Materials Science and Engineering C 61 (2016) 965–978

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Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Review

Antibacterial titanium surfaces for medical implants

S. Ferraris ⁎, S. Spriano
Department of Applied Science and Technology, Institute of Materials Physics and Engineering, Politecnico di Torino, C.so Duca degli Abruzzi 24, Torino 10129, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Bacterial contamination is a critical problem in different fields (ranging from everyday life to space missions, and
Received 4 June 2015 from medicine to biosensing). Specifically, in the case of medical implants, foreign materials are preferential sites
Received in revised form 16 November 2015 for bacterial adhesion and microbial contamination, which can lead to the development of prosthetic infections.
Accepted 28 December 2015
These problems can in turn lead to the necessity of a prolonged antibiotic therapy (which can last for years) and
Available online 31 December 2015
eventually to the removal of the device, with a consequent significant increase in the hospitalization times and
Keywords:
costs, together with a stressful, painful and critical situation for the patient. Commercially pure titanium and
Antibacterial its alloys are the most commonly used materials for permanent implants in contact with bone, and the preven-
Titanium tion of infections on their surface is therefore a crucial challenge for orthopaedic and dental surgeons. The prob-
Silver lem of the bacterial contamination of medical implants is briefly described in the first part of the present review.
Copper Then the most important inorganic antibacterial agents (Ag, Cu and Zn) are described, and this is followed by a
Zinc review of the reported attempts of their introduction onto the surface of Ti-based substrates.
© 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966
2. Bacterial contamination of medical implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966
3. Antibacterial agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 967
4. Surface modifications that can be introduced to obtain antibacterial metallic surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 967
4.1. The introduction of silver onto titanium surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 968
4.1.1. Ion implantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 968
4.1.2. Electrochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
4.1.3. In situ synthesis of silver nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
4.1.4. Ion exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
4.1.5. Sol–gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
4.1.6. Sputtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970
4.1.7. Plasma spray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970
4.1.8. Alloys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
4.2. Copper introduction onto titanium surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
4.2.1. Ion implantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
4.2.2. Ti–Cu alloys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
4.2.3. Sputtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
4.2.4. Sol–gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973
4.2.5. Chemical vapour deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973
4.3. Zinc introduction onto titanium surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973
4.4. Other strategies adopted to impart antibacterial properties to titanium surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974
5. Conclusions and future outlooks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 975
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 976

⁎ Corresponding author.
E-mail address: sara.ferraris@polito.it (S. Ferraris).

http://dx.doi.org/10.1016/j.msec.2015.12.062
0928-4931/© 2016 Elsevier B.V. All rights reserved.
966 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
1. Introduction
Bacterial contamination is a serious problem that can affect numer-
ous applications, from home and everyday life, to food handling and
storage, medical implants, clinical settings, biosensors and even to
space missions [1–8].
The case of medical implants is particularly critical since an infection
can develop in a patient who has a compromised health and immune
system. Moreover, implant materials are preferential sites for bacterial
adhesion. The development of a prosthetic infection actually involves
about 1% of all total joint replacements, but this percentage dramatically
increases in the case of revision surgery [9–11]. For example in the den-
tal field, peri-implantitis has been observed for up to 14.4% of implants
in the first 5 years, but a higher incidence has been documented for lon-
ger follow-ups and after a first septic failure [12]. This complication
leads to the necessity of a prolonged antibiotic therapy (which can last
for years) and eventually to the removal of the implant. The conse-
quence of these post-operative complications, and of the lack of effec-
tive treatments, is a significant increase in hospitalization times and
costs, together with a stressful, painful and critical situation for the pa-
tient. In addition it should be recalled that a significant increase in anti-
biotic resistant bacterial strains has been observed, and this has led to
the necessity of innovative prevention and treatment strategies [11].
A brief description of the bacterial infection of medical implants is
reported in the first part of the present paper. Then a review of the
scientific papers concerning the surface modification of titanium
substrates intended for medical implants is reported with a specific
focus on the introduction of inorganic antibacterial agents (Ag, Cu,
Zn). To the best of the authors' knowledge, these specific topics have
not been reviewed in the recent literature. In fact, the most recent re-
views deal with antibiotic biomaterials for dental applications [12]
and antibacterial coatings for titanium implants [13,14]. However, in re-
cent years, research has been focused on the investigation of alternative
antibacterial agents to antibiotics, as well as on surface functionalization
and modification, instead of coatings. This is due to the numerous draw-
backs related to the systemic use of antibiotics (development of antibi-
otic resistant bacterial strains) as well as to delamination and
bioresorption related to the coatings on implants [11,15].
The aim of the present review is to collect and compare the recent
researches on surface modification of titanium surfaces in order to con-
fer antibacterial properties by the introduction of inorganic antibacterial
agents, with a specific focus on metal ions (Ag, Cu, Zn).
