fully
implanted biomaterials (0.5±100% vs. 0.1±7%) [2].
Microorganisms can reach a biomaterials implant in
several ways at several time points, which determines the
properties of the biomaterials surface they will meet.
Airborne microorganisms, which can be present in the
operating theater, can reach the surface as early as before
the implantation [15, 16] and interact with a bare
substratum surface, not even covered with a conditioning
®lm. Also during insertion of the biomaterial, micro-
organisms from the skin can be pushed towards the
implant surface. Furthermore, microorganisms from the
skin can contaminate the operation wound and reach the
implant surface through diffusion, active movement or
hematogenous transport. Perioperative contamination is
believed to be the most common cause of BCI [17].
Generally, it is also assumed that microorganisms can
reach the implant via the hematogenous route at any time
after implantation, causing so-called hematogenous
infections. As shown in Table II skin infections, surgical
or dental interventions, pneumonia, abscesses, or
bacteriuria can cause temporal or chronic bacteremia
resulting in infections [18]. In addition, microorganisms
can translocate from the gastro-intestinal tract to other
body parts [19]. When the microorganisms survive in the
bloodstream, they can be transported to the biomaterials
surface, establishing an infection. In this respect it is
especially interesting to note that it has been proposed
that macrophages play a role in transporting microorgan-
isms to the biomaterials surface [20] as some strains are
capable to survive within macrophages [21]. As the
biomaterials surface elicits a foreign body reaction in the
®rst weeks after implantation, and in the case of chronic
in¯ammation also hereafter, macrophages are speci®-
cally attracted to the biomaterials surface thus potentially
transporting microorganisms to the biomaterials implant
or device [22]. As hematogenous infection can happen
anytime, biomaterials implants are sometimes called
``microbial time bombs''.
The etiology of BCI can provide information about the
origin of the infecting organisms. Table III shows the
organisms found in examples of studies with vascular
grafts, hip and knee arthroplasties. S. epidermidis and S.
aureus, primarily skin inhabitants, are the predominant
infecting organisms [14], followed by Gram-negative
bacilli like Escherichia coli and Pseudomonas aerugi-
nosa, primarily present in the gastro-intestinal and
urinary tract, streptococci from the mouth and pneumo-
cocci from the respiratory tract [18, 23]. Interestingly,
except in the case of airborne microorganisms, the
T A B L E I I Distant infectious foci of hematogeneously infected hip
[17] and knee arthroplasties [18]
Distant foci Hip n27(%) Knee n72(%)
Cutaneous region 19 39
Urinary tract 15 19
Respiratory tract 15 14
Oral cavity 30 7
Gastrointestinal tract 4 6
Septic arthritis 0 3
Abdominal abscess 0 1
Unknown 19 11
T A B L E I Incidences of infection of different biomedical implants
and devices adapted from Dankert et al. [2] and arranged according to
body site
Body site Implant or device Incidence (%)
Urinary tract UT catheters 10±20
Percutaneous CV catheters 4±12
Temporary pacemaker 4
Short indwelling catheters 0.5±3
Peritoneal dialysis catheters 3±5
Subcutaneous Cardiac pacemaker 1
Soft tissue Mammary prosthesis 1±7
Intraocular lenses 0.13
Circulatory system Prosthetic heart valve 1.88
Multiple heart valve 3.6
Vascular graft 1.5
Arti®cial heart* 40
Bones Prosthetic hip 2.6±4.0
Total knee 3.5±4
*From experiments in calves and sheep.
718

infecting organisms usually originate from the hosts
micro¯ora. There is evidence that the host might be
immuno-tolerant to some of these microorganisms,
which has been especially investigated for the intestinal
micro¯ora [24±26]. This might be an overlooked
virulence factor for causing BCI. As immuno-tolerated
microorganisms can survive longer in the blood without
being attacked by the hosts immune system, they have a
bigger chance to reach an implanted biomaterial.
