The Importance of Antibacterial Surfaces in Biomedical Applications

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The Importance of Antibacterial Surfaces in Biomedical

Applications
Chapter · January 2018

DOI: 10.1016/bs.abl.2018.05.001
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CHAPTER FOUR
The Importance of Antibacterial

Surfaces in Biomedical
Applications
Metka Bencina*, Tina Mavricx, jj, 1, Ita Junkar*, Aleksander Bajtx,
Aleksandra Krajnovicx, Katja Lakota{, Polona Zigon {
,

Snezna Sodin-Semrl {
, Veronika Kralj-Iglicjj and Ales Iglicx, #
*Jozef Stefan Institute, Ljubljana, Slovenia
x
Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia
{
Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia
jj
Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia
#
Laboratory of Clinical Biophysics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
1
Corresponding author: E-mail: tina.mavric@fe.uni-lj.si
Contents
1. Introduction 116
2. Metal Implants in Biomedical Applications 118
2.1 Dental Implants 118
2.2 Orthopedic Implants 119
2.3 Vascular Implants 120
2.4 Common Materials Used in Implantable Biomedical Devices 122
2.4.1 Titanium and Its Alloys 122
2.4.2 Stainless Steels 122
2.4.3 CobalteChromium Alloys 122
2.4.4 Tantalum Alloys 123
2.4.5 Silver and Its Alloys 123
2.4.6 Zirconia and Its Alloys 124
2.4.7 Diamondlike Carbon 124
3. Bacterial Adhesion and the Formation of Biofilm on Metal Surfaces 124
3.1 Dynamics of Biofilm Formation 128
4. Pathogens Binding to Vascular Implants 133
4.1 Microbiology and Risk Factors 133
4.2 Pathogenesis and Vascular Graft Infections 135
4.3 Endothelial and Smooth Muscle Cells Covering Vascular Stents and 137
Approaches to Limit Complications
4.3.1 Prevention of Smooth Muscle Cell Proliferation 137
4.3.2 Acceleration of Re-Endothelialization 139
Advances in Biomembranes and Lipid Self-Assembly, Volume 28

© 2018 Elsevier Inc.
j
ISSN 2451-9634
https://doi.org/10.1016/bs.abl.2018.05.001 All rights reserved. 115
116 Metka Bencina et al.
5. Mechanisms of Antibacterial Effect 140

5.1 Antifouling Effect/Passive Surfaces 141
5.1.1 Antiadhesive Polymeric Surfaces 141
5.1.2 Superhydrophilic/Superhydrophobic Surfaces 142
5.1.3 Nanorough Surfaces 145
5.2 Bactericidal Effect/Active Surfaces 145
5.2.1 Organic Antibacterial Agents 145
5.2.2 Inorganic Antibacterial Agents 149
6. Future Outlooks for Antibacterial Implantable Surfaces 151
7. Concluding Remarks 153
Acknowledgments 154
References 154
Abstract
Infections by pathogenic microorganisms present a serious concern in health care
sector, as such infections kill more people than any other cause. The bacterial infections
are commonly treated by antibiotics, but serious problems occur because of biofilm
formation, which highly reduce the effectiveness of antibiotic therapy. Moreover, the
overuse of antibiotics with excessively broad spectrum has increased the number of
antibiotic-resistant bacterial strains, which are life threatening for patients. The bacterial
infections due to implantable materials are connected with a high number of postsur-
gical complications, which cause immense health care costs because of prolonged
antimicrobial therapy, multiple surgical intervention, and even implant removal. Thus
there is an increasing demand to develop novel superior-quality medical implants that
would prevent bacterial adhesion, biofilm formation and at the same time provide appro-
priate cell support for specific application. The rapid growth of nanotechnology and its
immense potential in medicine has offered numerous opportunities for designing sur-
faces with superior properties, which will be used in new-generation medical devices.
In this chapter, some of the antibacterial implantable devices are summarized, with
emphasis on vascular implants. Bacterial adhesion and biofilm formation on implantable
materials is discussed. Furthermore, the bacterial infections associated with vascular im-
plants and the biological responses on these surfaces are presented in more detail.
Research focuses on the major antibacterial mechanisms, reviewing antifouling and
bactericidal approaches for the benefit of antibacterial surfaces.
1. INTRODUCTION
Biomedical devices have become essential in the human health care
system. The usage of orthopedic implants, stents, heart valves, vascular grafts,
and nonimplanted devices, such as orthopedic fixation screws, sutures, and
catheters, saves lives and restores quality of living. However, device-related
The Importance of Antibacterial Surfaces in Biomedical Applications 117
infections (DRIs) in implant surgery, as well as short-term biomedical

devices, present a huge problem that cannot be overlooked. In the United
States, nearly 2 million health care-associated infections occur each year and
almost 100,000 of them result in mortality [1e3]. The health care system
tends to minimize DRIs due to short-term biomedical devices, meaning
they are being constantly replaced with new ones. With implants, however,
the problem cannot be solved so easily. Biofilm formation on implantable
surfaces presents a serious threat, as in such cases the effectiveness of
antibiotic therapy is also highly reduced, and according to Gbejuade et al.
[4], more than 65% of all human infections are associated with biofilm.
Before the biofilm develops, bacteria need to adhere to the chosen surface.
The adhesion is performed through the contact of their membrane and
different membrane appendages. Both gram-positive and gram-negative
bacteria, which differ in structural properties of the lipid membrane, are
found to cause implant-related infections [5,6]. For a better understanding
of the differences in gram-positive and gram-negative bacteria, both
membranes are schematically represented in Fig. 1.
The patients undergoing surgical procedure have already compromised
health and lower immune system that in turn makes them more prone to
bacterial infections. The highest risk of infections is during the first 48 h after
implementation. Moreover, although sterilization can prevent early-stage
infections, delayed infections can occur months after the surgery, as all
individuals with implantable materials in the body and compromised
health are more prone to biofilm infections. The patient abolition remains
Figure 1 Schematic representation of gram-negative and gram-positive bacterial

membranes.
a problem to implant-related industries and health care institutions. Usually

in case of bacterial infections, antibiotic therapy is prescribed; however, its
success is mainly governed by the type of bacterial strains because of the
increase in antibiotic-resistant bacterial strains. According to statistics,
25,000 patients in Europe die each year because of antibiotic-resistant infec-
tions [7]. It was estimated that global material damages in the European
Union are around V1.5 billion per year [7].
Because of the fact that antibiotic-resistant microorganisms have become
a serious health issue, research into new and effective antibacterial agents
areas has emerged [8]. In order to minimize biofilm formation and bacterial
colonization, development of a biocompatible implant surface that can
withstand bacterial adherence is urgently needed, in addition to promoting
bacterial evasion and osseointegration [9].
2. METAL IMPLANTS IN BIOMEDICAL APPLICATIONS

2.1 Dental Implants
Dental implantology implies fixing a device in the maxillary or
mandibular bone aiming to replace a missing root and secondly, to support
a prosthetic element; the endosseous implant is in contact with at least three
different tissues: the buccal epithelium, the gingival connective tissue, and
the alveolar bone [10].
In dentistry, nanotechnology has been used mostly in the development of
restorative materials and many methods are being developed to treat diseases,
although clinical use must be carefully considered. Nevertheless, nanoparticles
promise better aesthetic traits (as they are more translucent) and improved
wear properties. With the understanding of their physical and chemical char-
acteristics, such as surface charge, the possibilities are plenty [11].
An indispensable part of a dental implant is its implantebone interface,
which allows the bone to grow and consequently the implant to connect
with the tissue. For successful dental implants, enhancement of protein
adsorption for cell adhesion must be achieved, as well as inhibition of
bacterial colonization and hence biofilm formation. Biomolecules that
enhance such integration include adhesion molecule (RGD)50 peptide,
alkaline phosphatase, extracellular matrix (ECM) proteins, and calcium
phosphate (CaP) coating. Regarding the surface properties of dental implants,
it is important to carefully calculate the desired forms so as to maintain a suc-
cessful implantebone integration. Osseointegration is achieved first by
mechanical anchorage and then by biological anchorage. In nano terms the di-
mensions and spaces between surface patterns are designed as to alter the cell
behavior. Changing the diameter of nanotubes from 30 to 70e100 nm has
induced the transition from adhesion to differentiation of cells to nanostructured
surfaces. A spacing of 58 nm between the nanotubes incited the formation of a
stable adhesion onto the bone and therefore large cell growth, whereas a spacing
of 108 nm slowed the process of cell proliferation. Regarding the bonee
implant contact, higher density and a curvature of 60 nm demonstrated higher
boneeimplant contact than a curvature of 120 nm [11].
2.2 Orthopedic Implants

The earliest known example of an implantation with osseointegration
reaches far back in history. Back then, iron was used as the only suitable
metal for this purpose. With the development of metal engineering in the
20th, but mostly in the 21st century, the popularity of such procedures
increased. Implants with primarily a mechanical function, e.g., mostly dental
and orthopedic implants, have achieved considerable success for the reason
that allows aging and injured individuals to retain their abilities [9].
The major reasons for failure of dental and orthopedic devices are aseptic
loosening and infection. As of 2015, about 7 million individuals in the
United States have received joint replacements. Within this group, infec-
tions occur in approximately 1.5% of total knee replacements and 1.6% of
total hip replacements. Treatments involve extended antibiotic therapy,
replacement of the infected implant, and at least one additional surgery.
Reinfection rates can be considerably higher, with estimates in the literature
ranging between 26% and 49% [9]. Because implants are designed to inte-
grate with the bone, which proceeds by adhesion of proteins to the implant
surface, osseointegration is interrupted by bacterial protein binding. Thin
bacterial alloys are formed on the surface of implants and cause infection
[9]. This leads to reoperation, replacement of the infected device, and
incurred costs for health care system [2].
Knee and hip replacements are the most routine orthopedic surgeries of
the 21st century. Arthroplasty surgeries (a surgery to replace a joint) enables
injured and aging individuals who suffer from osteoarthritis to retain their
physical functions and mobility and regain their independence on a daily
basis [2]. Orthopedic implants are mostly used in fracture fixation, tissue
reconstruction, spine fixation, and joint arthroplasties [12]. They can be
roughly separated into two groups: joint replacements and fixation
screws/discs (Fig. 2).
Figure 2 Example of three-dimensional printed titanium spinal cage implant. Kindly

donated by Ekliptik d.o.o; photo courtesy of Ita Junkar.
Prosthetic joint infection and surgical site infection remain the most
problematic, which both require an additional operation to remove the
infected implant. According to the data published in the literature, orthope-
dic implanteassociated infection rate is up to 9%, nearly doubling for
revision surgeries. According to Montanaro et al. [13] a rate of infections
in revision surgery is reported between 3.2% and 5.6%. It is considered
that 0.5%e2% of patients develop a biofilm orthopedic implante
associated infection in the first 2 years after surgery.
The most common pathogens of orthopedic implants that cause up to
two-thirds of infections are staphylococci (Staphylococcus epidermidis, methi-
cillin-sensitive Staphylococcus aureus, methicillin-resistant S. aureus [MRSA],
coagulase-negative staphylococci). Other bacteria associated with implant
infections are Escherichia coli and Pseudomonas aeruginosa [14e16].
2.3 Vascular Implants