2. Bacterial contamination of medical implants
Bacterial contamination of solid synthetic surfaces, which is a phe-
nomenon that has been widely documented, depends on the specific
characteristics of both the involved microbes and of the materials.
As far as bacteria are concerned, their filamentous proteinaceous ap-
pendages (e.g. Pili, flagella) can support adhesion to solid surfaces;
these appendages are, on a nanoscale, about 10 nm in diameter and
100 nm in length. Their dimension is comparable with some features
typical of innovative surfaces (nano-roughness and pattern produced
on synthetic surfaces in order to promote cellular adhesion and differ-
entiation) intended for medical implants and must be taken in consider-
ation in the design of that substrates in order to avoid bacterial
adhesion. Bacteria produce exopolymeric substances (mainly polysac-
charides and other macromolecules) which contribute to biofilm forma-
tion [16] and consequent permanent contamination of implanted
surfaces. A biofilm can be defined as a community of different micro-
organisms that are protected in a self-produced polysaccharidic matrix
(the EPS described above), and which are irreversibly attached to a sur-
face [17,18]. A biofilm protects organized bacteria from both fluid shear
stress and the action of systemic pharmacological therapies. Moreover,
it acts as a storage facility for nutrients and substances [17]. Moreover
bacteria live in the polymeric protective structure of the biofilm, as an
organized and co-operative community, as in a city, protecting them-
selves from external adversity [17,19].
Biofilm formation and development can be divided into a sequence
of events [17,18,19,20]:
1. A first reversible attachment of bacteria to the solid surface, mainly
due to cellular Brownian motion and microbe-surface interactions:
electrostatic, gravitational or Van der Waals forces, and the hydro-
phobic degree of the bacteria and of the matching biomaterial. In par-
ticular the roughness and surface topography have a great influence
at this stage.
2. Bacteria accumulation and irreversible attachment, which are medi-
ated by specific molecular and cellular interactions (adhesion pro-
teins, proteinaceous appendages and EPS production)
3. Biofilm maturation, the formation of bacterial microcolonies and the
entrapment of planktonic cells in the EPS
4. The detachment of some bacteria for the colonization of new surfaces
As an example dental plaque is a biofilm typical of natural tooth and
dental implants [12]. When it colonizes the dental implant, generally
through the transmucosal abutment, and lead to peri-implantitis devel-
opment, it constitutes one of the major causes of implant failure [12]. In
particular a dynamic biofilm composition has been reported for dental
implant surfaces [21]: the first bacteria that approach the surface
are generally Streptococci and Actinomyces species, whose adhesion is
affected to a great extent by the surface characteristics. Later, during bio-
film maturation, other bacterial strains, such as Fusobacterium nucleatum
and Prevotella intermedia (firstly) and Porphyromonas gingivalis,
Capnocytophaga gingivalis and Actinomyces israelii (later), appear, but
they are influenced less by material surface properties. Furthermore it
has been observed that implant surface properties (roughness, hydrophi-
licity, chemical composition and even sterilization method) affect the
early colonization of the material but not biofilm maturation directly
[21]. On the other hand the type and amount of early surface contamina-
tion significantly influence the consequent plaque development on im-
plant surface [21].
Specifically the main pathogens involved in orthopaedic and
cardiovascular prosthetic infection are Staphylococcus aureus and Staph-
ylococcus epidermidis; the former is the main colonizer of metallic im-
plants, while the latter is the main colonizer of polymeric surfaces [18,
6].
Since bacteria are 0.5–2 μm-sized cells, they can be modelled as
colloidal particles that interact with solid surfaces [16]. However, this
simplification seems to be too approximate. The main problems of this
simplification are related to the heterogeneous structure of the mi-
crobes and to their above cited complex features, which mediate bacte-
ria–surface interactions (filamentous proteinaceous appendages and
produced exopolymeric substances). At the same time, the surface char-
acteristics of solid surfaces exposed to a biological environment change,
due to protein absorption and can favour bacterial adhesion if compared
to bare inorganic ones.
As far as the features of the surfaces are concerned, their chemical
composition, charge, wettability and roughness are the main factors
that can affect the interaction with bacteria [20,21]. For example
roughness, in the tenth of micron range, has been reported to dramati-
cally increase bacterial adhesion [22,23], while lower Ra values than
0.2 μm, as well as surface nano-textures, do not improve biofilm forma-
tion [24,25,21]. Moreover it has been reported that rough and/or
hydrophobic surfaces lead to higher bacterial colonization [21]. As an
example calcium phosphate coatings and roughened dental implants
have shown increased bacterial contamination and peri-implantitis
development [12].