3.2. Microbial adhesion
When microorganisms have reached the biomaterials
surface, initial microbial adhesion can occur. Microbial
adhesion is mediated by speci®c interactions between
cell surface structures and speci®c molecular groups on
the substratum surface [14], or when viewed from an
overall, physico-chemical view-point by non-speci®c
interaction forces, including Lifshitz±Van der Waals
forces, electrostatic forces, acid±base interactions, and
Brownian motion forces [27]. Speci®c interactions are in
fact non-speci®c forces acting on highly localized
regions of the interacting surfaces over distances smaller
than 5 nm, while non-speci®c interaction forces have a
long-range character and originate from the entire body
of the interacting surfaces, as is shown in Fig. 1 [28].
Upon approach of a surface, organisms will be attracted
or repelled by the surface, depending on the resultant of
the different non-speci®c interaction forces. Thus the
physico-chemical surface properties of the biomaterial,
with or without conditioning ®lm or epithelial cells, and
microorganisms play a major role in this process.
The conditioning ®lm on the biomaterials surface (and
on the bacterial cell surface) plays an important role, as it
changes the physico-chemical properties of the inter-
acting surfaces. Most proteins are capable of reducing the
adhesion of microorganisms. Albumin is a strong
adhesion inhibitor, for unknown reasons, although
changes in hydrophobicity and sterical hindrance are
proposed mechanisms [14]. Fibronectin and ®brinogen
have been shown to promote the adhesion of S. aureus
and certain S. epidermidis strains, which is mediated by
speci®c adhesive cell structures directed to these proteins
[14, 29].
The adhesion mechanism of late hematogenously
transported microorganisms is unclear, as by that time
the biomaterial will be covered with host derived cells,
which will decrease the adhesion probability of
microorganisms [1]. Hematogenous infections are,
besides with intravascular prostheses, almost exclusively
reported with orthopedic implants [17, 18]. Orthopedic
implants are usually not completely integrated within
host tissue, as they consist often of metal parts, which are
not easily colonized by host tissue cells. The uncovered
metal surface can be colonized readily by microorgan-
isms. Another explanation could be that the repeated
hinging of orthopedic implants, for example in knee
prostheses, can cause cell damage, providing adhesive
sites for microorganisms [1].
When the microorganisms have adhered to a bioma-
terials surface they are protected against phagocytosis, as
the microorganism and biomaterial together are too large
to ingest. Furthermore Zimmerli and coworkers reported
that the activity of phagocytes and polymorphonuclear
leukocytes is decreased in the presence of a biomaterial
[30, 31]. After adhesion to biomaterials most micro-
organisms start secreting slime and embed themselves in
a slime layer, the glycocalix, which is an important
virulence factor for BCI and which explains the
extraordinary prevalence of slime producing S. epider-
midis in BCI [14]. The glycocalix provides protection
against humoral and excreted cellular immune compo-
nents, as these cannot readily diffuse through the slime
layer [4] and once a glycocalix has formed a BCI with all
its complications, including ultimately removal of the
implant, seems almost inevitable. However, to do real
damage the adhering microorganisms ®rst have to grow.
T A B L E I I I Microbial etiology of vascular graft [23] and hematogenous orthopedic implant [17, 18] infections
Germ Vascular grafts (%) Orthopedic implants (%)
Abdominal n17Inguinal n60Popliteal n8Hip n27Knee n72
S. aureus 14 40 33 52 52
S. epidermidis 7131744
Streptococci 14 8 25 22 14
Pneumococci n.d. n.d. n.d. 7 6
E. coli 42 9 0 7 11
Proteus species 3 11 0 4 4
other Gr. neg. bacilli 0 8 17 4 4
Other bacteria 10 5 0 0 1
Candida species 3 1 0 0 0
Unknown 7 5 8 0 0
n.d. not determined. Values for nindicate number of patients.
Figure 1 At separation distances of 450 nm, only attractive Van der
Waals forces occur. At 10±20nm, Van der Waals and repulsive
electrostatic interactions in¯uence adhesion. At 55 nm, short-range
interactions can occur, irreversibly binding a bacterium to a surface.
Adapted from Busscher and Weerkamp [27].