Coronary artery disease is the main cause of death and represents approxi-
mately 31% (17.7 million people) of all global deaths in 2015, as reported
by the World Health Organization. A coronary stent is the most prevalent
procedure for this disease and a bare-metal stent is used in most cases
(Fig. 3). However, such stents may lead to an in-stent restenosis in approx-
imately one-third of cases [17]. If reendothelialization is delayed after stent
implantation, platelets gradually adhere to the stent walls and/or the smooth
muscle cells in the tunica media excessively proliferate inside stents. Blood
Figure 3 Self-expandable vascular stent made of nickeletitanium alloy (Nitinol). Kindly

donated by Rontis AG; photo courtesy of Ita Junkar.
flow through the supported vessel is therefore impaired because of thrombosis

and/or intimal thickening. Consequently, in situ reendothelialization has
attracted attention as a strategy to solve in-stent restenosis and late thrombosis
[18]. Furthermore, infections are a universal problem, affecting the success of a
vascular stent. S. aureus is a highly adaptable microbial pathogen that is consid-
ered to be the leading cause of various community- and hospital-acquired
infections, which have risen in accordance with the number of performed
implantations of vascular stents. MRSA is considered to be amongst the
most problematic strains [19]. Endovascular therapies are increasingly being
applied in the management of infrainguinal ischemia. Stents are used primarily
or selectively after a suboptimal technical result following angioplasty. Most
common stents used in femoropopliteal arteries are balloon expandable stents;
however, few published series document the superiority of nickeletitanium
alloy (Nitinol) stents because of their self-expandable form and ability for
crush recovery [20]. A more detailed description of the pathogens binding
to the vascular implants is presented in Section 4.
2.4 Common Materials Used in Implantable Biomedical

Devices
2.4.1 Titanium and Its Alloys
Titanium is known for several beneficial properties in medical applications:
good corrosion resistance, which is a consequence of a stable oxide layer
formation; absence of tissue toxicity and allergic reactions; good strength,
a factor for the device safety; and low elastic modulus [21]. In contrast to
cobalt-chromium-molybdenum (CoCrMo), titanium has almost no allergic
or immunogenic reactions. Moreover, because of the rapidly forming tita-
nium dioxide, its implant-to-bone connection is stronger than that obtained
using stainless steel. Titanium’s high affinity to oxygen contributes to rapid
reparation of disruptions and damages on the implant. The biocompatibility
of titanium can be increased by coating with bioactive titanium dioxide in
nanoscales [22].
Titanium is mostly used in implant industry in two forms: as a pure tita-
nium for low mechanical loads or as Ti-6A1-4V alloy for high mechanical
loads [21]. Titanium is currently the prevalent material used in dental
implantology; however, it is being replaced with zirconia and its alloys.
2.4.2 Stainless Steels

Stainless steels can be specified by their chemical composition, as well as
microstructure. In implant industry, austenitic steels are used for their
sustainability. They are also widely used for nonimplantable medical equip-
ment and devices because of their good corrosion resistance [23].
Corrosion resistance of steels depends on the chromium content. Corro-
sion-resistant steels contain more than 12% of chromium, resulting in the
formation of a thin and chemically stable oxide layer that leads to better anti-
bacterial activity [24]. However, chromium is not the only substance that
contributes to corrosion resistance. Nickel (Ni) and molybdenum (Mo)
are also known for these properties [24].
Because of the content of nickel (Ni), which is toxic to human body, as
well as for mechanical reasons such as crevice corrosion, its usage in tempo-
rary devices is limited. However, because of its low price, in comparison to
other metallic biomaterials, it is still used in combination with cobalte
chromium and titanium alloys [24].
2.4.3 CobalteChromium Alloys

There are two types of CoCrMo alloys that have been developed specifically
for joint replacements. These are CoCrMo (ATSM F75 and F76) and
CoNiCrMo (ATSM F562). “Co-based alloys usually contain 26e30 wt.%

of Cr, which favorably influences their biocompatibility and corrosion
resistance via a strong tendency to create a thin Cr2O3 surface oxide
film” [23].
Nevertheless, once implanted in vivo, CoCrMo implants perform under
tribological contacts and loads while being lubricated by corrosive body
environment, i.e., biological fluids. This leads to the release of metal particles
(caused by wear) and metal ions (Co and Cr) from CoCrMo alloy, which
results in implant loosening, cytotoxicity, and immunological reactions,
overall leading to implant failure [25]. To reduce that, CoCrMo implants
are manufactured via additive manufacturing methods, which improve
their tribochemical performance. In addition, these methods empower
manufacturing of customized (patient-unique) devices, which are highly
needed among patients [26].
2.4.4 Tantalum Alloys

Tantalum is known for its high affinity to oxygen, resulting in the develop-
ment of a thick, stable, and microbe-protective Ta2O5 layer. Because of this
it is corrosion resistant, which has led to its massive usage in orthopedic
fields. However, primarily because of its high price, as well as high density
and high/low elasticity modulus properties, it is not considered to be suitable
for fabrication of large implants [23,27]. Because of its beneficial attributes, it
is used in joint and bone components exposed to high loads and components
requiring bone ingrowth [23,27].
2.4.5 Silver and Its Alloys

Silver is known for its beneficial antimicrobial properties since the first great
civilizations (Egyptians, Romans, Greeks). Back then, it was used for wound
dressings and burn bandages [28].
In the implant industry, silver coatings are used in the form of nanopar-
ticles. During the ion dissociation, Agþ ions from pure silver or even better,
AgCl2 interface with bacteria, leading to bacteria’s death. The problem
arises when silver ions interact with human cells [28]. Silver molecules
disrupt cell membranes; inhibit the metabolism of sugar and oxidative
enzymes in bacterial cells, specifically Streptococcus mutans and Lactobacillus
acidophilus; and induce proton leakage from membranes, resulting in cell
degeneration [11,29]. Silver is also in use in other osseo implants for its
impact on the E. coli respiratory chain and interference with its DNA
replication [29].
2.4.6 Zirconia and Its Alloys

Research of zirconia shows promising features for biomedicine, as it is
durable, osseoconductive, and resistant to corrosion. Several studies con-
ducted with ZrO2 have presented data of noncytotoxicity and noninflam-
matory effects of stated zirconia powder [29]. It is a bioinert material;
therefore, encapsulation by connective tissue is faint and the release of
residues is almost undetectable. Moreover, zirconia is known to be osseo-
conductive, which means that this ceramic facilitates bone formation
when in contact with it [10].
2.4.7 Diamondlike Carbon

Diamondlike carbon (DLC) has been proposed for use in blood-contacting
devices such as electrosurgical devices, artificial hearts, mechanical heart
valves, and artery stents. Various reports have shown that the anticoagulation
property of DLC is related to the bonding structure, hydrophobicity, and
smooth surface. A DLC film is suitable to prevent blood clotting and is
therefore appropriate to battle restenosis. DLC has excellent mechanical
and biological properties, and its traits can be augmented by the addition
of other substances. Nano-Cu particles, for example, diffuse into the film
and successfully kill bacteria by destroying their cell membranes. The a-C:
H films with Cu content have antibacterial properties against both E. coli
and S. aureus. Thus the a-C:H/Cu film can be considered a promising anti-
bacterial coating for applications in biomedical science and for minimally
invasive surgery devices [30].
3. BACTERIAL ADHESION AND THE FORMATION OF