Moreover, protein absorption from biological fluid leads to changes
in the original surface characteristics of the biomaterials, and conse-
quently influences bacterial adhesion. For example it has been observed
that a greater surface absorption of plasma proteins enhances bacterial
 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
adhesion [26]. Furthermore absorption of fibronectin can favour micro-
bial attachment, while albumin absorption can inhibit it [20,6].
In particular increased bacterial adhesion has been documented for
inert metallic surfaces (e.g. steel), compared to more reactive ones
(e.g. titanium), this phenomenon can be explained by the lower tissue
integration of inert substrates [6]. As widely reported in the literature,
after implantation, a sort of “race for the surface” starts between cells
and bacteria: the consequence is a lower bacterial contamination of sur-
faces that are easily colonized by cells [6,27].
A slightly higher bacterial contamination (Escherichia coli) has been
observed for Ti6Al4V, compared to the T6Al7Nb alloy [28]. Experimental
data comparing bacterial contamination cp Ti and Ti 6Al4V alloy are not
extensively reported in literature, a study related to surface modifica-
tion of grade 2 and grade 5 [29] Ti did not evidence important difference
between the bacterial contamination of the two substrates.
Finally a certain ability to reduce bacterial colonization has been re-
ported for several pure metals, and a rank of “antibacterial” metal has
been proposed: gold, titanium, cobalt, vanadium, aluminium, chromium
and iron [21], even though the effective antibacterial behaviour of these
surfaces has not yet been univocally demonstrated. Moreover, a distinc-
tion should be made between the anti-adhesive, bacteriostatic, bacteri-
cidal and cytotoxic effects of these elements.
3. Antibacterial agents
Bacterial infections are usually treated and prevented by the local
and systemic application of antibiotics. The main problems related to
antibiotic therapies are the increasing development of antibiotic resis-
tant bacterial strains (e.g. Meticilin Resistant Staphylococcus aureus —
MRSA) and their narrow spectrum of activity, which results in the diffi-
culty of treating infections caused by several bacteria. The development
of antibiotic resistant bacterial strains has recently been defined as
“global threat” to human health that needs to be addressed [30].
In this context, an increasing interest in alternative antibacterial
agents has been witnessed. In particular inorganic antibacterial agents
can represent a challenging strategy to fight bacterial infections, with-
out the use of antibiotics.
It must be evidenced that most of the reported studies were con-
ducted using planktonic cells and they do not investigate biofilm forma-
tion and growth: this is a not negligible limit when clinical implications
have to be deducted from lab data. This is in agreement with the usual
procedure of preliminary works: a general standard procedure for the
evaluation of antibacterial activity of biomaterials has not been defined
yet.
Metal ions, salts and nanoparticles are part of one of the most impor-
tant classes of inorganic antibacterial agents. Among these agents, silver
has long been known and used as an antibacterial agent [31]. Moreover
an effective antibacterial effect has also been pointed out for other metal
ions, such as copper and zinc.
The mechanisms of action of metals against bacteria have been re-
cently reviewed [32].
For example silver ions can effectively bind to thiol groups, a charac-
teristic of many proteins, and play a structural and functional role in the
bacterial cells, thus causing the death of microorganism. Ag ions can
alter the function and structure of proteins in the bacterial cell wall,
and lead to its consequent rupture. Moreover, they can bind and alter
several enzymes, which are crucial for cellular respiration and metabo-
lism. Finally, they can interfere with DNA through cell division and rep-
lication [33]. The multiple actions of silver ions explain the reduced
resistance development, compared to antibiotics, and also their broad
spectrum of activity, which includes many bacteria, as well as fungi
and yeasts [31,33]. An analogous mechanism of action has been de-
scribed for other metal ions (e.g. Cu, Zn) [34,35].
Increasing interest in silver nanoparticles, as antibacterial agents,
has been shown [36,37,38]. Ag-nanoparticles have a large surface area,
which allows a high silver ion release. Moreover, they can directly
967
bind to the bacterial cell membrane and are also able to penetrate the
cell. The antibacterial activity of silver nanoparticles mainly depends
on their dimension and shape [31,36]. On the other hand the toxicity
of these nanoparticles is currently under investigation; it should be
underlined that few systematic studies have focused on Ag-
nanoparticles, despite their widespread application [37,38].
The antibacterial activity of silver ions [39] and nanoparticles [40,
41], and their mechanism of action, have been demonstrated in vitro
on gram-positive (S. aureus) and gram-negative (E. coli) strains. Numer-
ous studies report values of the Minimum Inhibitory concentration
(MIC) of silver ions against bacteria (including S. aureus and E. coli)
and few ones consider also copper and zinc ones [42,43,44,45]. A signif-
icant variability in the values can be registered among the various stud-
ies; however a difference of two orders of magnitude in the MIC of Ag+
compared to the Cu2+ and Zn2+ ones is generally reported. A compari-
son between MIC values of Ag+, Cu2+ and Zn2+ and the corresponding
Lethal Doses 50 (LD50) values (for L929 mouse fibroblasts cells) [46] is
summarized in Table 1.