719

3.3. Microbial growth
Initial microbial growth, i.e. the growth in the ®rst hours
after microbial adhesion, is another important factor for
the outcome of BCI, as bio®lm organisms become more
resistant to the immune system while the bio®lm
matures. Growth of sessile microorganisms has been
mainly studied in vitro [32]. Barton et al. [33] found that
growth of sessile P. aeruginosa, S. epidermidis and E.
coli depended on the biomaterial involved. Only for P.
aeruginosa they could ®nd a correlation between
physico-chemical properties of the biomaterials surface
and initial growth rates. Also proteins in the conditioning
®lm can in¯uence the growth rates of adhering
microorganisms. Poelstra et al. found that growth rates
of P. aeruginosa decreased in the presence of pooled
immunoglobulin G [34].
Van Loosdrecht et al. [35] concluded that adhesion of
bacteria does not directly in¯uence their metabolism and
growth yield. Changes in growth rate due to adhesion of
bacteria were suggested to be mainly the result of
changes in nutrient availability. Depending on the
amount of adsorbed nutrients and whether adsorption is
easily reversed, growth rates of adhering bacteria can be
decreased or increased with respect to the growth of their
planktonic counterparts. Another mechanism, causing a
decrease in the growth rates of E. coli after adhesion was
proposed to be the strong attraction to positively charged
biomaterials surfaces [36].
There is little information about microbial growth in
vivo. Barth et al. followed the number of bacteria for 48 h
on subcutaneously implanted polymeric and metal
biomaterials in rabbits, after inoculation at the time of
implantation [37]. They found, as is shown in Fig. 2, that
the number of non-slime producing S. epidermidis
decreased directly after implantation, probably due to
action of the host immune system, while the number of
slime producing S. epidermidis and S. aureus increased
in the ®rst 8±12 h to a maximum, whereafter their
number decreased again. Here also materials differences
played a role. S. aureus grew faster on the metal, while S.
epidermidis grew faster on polymeric biomaterials. An
important difference between in vitro and in vivo
experiments is the presence of the immune system.
Although unprotected bacteria are eradicated by the
immune system, it has been reported that those
microorganisms that do survive accelerate their growth
rates under in¯uence of cytokines excreted by macro-
phages [38].
3.4. Consequences of BCI
The presence of a microbial bio®lm on biomaterials
impairs the function of the implant or device and/or
worsens the clinical state of the patient. Examples with
non-implanted devices are voice prostheses, which are
situated between the trachea and the upper digestive
tract, as is shown in Fig. 3, or urinary tract catheters. The
action of the voice prosthesis is impaired by bio®lm
formation, because microorganisms block the valve
mechanism, or cause leakage of food into the trachea
[39]. As a consequence the prosthesis has to be replaced
every 4 months on average [40]. Urinary tract catheters
rarely escape colonization by microorganisms causing
blockage or, more seriously, bacteriuria [41]. Infections
of indwelling catheters, like for example central venous
catheters, often results in bacteremia which can cause
sepsis and endocarditis. Totally implanted prostheses
have lower rates of infections, but the consequences are
often more serious. Especially infections of implants in
the circulatory system, i.e. prosthetic valves and vascular
grafts, yield a high mortality (70% and 50% respectively
[42]). Infection of deep tissue implants, for example
Figure 2 The numbers of colony forming units per disk of (a) PMMA,
(b) UHMW-PE and (c) titanium vanadium aluminum alloy after
different in vivo implantation times. SE-360 (black) is a slime
producing and SP-2 (gray) is a non-slime producing strain of S.
epidermidis. White bars represent S. aureus. Note the decline in
adhering non-slime producing S. epidermidis in time. Adapted from
Barth et al. [36].
Figure 3 The voice prosthesis (a) and anatomy after total laryngectomy
(b). The voice prosthesis is placed in the tracheo-esophageal shunt, an
area which is heavily inhabited by microorganisms.
720

orthopedic implants or mammary prostheses, will usually
result in less serious complications like pain, swelling
and loosening of the implant, although mortalities up to
20% are reported with orthopedic implants [18, 43, 44].
The clinical signs of deep implant infections are reported
to appear up to a year after microbial seeding [45].