BIOFILM ON METAL SURFACES
The perseverance of microorganisms in some of the most rigid envi-
ronments, and their endurance despite unfavorable changes in the surround-
ings, is the testament to their adaptivity [31]. A very efficient form of
adaptation to harsh conditions is biofilm formation. This is a colony of
microorganisms (bacteria, algae, fungi, and protozoa) that are attached and
layered over almost any substrate where the cells are nested in self-excreted
ECM [32e35]. Interestingly, this kind of microorganism cell accumulation
in the human body has been reported already in 17th century by observation
of dental plaque under the predecessor of the microscope [36]. Among
microorganisms, bacteria are the prevalent biofilm-forming species, as
over 95% of bacterial populations are able to form films [37,38]. The
detrimental effect and obtrusive nature of biofilm formation can be found in

several fields, such as biomedical implants and tools [39], food-processing fa-
cilities [40], marine-related industry [41], and common households [42].
However, one must also take into consideration the fields where biofilm
occurrence is indispensable and that offer a significant advantage, e.g., in
environmental remediation [43] and agriculture [44]. However, for the
benefit of this chapter, the development of such a film is detrimental; it is
to blame for a significant amount of infections, greatly decreases the
longevity of implanted materials, and represents a serious health threat to
the patients [45e47].
The biofilm community forms a multicellular body, where the differen-
tiation of cells taking over different roles increases the adaptation, eases the
intercellular communication, and separation of tasks, which all insures better
efficiency and effective use of scarce resources [48,49]. Thus even though
cells in the biofilm proliferate from a single cell, they are not mere duplicates
of the original cell but show distinctly different phenotypical features, owing
to different gene expression, which is dictated by the environment-specific
assignment of the cell, i.e., depending on the amount of nutrients, oxygen,
or fluids that are available in the gradient of the biofilm [34,50,51]. This con-
firms the evidence that almost all bacteria are proficient in film formation,
but there is prominent heterogeneity in the formation of the ECMs [52].
Complexity and role differentiation of the biofilm ensures its resistance.
Heterogeneity of the biofilm is also derived from its establishment; a biofilm
formed from several spatially divided bacterial microcolonies as its basic units
can be established by different species of bacteria. Through the growth of
the colonies the biofilm spreads over the surface, and other bacterial species
and even fungi can adjoin onto this growing mass [36,53,54]. Therefore
we not only have specific gene expression within the species but also
have the heterogeneity of specific genes across different species [54,55]. Cells
enclosed in the biofilm can persevere by establishing a defense system
secreting extracellular material, also known as slime or slimy secretion,
which consists mainly of insoluble polysaccharides and proteins, as well as
lipids, nucleic acids [56e59], flagella, and pili [60]. The composition,
biochemical pathway expression, and shape largely depend on the bacterial
species present. More and more evidence is revealed that speaks of complex
preconceived protein localization with specific interaction between compo-
nents to ensure the survival of the colony [61]. Moreover, the cells that fail
to produce exopolysaccharides are not able to form complex architectures
and are easily detached from the surface [62].
As mentioned earlier the polymeric building blocks partake different

shapes and sizes, which are largely dependent on the species of bacteria
that secret them. We have summarized the components and roles of the
biofilm ECM, also known as the glycocalyx coating or extracellular poly-
meric substance (EPS), in the scheme in Fig. 4A. The ECM represents
over 70% of the biofilm volume and thus has several important roles to
enable the prosperity of the bacterial microcolonies nestled inside [63].
ECM is essential for biofilm adhesion and thus for the adherence between
the cells, as well as for the adherence of the mature biofilm to the substrate.
In this manner, it offers mechanical stability to keep the growing community
of cells grounded [64]. It also provides the architectural support to the cell
community to grow into three-dimensional expansions of several small
microcolonies. Furthermore, the three-dimensional architecture is impor-
tant for the development of the sustenance system throughout the biofilm.
The ECM has the ability to harvest the nutrients from the biofilm surround-
ings via charged polysaccharide groups on its surface and to transport them
throughout the matrix for additional cell alimentation [65]. It has been
shown that the pronounced diversified topography, which is visibly
protruding from the surface of a mature biofilm, conceals a rudimentary
“circulatory system” [63,66]. The biofilm-resident bacteria are dependent
on the flow of nutrients, oxygen, and biofluids (or water) from the biofilm
surroundings [52], because as the film progresses, it can expand into several
Figure 4 (A) Schematic representation of different extracellular matrix (ECM)

component macromolecules together with the tasks attributed to the ECM. (B) Repre-
sentation of different layers of a mature bacterial biofilm. Adapted from H. Rohde,
S. Frankenberger, U. Za €hringer, D. Mack, Structure, function and contribution of poly-
saccharide intercellular adhesin (PIA) to Staphylococcus epidermidis biofilm formation
and pathogenesis of biomaterial-associated infections, Eur. J. Cell Biol. 89 (2010) 103e111.
layers of cells. For this purpose, ECM channels are formed to supply nutri-
ents to the cells inside the biofilm. The cells near the substrate (base film) and
the cells that do not reside near such channels still have the distinct disadvan-
tage of living in straitened circumstances for nutrition, water, and oxygen
availability [55,66]. The specific conditions of the biofilm thus dictate the
direction of bacterial microcolony development, such as anaerobic or
aerobic adaptation due to oxygen gradient formation. Regarding both
lack of oxygen and nutrients the biofilm can also host dormant bacteria [67].
In addition to these roles the protective encapsulation allows the preser-
vation of microorganisms in the biofilm against outside environmental stress
and desiccation. Therefore unlike in the planktonic or suspended state,
biofilm-based bacteria can build not only tolerance to innate and adaptive
immune responses but also tolerance and resistance to several antibiotics
or biocidal agents because the biofilm formation offers them protection
against unfavorable chemical and physical conditions [65,68]. The ECM
harbors specific mechanisms to battle the onset of antibiotics or other disin-
fectants and antibacterial agents. For example, not all bacterial species are
resistant to antibiotics, but the formed biofilm architecture can delay the
protrusion of drugs into the ECM through its ion-exchanging capabilities
[55]. Additionally, even if the antibiotic penetration is successful, the biofilm
architecture establishes an oxygen gradient formation (spatial limitation),
which consequently subdues the metabolic activity; therefore, cells disregard
the antibiotic presence, do not metabolize the drug, and are thus unaffected
by it [69]. Also, specific antibiotic-inactivating enzymes (b-lactamase, form-
aldehyde lyase, formaldehyde dehydrogenase) can be embedded and
concentrated in the ECM [55]. Even cation-chelating extracellular DNA
is reported to contribute to the antibiotic inefficiency over the biofilm layer
[70]. Adding to the antibiotic resistance is also an elaborate ECM-mediated
cell-to-cell communication termed “quorum sensing”. This mechanism
holds control over gene expression. The precondition for this mechanism
to manifest itself is the required density of cells in the biofilm. This does
not mean the cells can sense each other’s presence, but that they are suscep-
tible to the increased levels of signaling molecules in their surroundings.
Once the threshold is passed, bacteria in the biofilm communicate by
horizontal transfer of genes (as opposed to vertically parent/offspring)
[71e73]. In this respect the horizontal gene transfer allows for the spread
of antibiotic resistance genes throughout the colony [55].
Fig. 4B is a schematic representation of a mature biofilm over a substrate
consisting of three layers: conditioning film, biofilm base, and surface film
[47,74]. The biofilm base is denser, whereas the surface film is characterized
by a looser layer of matrix-based bacteria, which can extend into the sur-
rounding medium [55]. The protrusion of colonies depends on the shear
forces present in the environment; when shear forces are low, the growth
of the biofilm towers in the form of a mushroom into the surroundings.
On the other hand, when the shear forces are dominant, the colonies
grow in an elongated form that is able to withstand pressure [63].
3.1 Dynamics of Biofilm Formation

In order to produce a biofilm, bacteria first need to adhere to the substrate.
By many researchers, this step is regarded as decisive in biofilm formation.
The source of first colonizers is a liquid with resident planktonic bacteria.
The adhesion of these cells is not final; in the first stage, bacteria can be
detached from the surface. The first colonizers’ adhesion dynamics is influ-
enced by several factors, which will be discussed later. The adsorption in the
first stage is weak and does not persist for long; if the adhesion conditions are
unfavorable, this stage is referred to as reversible adsorption [54]. The second
stage of biofilm formation is the nonreversible stage, where microcolonies
start to form and the bacteria intensely secrete EPSs. In the third and fourth
stages, microcolonies or multilayered clusters are shaped and the film is in the
process of maturation across the several microcolonies that had indepen-
dently arisen on the surface, constructing its three-dimensional structure.
Here, other species of bacteria from the environment join the growing
biofilm [53,54]. In the final fifth stage, the biofilm reaches maturation;
this leads to the release of planktonic bacteria, which allow the cycle to
continue. In Fig. 5, the biofilm progression on a substrate is schematically
depicted. We can see the progression of growth of the biofilm through
the several stages. The first stage, the stage of adsorption, is very fast and takes
place within seconds [36,75,76]. In the second, nonreversible stage and the
third stage, the microcolonies grow exponentially and the process takes
minutes to hours. In the final maturation phase, the growth is not as pro-
nounced and becomes stationary. Within days, the mature biofilm life force
can start to decline, but the shed cells warrant bacterial persistence [36].
The first interactions between the bacterial cells and the adhesion surface
can be either specific or nonspecific. When bacteria are in the planktonic
form and freely moving in the medium (e.g., water or blood), they first
interact with cells by long-range interactions, which are nonspecific and
take effect in distances over 50 nm. These interactions convey the bacteria
closer to the material surface (e.g., electrostatic attraction/repulsion,
Figure 5 The progression of biofilm formation through different stages and time.
Reused with permission from J.G. Thomas, L.A. Nakaishi, Managing the complexity of a
dynamic biofilm, J. Am. Dent. Assoc. 137 (2006) 10Se15S.
Brownian motion, van der Waals attraction forces). Specific interactions

(hydrogen or other chemical bonding and ionic, dipole, and hydrophobic
interactions) are distinctly localized, located in the radius less than 5 mm
from the surface, and prevail once the cells are within the required distance
[77,78]. Here, it is necessary to emphasize that both specific and nonspecific
interactions have the same physio-chemical origin, the differentiation actu-
ally lies in the recognition of the specific highly localized molecular group
interactions or specific events between parts of the entities in contact,
whereas nonspecific ones refer to the interactions of whole entities, without
setting out particular points of interaction or contact [79].
Regarding the surfaceebacteria interactions, there are two factors to be
taken into consideration: the material surface characteristics and the charac-
teristics of bacteria [80]. Additionally, the medium or serum where these
interactions take place also influences the course of the adhesion [79]. All
these influence the physiochemical interactions and determine the path of
bacterial adhesion. Knowing the parameters relevant to cell attachment pro-
motion could lead to the prevention of the resilient biofilm formation.
Typically, the general environmental factors influencing bacterial behavior
are pH and the concentration of certain electrolytes, temperature, flow
conditions, and the possible antibiotic occurrence in the environment