Moreover the antibacterial activity of metallic or oxide nanoparticles
(e.g. Ag, Ag/Au coated iron oxides, ZnO, CuO, TiO2, Al2O3, and Ga)
against various bacterial strains has been demonstrated [47,48].
Finally Minimum Inhibitory Concentrations of metallic and oxide
nanoparticles against different bacterial strains, including S. aureus
and E. coli have been reported in the literature [43,49,50]. MIC values
comprised in the range 3–180 μg/ml have been reported for Ag nano-
particles with 3–95 nm diameters [43,49,50]. As far as copper nanopar-
ticles (9–95 nm) are concerned values in the range 20–2500 μg/ml have
been reported [49,50] for the MIC. Slightly lower values can be found for
copper oxides nanoparticles (22–95 nm), in particular 100–5000 μg/ml
for CuO nanoparticles and 500–5000 μg/ml for Cu2O ones [50]. Finally
MIC in the range 2055–5000 μg/ml have been reported for ZnO nano-
particles (22–95 nm) [50].
These data underline the significantly higher antibacterial action of
silver, compared to copper and zinc, which can support the higher num-
ber of publication concerning silver enriched surfaces compared to Cu
and Zn enriched ones, as discussed in the following.
4. Surface modifications that can be introduced to obtain antibacte-
rial metallic surfaces
Various techniques have been explored in the scientific literature
and in industrial and clinical applications in order to obtain titanium
surfaces enriched with antibacterial agents. In particular three main cat-
egories of surface modifications can be considered for inorganic antibac-
terial agents:
1) surface enrichment of the inorganic antibacterial agents without the
introduction of a layer
2) growth of a surface oxide layer (TiO2) and its doping with the anti-
bacterial agent (in the same passage or in a second step)
3) deposition of a coating (TiO2 or foreign one, e.g. hydroxyapatite —
HAp) and its doping with the antibacterial agent (in the same pas-
sage or in a second step)
These strategies and the main techniques used for their application
are briefly schematized in Fig. 1. The literature references for the report-
ed strategies are cited and compared in the following paragraphs.
Table 1
Minimum Inhibitory Concentrations (MIC), against various bacterial strains (including
S. aureus and E. coli) and Lethal Doses 50 (LD50) for L929 mouse fibroblast cells.
MIC [μg/ml] LD50 [mmol/l]
Ag+ 0.03–8 [42,43,45] 3.5 ∗ 10−3 [46]
Cu2+ 256–448 [44,45] 2.3 ∗ 10−1 [46]
Zn2+ 768 [45] 3.6 ∗ 10−3 [46]
968 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
Fig. 1. Strategies and main techniques for the introduction of inorganic antibacterial agents onto titanium surfaces.
4.1. The introduction of silver onto titanium surfaces
Extensive literature is available on this topic, but the comparison be-
tween the various research work results quite difficult because of the
different test methodologies employed (release media and times, bacte-
rial strains, cellular type and culture conditions). Depending on the
employed technique, silver has been introduced as both ions and as
metal nanoparticles. All the reported materials have resulted in antibac-
terial behaviour, but the mechanisms and times of action are different,
according to whether ions and/or nanoparticles are involved: a system-
atic comparison of the various actions of ions and nanoparticles will be
of particular interest in future works, since it has not yet been fully in-
vestigated. Biocompatibility, with respect to osteoblasts, depends on
the amount of silver released. Moreover it should be considered that
dental and orthopaedic applications suffer from similar problems
concerning bone-implant contact, but they differ significantly
concerning the required time of action (much longer than 1 month,
for dental implants), as well as soft tissue-implant interaction (the
gum is in contact with the collar of dental implants).
Either a higher hydrophobicity or hydrophilicity has been obtained,
after silver addition, because the various modifications proposed pro-
duced surfaces with different roughness and surface composition of
the final material. Roughness magnitude and topography, as well as sur-
face chemical composition are the main factors affecting surface
wettability.
4.1.1. Ion implantation
Ion implantation has been employed for the introduction of silver
ions onto the surface of steel, c.p. titanium and Ti6Al7Nb and Ti6Al4V al-
loys [51,52]. Depending on the ion dose (up to 2 ∗ 1017 ions/cm2) an in-
creasing silver content can be introduced on the metallic surfaces [51,
52]. Antibacterial metals have been obtained against S. aureus [51,52]
for ion implantation in the range 1 ∗ 1016 ions/cm2–2 ∗ 1017 ions/cm2
[51,52]. Biocompatibility for osteoblasts cells has been verified for Ag
implanted (b1 ∗ 1017 ions/cm2) Ti6Al4V surfaces [52]. Finally an
improvement/maintenance in wear/corrosion resistance has been
observed for Ag ion implanted metals [51].