Apparently the infectious bio®lm can stay silent for a
long period, and probably a signi®cant part of the
infections is never recognized. Recent investigations at
our laboratory revealed that the so-called aseptic
loosening, i.e. loosening of an orthopedic implant
without microorganisms present, is often diagnosed
falsely as aseptic. As standard microbiological techni-
ques are used to test for the presence of infectious
microorganisms, slow growing bio®lm organisms often
remain undetected. Similarly, it has been reported that
®ve of 28 removed scleral explants were covered with a
bio®lm, while clinical signs of infection were only
present in one out of these ®ve cases [46]. Thus the
incidence of problems associated with BCI is possibly
higher than generally assumed.
4. Treatment and prevention of BCI
4.1. Treatment
Treatment of an established BCI is dif®cult, as the
minimal inhibitory concentration (MIC) of antimicrobial
agents, necessary to kill the microorganisms, is sig-
ni®cantly higher for microorganisms in a bio®lm than for
planktonic ones [4, 5]. As antibiotics have little effect on
BCI, the standard procedure for infected orthopedic
prostheses is the removal of the implant and implantation
of an antibiotic releasing device at the implant site. A
new prosthesis is inserted when the implant site is free of
microorganisms, usually six months later. For many
implants, especially those in the circulatory system,
removal of the implant is dangerous, and a high mortality
is associated with these infections. Much research has
been done to make bio®lm bacteria more susceptible to
antibiotics. Ultrasonic treatment of the infecting bio®lm,
for example, has been shown to enhance the action of
antibiotics towards these bio®lm bacteria [47]. Also
application of an electrical ®eld yields enhanced effects
of antibiotic treatment, the so-called bioelectric effect
[48]. The application of these techniques in patients
could facilitate the treatment of BCI with antibiotics in
the future.
4.2. Prevention
Surgeons take considerable effort in preventing the
contamination of implants with microorganisms during
implantation. Although application of prophylactic
antibiotics and better operation hygiene has reduced the
incidence of BCI the last four decades, still a signi®cant
number of patients suffer from these infections.
Different strategies seem useful to prevent BCI. In
general it is aimed to reduce the attractive force between
bacteria and biomaterials surface by optimizing the
physico-chemical surface properties of the biomaterial.
Bacterial adhesion is low, for example, on extremely
hydrophobic surfaces [49, 50], while also more nega-
tively charged biomaterials attract less bacteria [51].
Albumin and heparin coatings have shown to decrease
the adhesiveness of biomaterials [52].
However, microorganisms always seem to be able to
adhere to some extent to solid materials. Moreover, when
proteins are present they can cover an anti-adhesive
biomaterial, and be anchors for microorganisms to
adhere to. Another approach to prevent bio®lm formation
is to prevent the growth of adhering microorganisms.
This can be achieved by application of antimicrobial
agents near the biomaterials surface. One way to do this
is the design of antibiotic releasing biomaterials.
Examples are gentamicin-loaded bone cement and
silver-loaded catheters [53, 54]. A disadvantage of
these applications is that they usually only work for a
few days to weeks, as the amount of antibiotic that is
actually released is extremely limited and does not
exceed 15% of the total amount incorporated [55]. A
more dangerous problem with antibiotic releasing
materials and the low dose actually released is the
development of antibiotic resistant microbial strains [56].
A better approach would be to couple the antimicrobial
agent covalently onto the biomaterials surface, while
maintaining its activity. As in this approach the
antimicrobial agent can only reach the outside of the
microbial cells, it can only be employed with antibiotics
working at the level of the cell wall or membrane.
Polymers with incorporated quaternary ammonium
groups have shown such antimicrobial activity in vitro
[57, 58], thus these compounds might have the required
properties.
References
1. A. G. GRISTINA,Science 237 (1987) 1588.
2. J. DANKERT,A. H. HOGT and J. FEIJEN,CRC Crit. Rev.
Biocompat.2(1986) 219.
3. J. D. DENSTEDT,T. A. WOLLIN and G. REID,J. Endourol. 12
(1998) 493.
4. J. W. COSTERTON,P. S . ST EWAR T and E. P. GREENBERG,
Science 284 (1999) 1318.
5. P. GILBERT,J. DAS and I. FOLEY,Adv. Dent. Res. 11 (1997)
160.