[37,77]. When we consider the implant’s final residence in the human
body (with the exception of dental implants), the environment is considered
sterile, e.g., blood, cerebrospinal fluid, bone, bone marrow, joint fluid,
internal organs, and thus the infection-inducing bacteria have to enter this
environment at the time of insertion during the operating procedure [81].
Excluding the postoperative antibiotic treatment, the newly implanted
material environment is relatively beneficial to pathogen growth. Further-
more, rarely is the material used for different applications not affected by
its surroundings. Thus when any kind of substrate is subjected to a nonsterile
environment, (macro)molecules that are found near the implanted material
adsorb onto the surface, forming the so-called conditioning film [82]. Con-
ditioning film is composed of organic (mainly proteins) and inorganic
components, which are brought into the vicinity of the film by, for example,
flow movement [83,84]. Adhesion parameters in such conditions are of
course different from those in sterile surfaces, as better adhesion and nutrient
supply are provided [37].
Material characteristics in the absence of conditioning film represent a
particular aspect of influence on bacterial adhesion. Among the relevant
factors are hydrophilicity, surface topographical features, chemistry, and
charge [85e88]. Surface roughness is generally accepted as an important
factor with respect to bacterial adhesion. On initial consideration, ultra-
smooth surfaces are regarded as less favorable for adhesion and consequent
biofilm formation in comparison to surfaces consisting of imperfections
(cracks, pores, dislocations) or grain boundaries [89,90]. Increased roughness
offers better adhesion opportunities because of enhanced surface area, addi-
tional adhesion sites, and the protection from the environment, mainly from
shear forces [53,89]. Nevertheless, the roughness issue is not that straightfor-
ward. While it can promote bacterial adhesion, the factors must be consid-
ered more closely. There have been reports that consider pristine surfaces,
which lack any biological substrate, to be less favorable for adhesion, and
it is on the pioneer cells to set the conditions for the biofilm to develop
[91]. However, certain bacteria such as P. aeruginosa, S. aureus, and S. epider-
midis are known to easily bind also to stainless steel and titanium orthopedic
screws [47]. On the other side, the distinction between micro- and nano-
sized surface roughness has to be made. Prokaryotic cell size ranges from
100 nm to 5 mm. If the surface disturbances are in the range that can accom-
modate the bacteria between them, the bacteria will take advantage of this
and maximize the contact area between themselves and the surface to have
Figure 6 The comparison of bacterial spatial configuration on surfaces with (A) micro-
and (B) nanostructured topography [85].
the best possible grip [92e95]. The relationship between the bacterial size
and the surface roughness is depicted in Fig. 6A. As opposed to results
considering micrometer roughness, the reports on experimental results
considering the nanoscale-textured surfaces are diverse. Some authors indi-
cated that nanosized roughness is advantageous because it decreases bacterial
adhesion. The two predominant reasons supporting this argument were that
a decrease in contact points between bacterial cells takes place (Fig. 6B) and
that the contact forces from the large area of the nanostructured surface repel
bacterial cells [53,85,87]. Other authors, however, reported that nanosized
surface roughness is beneficial for bacterial adhesion [90,96,97]. The ambi-
guity regarding the roughness of surfaces is not surprising, as it depends on
nanosized surface [98], physical configuration [77,99], different bacterial
strains [53] and their contrasting cell characteristics, membrane rigidity,
metabolic activity, and production of EPSs [90]. Bacterial characteristics
are actually of great importance in terms of adhesion, as bacteria are known
for their adaptive features.
Friedlander et al. [100] have initially observed a decrease in bacterial
adhesion to nanostructured surface, in comparison to flat controls. Howev-
er, after prolonged exposure of the surfaces to bacterial cells, the adhesion on
nanostructured topographies was substantially promoted. Bacterial cells have
adapted by attaching themselves through flagella, the protein appendage,
which can be produced through specific gene expression by the offspring
of first colonizers as a response to dire adherence conditions, i.e., the time
lapse between reduced and promoted adhesion, in this particular case.
The irreversible phase is actually marked by the molecular and cellular inter-
action of specific bacterial surface structures or proteinous cell appendages
with the chosen surface [80,101]. This testifies to the incredible ability of
bacteria to adapt to new environments, because they can use different
surface appendages to protrude the nanosized crevices. Besides flagella, bac-
teria can aid their adhesion also with pili or fimbriae. These protein bacterial
membrane protrusions are able to gain access into the nanosized spaces and
achieve a better grip; thus, some of the nanostructured surfaces actually offer
good relief and numerous contact points for first colonizers to adhere to.
Actually, it was shown that bacteria are able to produce distinct adhesion
strategies depending on the topography they are adhering to [53,101].
Lorenzetti et al. [85] have concluded their observations with remarks that
topography had prevailed over the effect of surface charge and hydrophilic-
ity in terms of bacterial adhesion. Wettability, in general, is cited as a factor
affecting adhesion, where hydrophobic surfaces are less advantageous owing
to the repulsion; bacteria in aqueous media have a net negative surface
potential [80,86,102]. On the other hand, there is evidence of hydrophilic
cells adhering strongly to hydrophilic surfaces and, by analogy, hydrophobic
cells to hydrophobic surfaces with the important consideration of the adap-
tivity of bacteria and alteration in their surface charge, according to the
environmental conditions [103e106]. Yuan et al. [86] performed experi-
ments considering wettability conditions affecting the growth of E. coli.
Fig. 7 schematically presents different wettability conditions influencing
bacterial adhesion. In Fig. 7A the surface is superhydrophobic (very high
water contact angle [WCA] of 105 degrees), the water in which bacteria
reside, is repelled from the surface and bacteria are left to make contact
with the limited surface protrusions, which were wetted. The highest
bacterial adhesion was observed with moderate hydrophobicity (Fig. 7B),
where there was a higher wettability of the rough surface. Here, the protein
Figure 7 Different interactions between the bacteria Escherichia coli (black elements in
AeC) and the rough surface in the context of wettability [86]. LB CA: Contact angles of
LuriaeBertani medium. Published by The Royal Society of Chemistry.
appendages of E. coli have an important role in adhesion by providing

adequate attachment of the cells to the surface. In contrast, in Fig. 7C, com-
plete wettability can be observed but bacterial adhesion is lowest. This is due
to the repulsive interactions between bacteria and the surface, despite the
increased wettability.
Hitherto, no rule has yet been found with regard to the promotion or
prevention of the adhesion of bacteria to nanorough surfaces. Several factors
were found to influence the adhesion of bacteria onto nanostructured
surfaces. Finding an appropriate set of surface conditions that decreases
adherence of a certain strain does not necessarily mean the same conditions
would also repel other strains. Further research is definitely needed to help
address the answers to these complex problems.
4. PATHOGENS BINDING TO VASCULAR IMPLANTS

Infections involving vascular graft prostheses are infrequent but devas-
tating complication of reconstructive vascular graft surgery and are associated
with high morbidity and, in some situations, mortality [107]. Underlying
comorbidities, such as diabetes mellitus or being immunocompromised,
increase the risk of infection and serious infection-related complications.
Vascular graft infections (VGIs) can be categorized broadly into those that
occur in an extracavitary location, primarily in the groin or lower extrem-
ities, or in an intracavitary location, primarily within the abdomen or less
commonly within the thorax. The incidence of VGI ranges from less than
1% for abdominal aortic grafts to 2.6% for lower extremity grafts to approx-
imately 6% for infrainguinal vascular grafts [107e109]. Some authors suggest
that the rate of infection may be increasing [110,111]. The rate of infection
in prosthetic arteriovenous hemodialysis grafts is approximately 3.5%
[112,113]. Medical as well as economic costs of VGI are extensive. The
overall mortality related to VGI has been reported to be between 13%
and 58%, and the amputation rate in survivors varies between 8% and
52% [109]. Similarly, estimates suggest that 20%e36% of all deaths in
hemodialysis patients are related to infectious complications [113,114].
4.1 Microbiology and Risk Factors

The microbiological cause of VGI has evolved over the years [5,107].
In early published studies, S. aureus was the predominant microorganism
recovered [5,115]. Improvements in surgical techniques, administration of
Figure 8 Microbiology of prosthetic graft infections [116].
prophylactic antistaphylococcal therapy, and other factors have resulted in a

changing microbiological epidemiology. Surgeries performed in patients
with complicated vascular anatomy and in patients with several underlying
comorbidities, increased frequency of emergency procedures, and changes
in hospital flora have all contributed to the changing spectrum of infection.
A more diverse microbiological spectrum of infection includes multidrug-
resistant strains, polymicrobial infection, and Candida species. Gram-positive
cocci account for at least two-thirds of VGIs. Infections caused by coagulase-
negative staphylococci are more common than those caused by S. aureus,
with MRSA infections increasing in frequency. P. aeruginosa is now the
most common cause of gram-negative infections and accounts for at least
10% of VGIs [5,6] (Fig. 8).
Assessing risk factors that predict perioperative mortality and graft
patency is essential for selecting patients who would benefit from surgery
[117]. Omitting surgical reconstruction and endovascular intervention
may be preferable especially when multiple risk factors are present or in
the absence of critical limb ischemia. Infrainguinal revascularization proced-
ures are much more likely infected than aortic grafts [111]. The presence of a
groin incision is an especially important risk factor for infection. Other
commonly cited risk factors for VGI include diabetes mellitus, obesity, renal
failure, revision surgery (particularly revision within 30 days), and use of
Table 1 Risk Factors for Vascular Graft Infections
Presence of Groin Incision

Infrainguinal procedure
Wound-related complications (e.g., hematoma, poor wound healing)
Comorbid conditions (diabetes mellitus, obesity, chronic renal insufficiency)
Revision surgery, especially within 30 days
Emergent surgery
Use of prosthetic grafts
Prolonged operative time
Postoperative hyperglycemia
Perioperative infection at another site
prosthetic rather than native graft material [118] (Table 1). Prolonged
operative time is also associated with infection. Other investigators have
reported a link between VGI and perioperative infection at another site,
postoperative hyperglycemia, immunosuppression, and infected central lines
[115,119,120]. The lowest rates of infection following revascularization
have been seen with anatomic, autologous reverse vein reconstructions
[111]. For abdominal aortic grafts, endovascular repair does not appear
to have a lower risk of infection than open repair; however, the risk of
abdominal aortic graft infection is quite low with either approach [120].
4.2 Pathogenesis and Vascular Graft Infections