The Plasma Immersion Ion Implantation (PIII) process induces the
precipitation of silver nanoparticles (5–8 nm in diameter) onto titanium
substrates [53,54]. A change in the zeta potential, through a less
negative value (at about −40 mV at pH 7) than the control (at about
−100 mV at pH 7), as well as an increase in hydrophobicity and surface
nano-hardness have been observed after these treatments [53,54]. Al-
most no Ag release (b10 ppb in 60 days) has been observed for these
surfaces, but significant antibacterial behaviour (95–100% reduction)
against E. coli and S. aureus [53,54] and good tolerability, by the osteo-
blasts, have been documented [53]. Considering the negligible Ag+ re-
lease observed for these surfaces electrostatic interactions and
microgalvanic phenomena have been suggested in order to explain
the antibacterial action, however, the mechanism of this action still re-
quires further investigation.
Titanium oxide layers, doped with silver, have also been obtained by
means of ion beam assisted deposition (IBAD), performed in an oxygen
atmosphere, on titanium substrates, polished [55] or plasma spray coat-
ed [56], using silver and titanium targets [55]. Metallic silver has been
introduced into the oxide layer (constituted by TiO, Ti2O3 and TiO2)
during the IBAD process. An increase in surface tension and energy
has been recorded for higher Ag concentrations. The realization of dif-
ferent sizes of Ag nanoparticles (4–25 nm), by means of the IBAD tech-
nique [56], has pointed out better antibacterial behaviour against E. coli
and S. aureus for particles larger than 5 nm, together with a good toler-
ability by the osteoblasts. Considering the negligible silver release ob-
served for these surfaces (b10 ppb Ag cm2 after 70 days in water,
analogous to the one cited before for PIII process [53,54]) the antibacte-
rial activity (in the dark) has been attributed to an electron storage
capability of silver nanoparticles [56].
Moreover ion beam assisted deposition has been employed to obtain
Ag-doped hydroxyapatite, which is used as a coating on titanium sub-
strates, with increasing crystallinity from the top (surface) to the bot-
tom (substrate interface) of the coating [57]. Silver nanoparticles of
about 5–30 nm were dispersed through the coating. The less crystalline
portion of the coating can be rapidly dissolved in solution, while the
more crystalline part leads to stability of the surface layer and acts like
a silver reservoir. Accordingly, the release of silver into water it initially
resulted in a high value (from 0.4 to 0.8 ppm in 24 h and from 0.78 to
1.70 in 172 h, depending on the silver content introduced) and it then
it decreased (for up to 15 days). The introduction of 3 wt.% of silver
allowed a surface to be obtained that was antibacterial against
S. aureus and biocompatible for osteoblast cells.
 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
4.1.2. Electrochemical techniques
A titanium oxide enriched in silver has been obtained by means of
plasma electrolytic oxidation (PEO) on Ti6Al7Nb, in an electrolyte
based on calcium acetate/calcium glycerophosphate, containing com-
mercial Ag nanoparticles (7–25 nm) [58,59]. Silver nanoparticles were
introduced onto the surface of the oxide, into its pore walls and embed-
ded throughout the entire coating thickness. An increase in surface
roughness and wettability was detected, after the surface modification
process [59]. The silver enriched surface showed the ability to complete-
ly kill methicillin-resistant S. aureus after a culture of 24 h [59].
Moreover titanium oxide layers, enriched in silver, have been obtained
on Ti6Al4V alloy by means of anodic spark deposition, in an aqueous elec-
trolyte containing silver nanoparticles [60]. The silver was detected as
small particles (mainly metallic), distributed within a 100 nm thick sur-
face layer. The Ag ion release from the treated surface, in PBS, presented
a maximum at 24 h (1.2 ng ∗ g−1 ∗ mm−2) and then remained constant
for 15 days (0.4 ng ∗ g−1 ∗ mm−2). The silver containing surfaces present-
ed effective antibacterial behaviour (complete killing after 12 h incuba-
tion, superior than pure silver) against gentamicin-resistant S. epidermidis.
Plasma Electrolytic Oxidation (PEO), in an electrolyte containing Ca,
P and Ag-nanoparticles, has also been performed on a plasma spray (PS)
titanium coating on a Ti6Al4V alloy [61] in order to obtain a surface layer
with interconnected micro- and nano-porosity and antibacterial activi-
ty. A TiO2 microporous (0.07–5 μm pore) layer, rich in Ca, P and silver
nanoparticles, was obtained. The coating follows the starting surface
macro-roughness of the PS coating. The Ca/P ratio was similar to that
of the hydroxyapatite one and the Ag nanoparticles were dispersed
both on the surface and in the pores of the coating.