6. H. T. SPIJKER,R. BOS,W. VAN OEVEREN,J. DE VRIES and
H. J. BUSSCHER,Coll. Surf. B: Biointerf. 15 (1999) 89.
7. L. TANG and J. W. EATON,Am. J. Clin. Pathol. 103 (1995) 466.
8. A. REMES and D. F. WILLIAMS,Biomaterials 13 (1992) 731.
9. T. E. MOLLNES,Vox Sang. 74 S2 (1998) 303.
10. J. M. ANDERSON,ASAIO Trans. 34 (1988) 101.
11. S. H. DOUGHERTY and R. L. SIMMONS,Curr. Probl. Surg. 19
(1982) 217.
12. J. H. JACKSON and C. G. COCHRANE,Hematol. Oncol. Clin.
North Am. 2(1988) 317.
13. S. D. ELEK and P. E . CON E N,Br. J. Exp. Pathol. 38 (1957) 573.
14. G. D. CHRISTENSEN,L. M. BADDOUR,D. L. HASTY,J. H.
LOW RA NC E and W. A. SIMPSON, in ``Infections Associated
with Indwelling Medical Devices'', edited by A. L. Bisno and
F. A. Waldvogel (American Society of Microbiology, Washington
DC, 1989) p. 27.
15. O. M. LIDWELL,E. J. LOWBURY,W. W H Y T E ,R. BLOWERS,
S. J. STANLEY and D. LOWE,Br. Med. J. Clin. Res. Ed. 285
(1982) 10.
16. J. CHARNLEY,Clin. Orthop. 87 (1972) 167.
17. A. AHLBERG,A. S. CARLSSON and L. LINDBERG,ibid. 137
(1978) 69.
18. S. BENGTSON,G. BLOMGREN,K. KNUTSON,A. WIGREN
and L. LIDGREN,Acta Orthop. Scand. 58 (1987) 529.
19. P. A. VAN-LEEUWEN,M. A. BOERMEESTER,A. P.
HOUDIJK,C. C. FERWERDA,M. A. CUESTA,S. MEYER
and R. I. WESDORP,Gut 35 (1994) S28.
721

20. E. M. MORA,M. A. CARDONA and R. L. SIMMONS,Arch.
Surg. 126 (1991) 157.
21. C. L. WELLS,M. A. MADDAUS and R. L. SIMMONS,ibid. 122
(1987) 48.
22. W. GU O,R. ANDERSSON,A. LJUNGH,X. D. WANG and S.
BENGMARK,Scand. J. Gastroenterol 28 (1993) 393.
23. G. PRINTZEN,Injury 27 S3 (1996) SC9.
24. R. DUCHMANN,E. SCHMITT,P. KNOLLE,B. K. MEYER-
ZUM and M. NEURATH,Eur. J. Immunol. 26 (1996) 934.
25. M. C. FOO and A. LEE,Infect. Immun. 6(1972) 525.
26. R. D. BERG and D. C. SAVAGE,ibid. 11 (1975) 320.
27. C. J. VAN OSS,Biofouling 4(1991) 25.
28. H. J. BUSSCHER and A. H. WEERKAMP,FEMS Microbiol.
Rev. 46 (1987) 165.
29. Y.H. AN and R. J. FRIEDMAN,J. Biomed. Mater. Res. 43 (1998)
338.
30. W. ZIMMERLI,P. D. LE W and F. A. WALDVOGEL,J. Clin.
Invest. 73 (1984) 1191.
31. W. ZIMMERLI,F. A. WALDVOGEL,P. VAUDAUX and U. E.
NYDEGGER,J. Infect. Dis. 146 (1982) 487.
32. G. G. GEESEY and D. C. WHITE,Annu. Rev. Microbiol. 44
(1990) 579.
33. A. J. BARTON,R. D. SAGERS and W. G . PI TT,J. Biomed.
Mater. Res. 32 (1996) 271.
34. K. A. POELSTRA,H. C. VAN DER MEI,B. GOTTENBOS,
D. W. GRAINGER,J. R. VAN HORN and H. J. BUSSCHER,
ibid. 50 (2000) 224.
35. M. C. M. VAN LOOSDRECHT,J. LYKLEMA,W. N OR DE and
A. J. B. ZEHNDER,Microbiol. Rev. 54 (1990) 75.
36. G. HARKES,J. DANKERT and J. FEIJEN,J. Biomater. Sci.
Polym. Ed. 3(1992) 403.