Part of the difficulty in treating bacterial infections is the fact that many
involve the formation of a biofilm at some stage [121]. The host response
to biofilm infection can inadvertently increase biofilm mass, as platelets
and fibrin migrate and attach to the site of infection [122]. First, bacteria
in a freely moving, planktonic state adhere to an artificial surface. Once
they have adhered to a surface, the individual cells begin multiplication to
form microcolonies. Development and maturation then occur (Fig. 9).
Bacteria produce an extracellular polymeric matrix that is the hallmark of
a biofilm and may contain proteins, DNA, and polysaccharides [68]. Bio-
films are a polymeric matrix produced by microorganisms that expand
and mature as they adhere to surfaces, and they play an essential role in
VGIs and other implant infections. When bacteria exist as a biofilm, they
are significantly less susceptible to antibiotics. This is a result of metabolic
changes in cells within the biofilm and structural features influencing drug
permeability [123]. Also, within the biofilms, microorganisms display
phenotypic phase variation between an embedded sessile form and a
Figure 9 Biofilm contamination of catheters can lead to bacteremia. (AeD) The stages
of colonization of a catheter where planktonic cells attach, form microcolonies, and
then form a mature biofilm. Reprinted with permission from G. Hughes, M.A. Webber,
Novel approaches to the treatment of bacterial biofilm infections, Br. J. Pharmacol. 174
(2017) 2237e2246.
free-floating planktonic state [124]. The sessile form is responsible for

adhesion and biofilm formation, whereas the planktonic form is responsible
for dissemination. Microorganisms in the sessile stage are comparatively
phenotypically inert but exhibit complex metabolic changes and organized
behavior much different to what would be expected normally.
The likelihood of an implant becoming colonized or infected will vary
depending on the type and structure of the material involved, as well as
the microbes involved [109]. Sessile microorganisms within biofilms are
resistant to killing by both host immunity and antibacterial agents. The
mechanisms of this resistance are not fully understood but appear to be
due to multiple factors. Artificial implants stimulate a chronic low-grade
inflammatory response that not only results in an immune-incompetent
zone that impairs phagocyte bactericidal capacity but also leads to peri-
implant tissue damage and increases the likelihood of implant failure and
infection [124]. Microorganisms in biofilms have been shown to stimulate
antibody production, but the humoral immune response is ineffective in
killing biofilm-encased organisms [125].
Prosthetic grafts can become colonized via direct intraoperative bacterial

contamination of the vascular graft, which is considered to be the most com-
mon cause. The second most common cause of contamination of vascular
grafts is the spreading of the infection from the contiguous site, such as a
surgical wound infection or an intra-abdominal or pelvic abscess, to other
areas. Less commonly, VGI results from bacteremia via hematogenous seed-
ing. In the 1980s, a surgeon A.G. Gristina published a paper on “A Race for
the Surface” proposing that colonization of implants with host cells decreases
the possibility for colonization with bacterial cells [126]. In concordance
with this, the risk of hematogenous spread of VGI is highest in the early
postoperative period (<2 months) and decreases over time because of partial
endothelialization of the graft [107]. Different strategies/approaches were
used to prevent bacterial colonization of implants, including antiadhesive/
antiadsorptive approaches, antibacterial coatings (inorganic, organic), and
multifunctional nanostructured surfaces and coatings [127].
4.3 Endothelial and Smooth Muscle Cells Covering Vascular

Stents and Approaches to Limit Complications
Vascular stent implantation has been an important strategy to treat cardiovas-
cular disease. However, in-stent restenosis and late thrombosis are serious
complications affecting the success of treatment. There are several reasons
attributing to these complications: proliferation of vascular smooth muscle
cells (VSMCs) and slow endothelialization, whereby chronic inflammation
along with delayed wound healing and adhesion/activation of platelets and
fibrinogen also play prominent roles.
Bare-metal stents were first used to enable artery closure, but their
efficacy was severely hampered by proliferating VSMCs and the resultant
neointimal hyperplasia was the only mechanism responsible for restenosis
after metal stent placement. Drug-eluting stents (DESs) proposed a solution
by coating stents with polymers to elute drugs targeting VSMC prolifera-
tion. This approach used since 2002 resulted in a substantial attenuation of
in-stent restenosis. But antiproliferative properties of DESs impair and/or
delay also endothelialization, hence leading to late stent thrombosis. To pre-
vent this, new approaches have lately been sought, including prevention of
VSMC proliferation and acceleration of re-endothelialization.
4.3.1 Prevention of Smooth Muscle Cell Proliferation

While endothelial cells (ECs) compose a monolayer of intima, VSMCs are
located in the media layer of the vessel. Increased neointimal proliferation
represents the main reason for in-stent restenosis, with increased growth and
migration of VSMCs observed as the main reason for the changed vessel state.
Changes in the endothelial layer (inflammatory and/or thrombotic activation)
can trigger VSMC proliferation and migration and intimal hyperplasia.
DESs can elute antiproliferative drugs for prevention of VSMC growth.
The first generation of DESs was generated from biostable polymeric drug
carriers; however, antiproliferative effects proved to be problematic for
re-endothelialization (restenosis and stent thrombosis) and hypersensitivity
reactions. So the second generation of DESs from biodegradable polymers
covering the stent offered a safer alternative to the uncoated bare-metal
stents. Recently, completely biodegradable drug-eluting polymer stents
were developed (biodegradable in 6e24 months), which reduce the proba-
bility of long-term complications [128]. The drugs certified for in-stent
delivery compose of mTOR (mechanistic target of rapamycin) inhibitors
(e.g., everolimus, sirolimus, zotarolimus, pimecrolimus, Biolimus) targeting
phosphatidylinositol 3-kinases (PI3K)-Akt-dependent proliferation mecha-
nisms, while Taxol derivatives (e.g., paclitaxel) are mitotic inhibitors stabi-
lizing microtubules and thus blocking cell division in the G phase [129].
However, there are multiple issues to be considered when deciding
which drug is appropriate. For example, sirolimus is used as an immunosup-
pressive drug for the treatment of cytopenias, associated with autoimmune
lymphoproliferative syndrome [130], or after organ transplantation, espe-
cially following heart or kidney transplantation [131]. These patients have
a high risk of developing symptomatic coronary artery disease and often
require coronary stent implantation. A recent report investigated the effects
of systemic sirolimus application on neointima formation in a mouse model
of vascular injury [132]. This accepted mouse model for studies of postan-
gioplasty restenosis closely resembles the angioplasty procedure that injures
both the endothelium and vessel wall. The data showed that systemic appli-
cation of sirolimus prevented postangioplasty, neointima formation, and
vessel narrowing by inhibiting the inflammatory response to injury. How-
ever, and importantly, systemic sirolimus treatment also impaired endothe-
lial regeneration after angioplasty. So the report warns that extreme caution
should be paid to current approvals of new-generation DESs to reduce dual
antiplatelet therapy time to 3 months only, as safety data for this approval
have not been obtained in patients systemically treated with sirolimus or
in organ transplant patients.
Although mice may significantly differ from humans, the data from this
study clearly indicate the need for further clinical trials to determine the
optimal duration of antiplatelet therapy after coronary interventions in

patients undergoing systemic sirolimus treatment [132].
As antiproliferative effects are not cell-type specific, the need for newer
VSMC-specific antiproliferative drugs is currently leading the research.
Recently, an interesting approach to search for enzymes released by
VSMCs, but not ECs, was proposed to trigger drug release from stents,
leading to the identification of matrix metalloproteinase-9 as a potential
candidate [133].
4.3.2 Acceleration of Re-Endothelialization

In vessels, a healthy endothelial layer is responsible for antithrombotic and
antiadhesive characteristics, barrier functions for leukocyte migration, and
regulating inflammation and thrombosis/fibrinolysis. During the stenting
procedure, the endothelial layer can be destroyed. Re-endothelialization
consists of regrowth of the remaining endothelial layer, partly from circula-
tory endothelial progenitor cells (EPCs) and partly from bone marrowe
derived cells circulating in low levels in the bloodstream.
Changing the surface of the vascular implant with single-molecule or
multimolecule coating or layer by layer coating (with, e.g., heparin, fucoi-
dan, chondroitin sulfate, hyaluronic acid, gallic acid, laminins, fibronectin,
selenium catalyst nitric oxide release surface) has been used to mimic vessel
architecture and promote endothelialization [18,134].
Alternative approaches include incorporation of adhesion molecule
peptide sequences in a polymer base layer (such as laminin-derived RGD
sequence or fibronectin-derived REDV sequence), with huge efforts in
developing EC-type selective adhesion sequences [18,135].
Several approaches have been proposed for incorporating growth
factors to increase endothelialization (e.g., vascular endothelial growth
factor [VEGF]-eluting stents) [136], but as with antiproliferative agents,
cell-type specificity is not high. Dual DESs have been developed by
combining the mechanisms of one drug-preventing restenosis (e.g., anti-
proliferative substance) and another drug-preventing in-stent thrombosis
with antithrombotic action (e.g., hirudin, triflusal) or as endothelialization
promoters (e.g., estradiol, anti-CD34) [129].
Preseeding of EPCs to stents was proposed [137], with stents allowing for
the capture of EPCs developed using anti-CD34 antibody [138] or VEGF
receptor, VEGFR2. A few growth factors increasing adhesion of EPCs
(namely, brain-derived neurotrophic factor, stromal cellederived factor-1)
were also proposed for use as stent material [139]. However, the question
of correct differentiation of the attached cells remains (as CD34þ cells could
differentiate into other cell types, including VSMCs). Additional systemic
treatments to increase EPCs in circulation have been applied (e.g., statins,
erythropoetins) [139] in order to promote re-endothelialization.
Testing the biocompatibility and usefulness of the vascular stent material
in vitro may prevent later complications. So in vitro seeding of ECs and
VSMCs on stent materials in static or laminar flow conditions should be
performed. The latter also enables the simulation of conditions for DESs
and evaluation of the effects of inflammation and/or platelet/leukocyte
adhesion. Cytotoxicity of stent material testing, rate of proliferation, and
expression of molecules important for endothelial function (e.g., expression
of thrombocytes, leukocyte cell adhesion molecules, nitric oxide production
important for controlling thrombocyte/leukocyte activation, and VSMC
proliferation) are all important parameters to study and predict the effectivity
that stents could show in vivo.
5. MECHANISMS OF ANTIBACTERIAL EFFECT