Composite nanoparticles of TiO2 (23 nm) and Ag (4 nm) have been
obtained by means of nucleophilic reaction, and were then deposited
on titanium substrates by electrophoresis in an aqueous solution [62].
The optimization of the deposition parameters allowed homogeneous
surfaces to be obtained, with a limited number of cracks. The mechani-
cal properties of the surfaces were acceptable and comparable with
those reported in literature (HV ≈ 210 kg/mm2, E ≈ 29 GPa and
Kic ≈ 0.86 MPA m1/2). The surfaces presenting TiO2 and TiO2-Ag nano-
particles were able to induce apatite precipitation, after 1 week of
soaking in a simulated body fluid. However, a reduction in bioactivity
was observed when the silver content was increased. A decrease in
the availability of the Ti–OH groups, because of the formation of Ti–O–
Ag bonds, has been suggested as an explanation of this phenomenon.
Finally a Plasma Electrolytic Process (a combination of plasma elec-
trolytic oxidation and electrophoretic deposition) has been applied to
c. p Ti substrates, in order to obtain composite coatings (about 83 μm
thick), and made of titanium oxide and silver enriched hydroxyapatite
(Ag-HAp) [63]. The silver enriched hydroxyapatite was prepared by
adding 2.5 wt.% of silver to a HAp microwave synthesis, in order to ob-
tain antibacterial behaviour, without altering the bioactivity and bio-
compatibility of the HAp. Ag-HAp particles (60–70 nm) were added to
the electrolyte for the Plasma Electrolytic Process. The obtained coatings
improved the corrosion resistance of the surface, both at a physiological
(7.4) and an acidic pH (4.5, simulation of osteoclasts resorption). The
coatings were also bioactive and presented antibacterial behaviour
(inhibition zone) against E. coli.
4.1.3. In situ synthesis of silver nanoparticles
Silver nanoparticles have been obtained on different TiO2 substrates,
through a UV reduction in AgNO3 solutions [64,65,66,67]. The amount of
deposited nanoparticles depends on the concentration of the silver ni-
trate solution [65,66], while their size can be modulated by varying
the irradiation time [66].
For example the TiO2 nanotubes (130 nm diameter and 7 μm
length), enriched with silver nanoparticles (10–20 nm), showed a sig-
nificant Ag release in PBS: it is maximum in the first days (0.15–
0.45 ppm after 24 h depending on the silver content) and then slowed
down over 14 days (a half bacterial reduction after 14 days) [65]. The
969
rapid ion release and consequent Ag accumulation in the culture medi-
um induced a limited cytotoxic effect on the osteoblasts. The realization
of smaller nanotubes, the modulation of the oxidation reaction in water,
the deposition of a hydroxyapatite, polymeric or even “customized”
layer (able to favour ion release only in the presence of bacterial toxins)
have been proposed as possible solutions for the modulation of silver re-
lease and the reduction of the cytotoxic effect [65].
The presence of a nanotube layer (with or without silver doping) has
induced a significant improvement in substrate wettability (the contact
angle of titanium surfaces was 42.9° for water and 23.2° for
diiodomethane and it drops to 0° after nanotubes deposition) [67].
The antibacterial activity of these typologies of coatings has been
demonstrated against S. aureus [65,66,67] and E. coli [67], and has
been maintained for up to 30 days for TiO2 nanotubes containing Ag
nanoparticles at the highest concentration [65]. An increase in the anti-
bacterial activity with the nanotube diameter has been observed, with
an optimum value for 100 nm [67].
Furthermore silver nanoparticles, stabilized by citrate (6 nm obtain-
ed by means of a NaBH4 reduction of AgNO3), have been deposited,
through absorption, onto a polished titanium substrate [68]. The parti-
cles aggregated in multi-layer islands (100–300 nm). The surface cover-
age depended on the absorption time. A better adhesion to the substrate
was observed for the aggregated particles than for the isolated ones. Ef-
fective antibacterial behaviour (both reduction of bacterial adhesion
and bactericidal effect) against Pseudomonas aeruginosa was
demonstrated.
Finally alginate/chitosan polyelectrolyte multilayers, modified by
dopamine, have been obtained on commercially pure titanium, using
the layer-by-layer technique. These layers were enriched with silver
nanoparticles, using the reduction ability of dopamine on AgNO3 [69].
The thus obtained coatings were biocompatible for fibroblasts and anti-
bacterial against E. coli and S. aureus (inhibition halo of 2.85 ± 0.24 mm
and 2.13 ± 0.15 respectively).