37. E. BARTH,Q. M. MYRVIK,W. WAG N E R and A. G.
GRISTINA,Biomaterials 10 (1989) 325.
38. P. B. VA N WACHE M,M. J. A. VAN LUYN,A. W. DE WIT,
D. RAATJES,M. HENDRIKS,M. L. P. M. VERHOEVEN and
L. CAHALAN,J. Biomed. Mater. Res. 35 (1997) 217.
39. H. F. MAHIEU,H. F. SAENE,H. J. ROSINGH and H. K.
SCHUTTE,Arch. Otolaryngol. 112 (1986) 321.
40. F. A. VAN DEN HOOGEN,M. J. OUDES,G. HOMBERGEN,
H. F. NIJDAM and J. J. MANNI,ibid. 116 (1996) 119.
41. J. C. NICKEL,J. W. COS TERTON,R. J. MCLEAN and M.
OLSON,J. Antimicrob. Chemother. Suppl. A 33 (1994) 31.
42. K. H. MAYER and S. C. SCHOENBAUM,Prog. Cardiovasc.
Dis. 25 (1982) 43.
43. G. HUNTER and D. DANDY,J. Bone Joint Surg. Br. 59 (1977)
293.
44. R. H. FITZGERALD and D. R. JONES,Am. J. Med. 78 (1985)
225.
45. G. MANILOFF,R. GREENWALD,R. LASKIN and C. SINGER,
Clin. Orthop. 223 (1987) 194.
46. R. H. ASARIA,J. A. DOWNIE,L. MCLAUGLIN-BORLACE,
N. MORLET,P. MU NR O and D. G. CHARTERIS,Retina 19
(1999) 447.
47. A. M. REDISKE,B. L. ROEDER,M. K. BROWN,J. L.
NELSON,R. L. ROBISON,D. O. DRAPER,G. B.
SCHAALJE,R. A. ROBISON and W. G. P IT T,Antimicrob.
Agents Chemother. 43 (1999) 1211.
48. J. W. COSTERTON,B. ELLIS,K. LAM,F. JOHNSON and A. E.
KHOURY
,ibid. 38 (1994) 2803.
49. E. P. EVERAERT,H. F. MAHIEU,B. VAN DE BELT-
GRITTER,A. J. PEETERS,G. J. VERKERKE,H. C. VAN
DER MEI and H. J. BUSSCHER,Arch. Otolaryngol. Head Neck
Surg. 125 (1999) 1329.
50. J. TSIBOUKLIS,M. STONE,A. A. THORPE,P. GRAHAM,V.
PETERS,R. HEERLIEN,J. R. SMITH,K. L. GREEN and T. G .
NEVELL,Biomaterials 20 (1999) 1229.
51. A. H. HOGT,J. DANKERT and J. FEIJEN,J. Biomed. Mater.
Res. 20 (1986) 533.
52. J. R. KEOGH and J. W. EATON,J. Laborat. Clin. Med. 124
(1994) 537.
53. A. S. CARLSSON,G. JOSEFSSON and L. LINDBERG,J. Bone
Joint Surg. 60 (1978) 1059.
54. H. AKIYAMA and S. OKAMOTO,J. Urol. 121 (1979) 40.
55. H. VAN DE BELT,D. NEUT,D. R. A. UGES,W. SCHENK,J. R.
VAN H OR N,H. C. VAN DER MEI and H. J. BUSSCHER,
Biomaterials 21 (2000) 1981.
56. H. VAN DE BELT,D. NEUT,J. VAN HORN,H. C. VAN DER
MEI,W. SCHENK and H. J. BUSSCHER,Nat. Med. 5(1999)
358.
57. R. G. FLEMMING,C. C. CAPELLI,S. L. COOPER and R. A.
PROCTOR,Biomaterials 21 (2000) 273.
58. R. EL-KENAWY,F. I. ABDEL-HAY,A. EL-RAHEEM,R. EL-
SHANSHOURYand M. H. EL-NEWEHY,J. controled Release 50
(1998) 145.
Received 11 January 2001
and accepted 7 February 2002
722