Bacteria can readily colonize surfaces of biomedical devices, implants,
and vascular stents and can shield themselves with protective EPSs, i.e.,
biofilm. There is a strong need to reduce or prevent bacterial colonization
and biofilm formation on such surfaces, either by chemical surface modi-
fications or by topography adjustments. Antibacterial surface coatings
exploit (1) antifouling effect or (2) bactericidal effect that kills bacteria
before or after contact with the surface (Fig. 10) [140]. Antifouling strategy
includes an antiadhesive principle that prevents the attachment of bacteria
(fouling-resistant approach) and the reduction of the adhesion of bacteria
(fouling-release approach). Fouling-resistant coatings include passive poly-
mers, hydrogels, and superhydrophobic surfaces. Fouling-release coatings
are usually made of polymers that enable reversible changes in the surface
properties, e.g., wettability, charge, or topography [141]. Bactericidal stra-
tegies include the use of antibiotics and germicides, active cationic polymer
chains (killing of the attached bacteria), peptides, ion-releasing metals, qua-
ternary ammonium compounds, etc. Some coatings incorporate both these
mechanisms, for instance, antibiotic release from mesoporous TiO2 films
[142], which are both bacteria-repellent owing to the hydrophobic nature
and bactericidal. In the following sections, the mechanisms involved in
antibacterial performance of surfaces are discussed.
Figure 10 General mechanisms of antibacterial action [143].
5.1 Antifouling Effect/Passive Surfaces

5.1.1 Antiadhesive Polymeric Surfaces
Passive polymer coatings can contain one of the uncharged polymers such
as poly(ethylene glycol) (PEG), poly(2-methyl-2-oxazoline), polypeptoid,
poly(n-vinyl-pyrrolidone), and poly(dimethyl acrylamide) [144]. Passive
polymers prevent attachment of the microorganisms, especially adhesion of
S. aureus and S. epidermidis [145,146], to surfaces by hydrophilic/hydrophobic
repulsion, electrostatic repulsion, or low surface energy [144].
Owing to high chain mobility, large exclusion volume, and steric hin-
drance effect of a highly hydrated layer, PEG is one of the most extensively
studied antifouling polymer coating, which can resist biomolecule adsorp-
tion and attachment of microorganisms due to formation of steric barrier.
PEG is commonly grafted to the surface with a chemical anchoring group
or by in situ polymerization of polymer from a surface-bound/grafted
initiator [147]. Although the mechanisms of antibacterial repellence of
surface-immobilized PEG are not fully understood, studies indicate that
grafting density, chain length/thickness of the polymer layer, conformation,
and wettability properties of the grafted polymer play an important role in
resisting microorganism adhesion.
Despite excellent antiadhesive nature of PEG, development of antiadhe-
sive coatings against bacteria remains a challenging task because the bacterial
adhesion mechanism still surpasses the antirepellence strategies and helps
bacteria colonize the PEG-coated surfaces. A study by Zeng et al. [148]
showed that bacteria can adhere to the PEG-coated titanium surfaces with
polysaccharides and extracellular DNA; moreover, DNA could even cause
desorption of the PEG coatings from the surfaces. They suggest that the
Figure 11 Bacterial attachment on the conventional poly(ethylene glycol) (PEG)

coatings and high-density PEG brushes; the adhesion of bacteria by polysaccharide
or DNA adhesins is prevented on polymer brushes, whereas conventional PEG coatings
allow such adhesions. Reprinted with permission from G. Zeng, R. Ogaki, R.L. Meyer, Non-
proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush
coatings, Acta Biomater. 24 (2015) 64e73.
space between conventional polymer brushes enables the penetration of

DNA and its interaction with the underlying surface. The authors found
that this can be avoided by increasing the graft density, as shown in
Fig. 11, by forming a tightly packed and extended conformation of the
polymer that represents the steric barrier between the bacteria and the
surface. Such coatings can inhibit the adhesion of nonproteinaceous bacterial
adhesins such as polysaccharides and DNA.
5.1.2 Superhydrophilic/Superhydrophobic Surfaces

Structured surfaces that create unique surface characteristics such as superhy-
drophobicity and superhydrophilicity also exhibit microbial resistance through
antifouling effect. Such surfaces form a barrier between bacteria and materials
and prevent direct contact between them.
5.1.2.1 Superhydrophilic Surfaces

Superhydrophilicity of the surfaces can be achieved by various approaches,
for instance, (1) nanostructurization or (2) coating of hydrophobic surfaces
with hydrophilic counterparts.
Superhydrophilic layers of TiO2 nanomaterials are widely studied for
antibacterial applications. Younas et al. [149] developed an antibacterial
superhydrophilic barrier consisting of TiO2 nanoparticles on polyvinylidene
fluoride (PVDF)-based membrane. Cai et al. [150] prepared antibacterial

TiO2 nanofilms on sodaelime glass. The excellent superhydrophilic behavior
of TiO2 nanomaterials can be attributed to physical and chemical factors.
Junkar et al. [151] showed hydrophobic behavior of Ti foil in WCA
measurements, whereas nanostructured Ti foil, on which TiO2 nanotubes
were fabricated, was superhydrophilic. In another work, authors demon-
strated that the surface topography and roughness at the nano level substan-
tially affect the TiO2 surfaceewater droplet interaction, at least partially
because of water penetration into the porous structure [152]. The reason
for hydrophilic behavior of TiO2 nanotubes could also lie in the reaction
products, hydroxide compounds, on the surface of TiO2 nanotubes [153].
The hydrophilicity of TiO2 nanotubular surfaces can also be enhanced by
annealing [154] and ultraviolet (UV) radiation [155]. It has been reported
that the anatase crystal phase causes the formation of even more hydrophilic
surface of TiO2 nanotubes [156]. The effect of the crystal structure of the
material on its wettability is especially important if the samples are irradiated
with UV light before spell-out (WCA) measurements. It is known that
crystalline materials facilitate the mobility of photogenerated charge carriers
if exposed to light energy. However, the mechanism of the effect of photo-
induced surface superhydrophilicity on the TiO2 remains elusive [157]. It
has been suggested that the reason is in photocatalytic decomposition of sur-
face organic contaminants existing on the surface of metal oxides [158,159].
Another study proposed the hypothesis of desorption of weakly bonded
water molecules from the external surface hydrolayers by the thermal action
of light and increase of the surface hydrophilicity, which results from resto-
ration of the original structure of hydrated layers [160].
Another approach to render surface superhydrophilicity is by coating
the surface with polymers. One of the most common coatings used to
contribute to surface hydrophilicity is PEG [161]. Often various poly-
mers are coupled in order to create coating with multiple mechanisms
of antibacterial action. Gao et al. [162] prepared a blocklike copolymer
composed of fouling-resistant, fouling-release, and active bactericidal
part (shown in Fig. 12) that significantly diminished adhesion and
enhanced antibacterial activity against the gram-negative bacteria E.
coli and the gram-positive bacteria S. aureus. Fouling-resistant poly(PEG
methyl ether methacrylate) was responsible for improving hydrophilicity
of the PVDF membrane matrix, thereby reducing hydrophobic
interaction through the formation of a hydration shell. The poly(hexa-
fluorobutyl methacrylate) segment created low surface energy and poly
Figure 12 Multitasking antibacterial membrane matrix based on polyvinylidene

fluoride (PVDF). Schemes of prevention of adhesion are shown, including the unfavor-
able hydrophilicity (PEG-MA), antibacterial activity (PMTAC), and reduction of surface
energy (PHFBM).PEG-MA, poly(poly[ethylene glycol] methyl ether methacrylate);
PHFBM, poly(hexafluorobutyl methacrylate); PMTAC, poly[2-(methacryloyloxy)ethyl
trimethylammonium chloride]. Reprinted with permission from K. Gao, Y. Su, L. Zhou, M.
He, R. Zhang, Y. Liu, Z. Jiang, Creation of active-passive integrated mechanisms on mem-
brane surfaces for superior antifouling and antibacterial properties, J. Membr. Sci. (2017).
[2-(methacryloyloxy)ethyl trimethylammonium chloride] quaternary

ammonium salt served as an active antibacterial mechanism.
Similarly, Zhu et al. [163] developed antifouling and antibacterial poly-
ethersulfone membranes by the addition of poly(2-dimethylaminoethyl
methacrylate)-grafted silica (SiO2-g-PDMAEMA) nanoparticles, which
showed antibacterial activity against E. coli and S. aureus because of the
hydrophilic surface. Pan et al. [164] developed an antifouling and antibacte-
rial membrane consisting of Ag/SiO2-PVDF. Yang et al. [165] prepared
superhydrophilic salivary acquired pellicle (SAP) bioinspired tannic acid
(SAP3-TA) coatings on hydroxyapatite, which showed antibacterial and
antibiofouling properties against S. mutans.
5.1.2.2 Superhydrophobic Surfaces

Superhydrophobicity can reduce the adhesion force between bacteria and a
material [166]. When the surface energy of bacterial cells is smaller than the
surface energy of liquids in which cells are suspended, cells preferentially
attach to hydrophobic materials [167]. The mode of action of superhydro-
phobic surfaces to reduce bacterial adhesion is, however, still not clear,
because this is a relatively new topic [168].
Moreover, although superhydrophobicity is one of the factors affecting
bacterial adhesion, it is generally not sufficient to repel organic matter.
Therefore the addition of TiO2 nanomaterials to polymeric coatings is often

required to make surfaces antibacterial [169]. Li et al. [170] prepared hydro-
phobic liquid-infused porous poly(butyl methacrylate-co-ethylene dimetha-
crylate) surface with P. aeruginosa PA49-resistance. Tang et al. [171] showed
that adhesion of S. aureus is lower on superhydrophobic surfaces consisting
of TiO2 nanotubes treated with perfluorooctyl triethoxysilane than on
hydrophobic and hydrophilic TiO2 nanotubes. This is shown in Fig. 13.
5.1.3 Nanorough Surfaces