4.1.4. Ion exchange
Silver has been introduced onto titanium surfaces by means of an
ion-exchange process, after the formation of a sodium titanate layer in
a NaOH solution, through the exchange between Na+ and Ag+ ions
[70,71]. Silver acetate [70] and nitrate [71] were considered as sources
for the Ag+ ions. The surface introduction of silver, both in ionic and me-
tallic (nanoparticles) form, has also been recorded [70]. A consistent Ag
release was observed in a physiological saline solution (300–510 ppb),
in a PBS (320–440 ppb) and in a foetal bovine serum (64,000–
82,000 ppb), after 24 h. The significantly higher value in the serum
can be attributed to the high affinity between the Ag ions and the pro-
teins [70]. A faster silver release from the titanate, due to the presence
of silver ions, and a slower one from the metallic nanoparticles, have
been suggested for these surfaces [70]. Effective antibacterial behaviour,
against S. aureus [71] and Meticillin Resistant Staphylococcus aureus
(MRSA) [70], was observed. Bioactive behaviour (apatite forming ability
in simulated body fluid) was observed for all the treated surfaces, before
and after Ag-introduction [71].
Moreover the introduction of silver ions onto biocompatible [72] and
bioactive [73] glass/glass-ceramic coatings, deposited on titanium sub-
strates, has been considered. The possibility of tailoring the silver con-
tent, and consequently the antibacterial activity, by varying the ion
exchange condition, has been verified. Silver ion release in SBF up to
4 μg/mm2 has been obtained. Ag-doped coatings are antibacterial
against stock and clinically isolated S. aureus strains (2–5 mm inhibition
halos and 95–100% reduction of bacterial adhesion), bioactive (promote
hydroxyapatite precipitation in simulated body fluid) and biocompati-
ble (for osteoblasts cells) [72,73].
4.1.5. Sol–gel
The sol–gel technique has been applied to prepare TiO2–Ag compos-
ite nanoparticles [74], powders [75,76] or coatings; spin-coating has
970 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
been used on silicon [77] and titanium [78] substrates, while dip-
coating has been used on glass [79]. In most of the works [77,74,79,
75], titanium isopropoxide and silver nitrate were used as precursors
of TiO2 and Ag, respectively. Tetrabutyl titanate [78] and titanium sul-
phate [76] have also been considered as alternative sources for TiO2 syn-
thesis. In the sol–gel process, silver is introduced in the ionic form
(AgNO3) and converted to metallic nanoparticles during the calcination
process; Ag ions are larger than Ti ones, and, as a result, they cannot
enter the Ti crystal lattice and are reduced to metallic nanoparticles in
the thermal treatment [77,79]. They are also effective in inhibiting
TiO2 grain growth [79]. Some authors have witnessed the formation of
silver oxides after air annealing [74] or UV irradiation [77]. A noticeable
effect of silver on the crystallization of titanium oxide has also been
observed, and a stabilization of rutile, after air annealing, and of anatase,
after N2 annealing, has been observed [74]. Ion release tests in water
have shown that the main contribution occurs in the first hour
(0.4 ppm) and continues for up to 1 day (0.26 ppm), with an almost
complete release after 7 days (0.005 ppm) [77,76]. The antibacterial
activity of sol–gel TiO2–Ag composites was verified against E. coli (2.5–
7 mm inhibition halo, 100% viability reduction) [77,78]. An improve-
ment in TiO2 photocatalytic activity, due to the addition of silver, was
also evidenced [77,64,79].
The sol–gel technique has also been employed for the coating of
macroporous 3D titanium scaffolds with Ag-doped hydroxyapatite
[80]. Titanium scaffolds were acid etched and NaOH treated. A 30 μm
thick hydroxyapatite coating was obtained starting from a slurry con-
taining calcium nitrate tetrahydrate, phosphoiric pentoxide, ethyl alco-
hol and silver nitrate. The antibacterial effect against E. coli and
albusStaphylococcus increased with the silver content in the apatite
(N95% for 0.8 and 1.6 wt.%), while an opposite trend was registered
for biocompatibility, with respect to osteoblast cells. The introduction
of 0.8 wt.% of silver into the hydroxyapatite resulted in the best
compromise.
Finally in vivo tests, on a rabbit model, in infected surgical site by
local introduction of bacteria (S. aureus), have been performed for tita-
nium bone screws coated, or not, with a sol–gel coating of TiO2–Ag-
nanoparticles. It was observed that the coating resists the implantation
friction and appears unaltered after the implant in bone. The results re-
vealed the effective ability of coated screws to reduce biofilm formation
(no biofilm was observed on coated screws in contrast to uncoated
ones), with no accumulation of silver in the tissues (cornea, kidneys,
liver or brain analysed 28 days after implantation) [81].
4.1.6. Sputtering
The sputtering technique has been employed to deposit a TiO2 coat-
ing, containing silver nanoparticles, using Ag–TiO2 composite targets
[82,83], or Ag and Ti targets, and a reactive atmosphere [84,85]. Silver
was present in metallic form [82,83] in the sputtered layers. The intro-
duction of silver affected the crystalline structure of the film (some ru-
tile peaks were observed for 2.5 and 20% Ag). The hydrophylicity and
photocatalytic activity of the TiO2 films were significantly enhanced by
the introduction of silver (up to 5 vol.%), because of the better separa-
tion between the electrons and holes, the increase in oxygen anion rad-
icals (O−2 ) and the reactive surface centres represented by Ti
3+
[82,83].