It was also shown that surface roughness plays an important role in bacterial
attachment. Ohshima [172] suggests that bacteria can sense surface topog-
raphy and adjust the attachment mechanisms. Lorenzetti et al. [85] showed
that the bacterial attachment to TiO2-anatase coatings is mainly regulated by
surface topography, with only minor contributions from the wetting and
surface charge. These authors demonstrated that despite its high hydrophi-
licity, nanorough surface of such materials significantly reduces bacterial
adhesion because the voids in the surface do not offer full contact between
bacteria and the material. Despite being chemically identical, surfaces with
different topography differ in bacterial attachment.
5.2 Bactericidal Effect/Active Surfaces

According to the chemical composition, antibacterial surfaces with active
bactericidal effect can be differentiated into two groups: organic and inor-
ganic. Organic active surfaces generally consist of polymers and can be
classified into at least three groups by the mode of action (Fig. 14): (1)
biocidal polymers, (2) polymeric biocides, and (3) biocide-releasing poly-
mers [173]. Inorganic surfaces include metals/metal oxides or nonmetals,
such as selenium or graphene [174].
5.2.1 Organic Antibacterial Agents

5.2.1.1 Biocidal/Antibiotic Polymers
Biocidal polymers are antibacterial over the whole polymer chain. They
mainly consist of quaternary ammonium groups, shown in Fig. 15, that
generate quaternary ammonium compounds [175]. Other biocidal polymers
contain cationic biocides, such as phosphonium, tertiary sulfonium, and
guanidinium [144].
The mechanism of action of these polymers is via the positively charged
polymeric surface that is destructive for mostly negatively charged microbial
cells [175]. It is generally believed that the main antibacterial mechanism, by
Figure 13 Staphylococcus aureus adhesion after 2 and 4 h on hydrophilic TiO2 nano-

tubes, hydrophobic Ti foil treated with perfluorooctyl triethoxysilane (PTES), and super-
hydrophobic TiO2 nanotubes treated with PTES (10K). Adapted from P. Tang, W.
Zhang, Y. Wang, B. Zhang, H. Wang, C. Lin, L. Zhang, Effect of superhydrophobic surface of
titanium on Staphylococcus aureus adhesion, J. Nanomater. 2011 (2011) 2.
which such biocidal polymers directly kill microbes, is disruption of cell mem-
branes and complete loss of membrane function, with precipitation of intra-
cellular constituents [176]. Li et al. [177] prepared a hydrogel based on
dimethyldecylammonium chitosan (with high quaternization)-graft-PEG
methacrylate (DMDC-Q-g-EM) and PEG diacrylate that is able to attract
the sections of anionic microbial membrane into its internal nanopores, which
Figure 14 Mode of action of (A) biocidal polymers, (B) polymeric biocides, and
(C) biocide-releasing polymers [143].
Figure 15 Polymeric chains consisting of quaternary ammonium groups [143].
leads to microbial membrane disruption and microbial death. However,

Chindera et al. [178] proposed a new model for the antibacterial action of
polyhexamethylene biguanide, which enters microbes and causes the
so-called chromosome condensation, suggesting that disruption of cell
membrane is not the only mode of action of biocidal polymers.
Figure 16 Disruption of phospholipid bilayer of gram-positive bacteria via (A) the

polymeric spacer effect and (B) the phospholipid sponge effect [143].
According to Siedenbiedel and Tiller [143], two other possible mech-

anisms of antibacterial action of biocidal polymers exist, namely, poly-
meric spacer effect and phospholipid sponge effect, shown in Fig. 16.
The first mechanism suggests that the surface-grafted biocidal polymer
might penetrate gram-positive bacterial cell wall, reach the membrane,
and disrupt the phospholipid bilayer (Fig. 16A). The second mechanism
involves the disruption of the phospholipid bilayer by adsorption of
negatively charged phospholipids from the cell membranes of gram-
positive bacteria (Fig. 16B).
5.2.1.2 Polymeric Biocides/Antibiotics

Unlike biocidal polymers, polymeric biocides are functional polymers that
contain active specific segments only in the vicinity of their chains [179].
Attached biocide molecules can kill bacteria that adhere to the surface on
contact. Killing of bacteria in active ways can be achieved through electro-
static or biocidal interaction [144].
5.2.1.3 Antibiotic-Releasing Polymers

In biocide-releasing polymeric systems, polymers do not act bactericidal, but
serve as carriers for biocides/antibiotics. Polymers, such as poly(methyl
methacrylate), poly-L-lysine, PEG, dextrans, and cyclodextrin, can be
loaded with various inorganic or organic antibacterial active agents, such
as silver, gentamicin, tetraethylammonium bromide, and vancomycin

[144,180]. Active agents are incorporated into the polymers either by chem-
ical conjugation to premade polymers or by polymerizing an antibiotic-con-
taining molecule [181].
Suhardi et al. [180] published a study in which they prepared implants
made of ultrahigh-molecular-weight polyethylene embedded with anti-
biotic clusters. In such implants the drug is eluted over extended periods
of time and the mechanical strength of the material remains unchanged,
which do not require a two-stage surgery.
5.2.2 Inorganic Antibacterial Agents

The most studied metal nanoparticles that have shown antibacterial activity
are silver (Ag), gold (Au), and zinc (Zn). A nanoparticle can be defined as
any intentionally produced particle that has a characteristic dimension
from 1 to 100 nm and has properties that are not shared by non-nanoscale
particles with the same chemical composition [182]. The size and unique
properties of nanoparticles, such as increased specific surface area with
respect to particles of higher dimensions, make them good antibacterial
agents and drug(antibiotic)-delivery systems. Their size enables them to
penetrate through bacterial cell membrane and cause, among others, the
leakage of cellular materials [183]. Choi et al. [184] showed that 20-nm
Ag nanoparticles are able to penetrate a 40-mm E. coli biofilm within 1 h.
The high surface area of nanomaterials enables them to serve as antibacterial
delivery systems, which can improve the pharmacokinetics and therapeutic
index of a drug in comparison to the free form of a drug [185]. Such an
approach provides target drug delivery [186], sustained and controlled
release of the drug [187], and prolonged circulation lifetime of the drug
[188]. Besides, high surface area enables more bacteria-eliminating reactions
and interactions with bacteria to occur.
Despite the fact that exact mechanisms of antibacterial performance of
nanoparticles are still unclear, suggestions are as follows:
1. Accumulation and dissolution of nanoparticles inside or outside the
bacterial cell can disrupt membrane permeability and cellular respiration
[189,190].
Nanoparticles are able to attach to the bacterial cell wall through electro-
static interactions and disrupt the cell membrane [191,192]. Accumula-
tion of nanoparticles on the bacterial cell membrane can block the
respiratory system of the cell, whereas accumulation of nanoparticles

inside the cell causes the formation of aggregates that can damage the
cell membrane [193]. This can change membrane permeability and
release intracellular biomolecules into the cell surroundings [189].
2. Uptake of nanoparticles/dissolved ions in the cells can cause intracellular
ATP depletion, disruption of DNA replication, and DNA damage
[183,194].
Silver nanoparticles have a high affinity for binding with sulfur and phos-
phorus cell elements. Ag can therefore bind with the proteins in the cell
membrane, as well as with the DNA [195]. Ramamurthy et al. [196]
showed that silver nanoparticles cause DNA damage of E. coli. Also,
ZnO particles can penetrate through the cell membrane and interact
with the cell interior [197]. Mukha et al. [198] showed that the cell wall
is destroyed by the penetration of Ag nanoparticles, while further on,
also ROS are formed that inhibit ATP production and DNA replication.
3. Generation of reactive oxygen species (ROS) [199].
Zhang et al. [197] suggests that reasons for antibacterial performance of
ZnO nanoparticles, among others, lies in chemical reactions on the sur-
face and in the bacterial cell interior, with Zn2þ and reactive oxygen spe-
cies (ROS), such as H2O2, generated as a consequence of the presence of
ZnO. ZnO can block the cell membrane transport channels and damage
the membrane by abrasion. Similarly, dissolved silver cations interfere
with the thiol and amino groups of proteins, with nucleic acids, and
with cell membranes [200] and disrupt the bacterial cell membrane
permeability and cellular metabolism. In addition, depending on the
involved cell type, silver also contributes to the formation of ROS. Imani
et al. [201] demonstrated that uptake of TiO2 microbeads with nano-
structured surface leads to the generation of ROS inside the cancer cells
after illumination. A similar mechanism is responsible for the destruction
of bacterial cell wall after exposing the bacteria with attached nanopar-
ticles to light [202], as presented in Fig. 17.
4. Binding of nanoparticles with enzymes can cause their inactivation and
arrest of cellular respiration [203e205].
Nanoparticles can cause dysfunction of the respiratory electron trans-
port chain by inhibiting respiratory chain enzymes [206]. ZnO nano-
particles can inhibit the activity of a typical enzyme, b-galactosidase,
in S. aureus [207]. Similarly, nanoparticles such as SiO2, TiO2, ZnO,
and Ag mediated the inhibition of urease and DNA polymerase in
the same bacteria [208].
Figure 17 Bacterial cell damage caused by the generation of ROS in the presence of
nanoparticles after exposure to light.
6. FUTURE OUTLOOKS FOR ANTIBACTERIAL

IMPLANTABLE SURFACES
Surface coating techniques, such as ion implantation, electrochemical
anodization, ion exchange, solgel techniques, plasma spraying, and incorpo-
ration of metal ions such as silver, copper, or zinc, have been vastly studied
for the purpose of antibacterial material surface design [209,210]. With the
growing nanotechnology field, much of the recent research has been
devoted to the development of nanoparticles that are bound to the surface
of materials. Many scientific efforts have been undertaken for the synthesis of
ZnO, Cu, and Ag nanoparticles with biocidal properties, as already
described. Although the antibacterial properties of the surfaces have gained
more or less success by the mentioned procedures, the problem of cytotox-
icity and thus the inability to promote adhesion and growth of the desired
cell type (such as inhibited growth of osteoblast cells) is still not fully solved.
Furthermore, the drawbacks of many coating procedures are that they are
harmful to the environment, are expensive, and require long-lasting surface
treatment procedures, while the coating usually does not provide the desired
mechanical properties. Recently, also bioinspired approaches mimicking
the natural surfaces, which have over the years developed its fascinating
self-defense mechanisms to prevent bacterial colonization, have become
popular. Thus another very prospective approach is to alter surface

morphology and form a biomimetic surface that would prevent bacterial
adhesion, as well as limit bacterial proliferation and biofilm formation.
Although the bacterial cells are about 0.5e2 mm in size, they can interact
with the surface on the nanometer scale with their filamentous appendages,
as these appendages are about 10 nm in diameter and about 100 nm in
length. Some reports already showed that surface nanofeatures can signifi-
cantly influence bacterial adhesion. Additionally, it would be of great inter-
est to also improve the proliferation of the desired cell type, for example,
human osteoblast cells (bone-forming cells) in case of orthopedic implants
or ECs in case of vascular implants. Studies done on TiO2 nanotubes with
different diameters have already showed that nanotopography plays an
important role in bacterial adhesion [211] as well as in osteoblast adhesion
[212] and proliferation of ECs [213]. Similar results were also observed for
other nanostructured materials, as alteration in surface features on the nano-
meter scale significantly influenced bacterial adhesion of gram-positive bac-
teria [214]. The appropriately nanostructured/biomimetic surfaces would
present an intriguing way to prevent bacterial adhesion. Unfortunately, till
now, not enough systematic studies have been conducted to facilitate reli-
able information on appropriate topographic features (nanotopography/
microtopography) that would prevent bacterial adhesion. Probably, there
is also synergistic influence of other surface features such as surface chemistry
and wettability, which play vital roles in biological responses. For example,
titanium alloys tend to passivate its surface in air by forming an amorphous
nonstoichiometric oxide layer. This very thin film (2e6 nm thick) counts
for the bioinertness of the titanium implants when used in the body;
however, it is mechanically unstable and gets promptly covered by a
contamination film, even after cleaning in organic solvents and after sterili-
zation [215,216]. Massaro et al. [216] observed a large variation in the chem-
ical composition of commercial titanium implants because of contaminants
from the environment and/or fabrication processes. As surface contamina-
tion can influence the material bioperformances in vivo, rigorous quality
controls and/or preparation protocols should be applied during implant pro-
duction to deliver identical surfaces over time. Several surface strategies have
been proposed to remove this thin layer of carbon contamination [215] and
to mitigate the problem of bacterial attachment, among which different
passive surface finishing/modifications (i.e., superhydrophilic or superhy-
drophobic surfaces, nanorough surfaces, etc.) were employed [174]. One
of the interesting approaches for surface modification of biomaterials is
gaseous plasma treatment. Gaseous plasma not only easily removes contam-
inants from the surface but also modifies the topmost surface layer without
influencing its bulk attributes. Treatment of titanium by oxygen plasma has
already shown to be useful for improving cell differentiation and prolifera-
tion [212,213]. Plasma technologies are rapidly gaining importance in the
medical filed not only for surface treatment of biomaterials but also for direct
treatment of wounds and skin diseases, for teeth bleaching, and in cancer
therapy. Moreover, plasma modification is an environment-friendly process
that enables the modification of surface chemistry, morphology (on the
nanoscale), wettability, surface charge, and crystal formation of polymeric
materials [217,218]. Plasma surface modification could be used for the prep-
aration of antibacterial surfaces, not only for immobilization of antibacterial
coatings (ion sputtering, ion planting, plasma spraying, chemical vapor
deposition) but also to enable antibacterial properties on the surface of metal
implants by altering the surface nanotopography and chemistry. Such
changes should reduce the number of attached bacteria. For example, plasma
pretreatment of polymer suture significantly reduced bacterial attachment
[219]. Preliminary results already showed that Ti surfaces treated with radio-
frequency (RF) oxygen plasma at high power (1000 W) have antibacterial
influence against E. coli [220]. It was also shown that the surface of Ti alloy
(Ti-6Al-4V) treated with RF oxygen plasma at high power (1000 W)
reduces the adhesion of S. aureus [221]. In this case the surface morphology
was altered by ion bombardment and formation of a thicker oxide layer on
the surface. However, more systematic studies in this direction are needed in
order to develop new-generation surfaces with improved biological charac-
teristics not only for the prevention of bacterial adhesion and biofilm forma-
tion but also for improved proliferation of desired cell types needed for
specific applications.
7. CONCLUDING REMARKS
In this chapter the importance of antibacterial surfaces has been exam-
ined. Common features in the surfaces of biomedical implantable devices
have been reviewed. General biofilm formation mechanisms and pathogens
related to vascular implants have been summarized. Biomedical devices
including implants, usually made of metals, such as Ti and its alloys, stainless
steel, cobalt-chromium etc. and polymeric materials like PEG, often do not
offer full resistance to biofilm formation, which presents a serious problem to
public health. Millions of infections associated with biofilms are occurring

each year, and moreover, there is rapid emergence of antibiotic-resistant
bacterial strains. The infections most often involve coagulase-negative staph-
ylococci and P. aeruginosa, but infections caused by S. aureus are increasing in
frequency. Such infections are often associated with morbidity and also
mortality. There are also some suggestions that the rate of infection may
be increasing. Therefore implant-related infections present serious health
care burden, and in the recent years, many different approaches have been
proposed to lower the risk of infections from implantable devices or medical
tools. Modifications to the surface topography by nanostructuring the
surface or by using various coatings that inhibit adhesion of microorganisms
or act bactericidal are therefore developing rapidly. However, antibacterial
mechanisms of such surfaces are still poorly understood. Thus there is a
huge demand to develop new-generation implantable materials with
improved antibacterial or bacteriostatic surface properties.
ACKNOWLEDGMENTS
This work was supported by the Slovenian Ministry of Education, Science and Sport grant
“Public call for encouraging young investigators at the beginning of their career 2.0”, No.
5442-15/2016/18 and by the Slovene Research Agency grants No. P2-0232, P3-0314,
P3-0388, J2-8166, J2-8169, J2-8166, and J5-7098. The authors also acknowledge the
Ekliptik d.o.o, Teslova ulica 30, 1000 Ljubljana, Slovenia for the donation of the spinal
implant used in Fig. 2 and Rontis AG, Bahnhofstrasse 7, CH 6301 Zug, Switzerland for
the donation of the stent implant used in Fig. 3.
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The Importance of Antibacterial Surfaces in Biomedical

Chapter · January 2018

Metka Benčina Katja Lakota

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The Importance of Antibacterial

Advances in Biomembranes and Lipid Self-Assembly, Volume 28

5. Mechanisms of Antibacterial Effect 140

infections (DRIs) in implant surgery, as well as short-term biomedical

Figure 1 Schematic representation of gram-negative and gram-positive bacterial

a problem to implant-related industries and health care institutions. Usually

2. METAL IMPLANTS IN BIOMEDICAL APPLICATIONS

2.2 Orthopedic Implants

Figure 2 Example of three-dimensional printed titanium spinal cage implant. Kindly

2.3 Vascular Implants

Figure 3 Self-expandable vascular stent made of nickeletitanium alloy (Nitinol). Kindly

ﬂow through the supported vessel is therefore impaired because of thrombosis

2.4 Common Materials Used in Implantable Biomedical

2.4.2 Stainless Steels

2.4.3 CobalteChromium Alloys

CoNiCrMo (ATSM F562). “Co-based alloys usually contain 26e30 wt.%

2.4.4 Tantalum Alloys

2.4.5 Silver and Its Alloys

2.4.6 Zirconia and Its Alloys

2.4.7 Diamondlike Carbon

3. BACTERIAL ADHESION AND THE FORMATION OF

detrimental effect and obtrusive nature of bioﬁlm formation can be found in

As mentioned earlier the polymeric building blocks partake different

Figure 4 (A) Schematic representation of different extracellular matrix (ECM)

3.1 Dynamics of Bioﬁlm Formation

Brownian motion, van der Waals attraction forces). Speciﬁc interactions

conditions, and the possible antibiotic occurrence in the environment

appendages of E. coli have an important role in adhesion by providing

4. PATHOGENS BINDING TO VASCULAR IMPLANTS

4.1 Microbiology and Risk Factors

Figure 8 Microbiology of prosthetic graft infections [116].

prophylactic antistaphylococcal therapy, and other factors have resulted in a

Table 1 Risk Factors for Vascular Graft Infections

Presence of Groin Incision

4.2 Pathogenesis and Vascular Graft Infections

free-ﬂoating planktonic state [124]. The sessile form is responsible for

Prosthetic grafts can become colonized via direct intraoperative bacterial

4.3 Endothelial and Smooth Muscle Cells Covering Vascular

4.3.1 Prevention of Smooth Muscle Cell Proliferation

optimal duration of antiplatelet therapy after coronary interventions in

4.3.2 Acceleration of Re-Endothelialization

5. MECHANISMS OF ANTIBACTERIAL EFFECT

Figure 10 General mechanisms of antibacterial action [143].

5.1 Antifouling Effect/Passive Surfaces

Figure 11 Bacterial attachment on the conventional poly(ethylene glycol) (PEG)

space between conventional polymer brushes enables the penetration of

5.1.2 Superhydrophilic/Superhydrophobic Surfaces

5.1.2.1 Superhydrophilic Surfaces

ﬂuoride (PVDF)-based membrane. Cai et al. [150] prepared antibacterial

Figure 12 Multitasking antibacterial membrane matrix based on polyvinylidene

[2-(methacryloyloxy)ethyl trimethylammonium chloride] quaternary

5.1.2.2 Superhydrophobic Surfaces

Therefore the addition of TiO2 nanomaterials to polymeric coatings is often

5.1.3 Nanorough Surfaces