Silver release tests in artificial saliva and in PBS evidenced an Ag amount
higher than the threshold value necessary to obtain antibacterial behav-
iour (0.1 ppb); with a maximum being reached at 24 h [84,85]. An effec-
tive antibacterial action against S. aureus was verified, together with
biocompatibility for fibroblast cells [84,85].
The deposition of TaN–Ag coatings by means of sputtering has also
been considered on titanium substrates, using Ta and Ag targets and
an Ar or N2 flux [86]. Silver (up to 21.4 at.%) has been introduced in
the TaN coating in the form of well distributed Ag nanoparticles with di-
ameter comprised between 15 and 53 nm. An effective antibacterial ac-
tion has been demonstrated against S. aureus (N 90% reduction) together
with biocompatibility for fibroblasts cells [86]. Finally, Ag nanorods
have been obtained on a Ti layer, sputtered onto silicon, by means of
the sputtering deposition of an Ag film, a thermal treatment (700 °C)
and the application of a uniform electric field [87]. The capping of Ag
structures within a TiO2 layer was performed by means of sol–gel dip
coating in [87], in order to modulate the silver release; the largest quan-
tity of Ag ions was detected in the solution in the first 8 days (at about
550 nM/ml for both Ag nanorods and capped Ag nanorods) and the
quantity then reached a plateau only for the capped structures. Effective
antibacterial activity of these coatings was verified against E. coli (com-
plete killing of bacteria, 105 CFU/ml, in 1.75–2 h).
Moreover the sputtering technique has been employed for the
deposition of thin films (at about 600 nm) of hydroxyapatite (HAp)
and Ag-doped hydroxyapatite (Ag-Hap, 0.5 and 1.5 wt.% Ag) on titani-
um substrates [88], using pure HAp sintered tiles and silver strips as
targets. An optimal adhesion was obtained on plasma etched titanium
(0% damage by tape test), while a certain dissolution of the coating
was observed in PBS (Ag reduction to 0.15–0.25 wt.% after 4 weeks in
PBS). The Ag containing coatings reduced the adhesion of both
S. epidermidis (Gram-positive) and P. aeruginosa (Gram-negative) with
a greater effect on the latter, but the effect decreased after 48 h, proba-
bly due to partial coating delamination in the culture medium.
Finally ZrO2 and ZrO2 doped with silver or copper have been depos-
ited on titanium substrates, by means of twin-gun reactive magnetron
sputtering [89]. Cu and Ag entered the coating in both an oxide and me-
tallic form; when silver was deposited, silver nanoparticles appeared on
the surface. The sputtered zirconia was in monoclinic form and it main-
tained this structure after copper addition, while it became tetragonal
after silver addition. The presence of silver nanoparticles increased sur-
face hydrophobicity (at about 95° compared to the 80° of ZrO2): this
phenomenon was related to the antibacterial activity of the surface.
Both the Ag and Cu doped ZrO2 coatings demonstrated antibacterial ac-
tivity against S. aureus and Actinobacillus actinomycetemcomitans, with a
more pronounced effect for silver (at about 70% bacterial reduction for
Cu doped ZrO2 and 97% reduction for Ag doped one on both strains). A
moderate reduction of bacterial adhesion has been evidenced also for
ZrO2 coatings compared to bare titanium.
4.1.7. Plasma spray
Ag–TiO2 coatings have been obtained on titanium substrates by
means of air plasma spraying [90]. Metallic silver and silver oxide
(due to the oxidation developed in the air deposition process) were ho-
mogeneously distributed through the surface layer. The introduction of
silver did not alter the wettability or the biocompatibility of the TiO2
coating. The Ag release into water was at a maximum after 24 h (at
about 0.11 μg/(ml∗cm2)) and then slowly continued for 28 days. An ef-
fective antibacterial action against E. coli (100% reduction after 24 h)
was observed for these coatings.
Furthermore plasma spray titanium coatings, sprayed onto Ti6Al4V
substrates, have been treated in NaOH and in a calcification solution
containing Ca, PO4, Na and Ag, in order to obtain a silver enriched hy-
droxyapatite layer on the surface (silver content from 1.50 to
5.15 wt.%) [91]. The Ag was released in a simulated body fluid and
showed a maximum at 24 h (at about 0.4 ppm for the highest silver con-
centration). The antibacterial activity of the coatings was verified
against E. coli, P. aeruginosa and S. aureus (bacterial reduction higher
than 95% was registered for all coatings against the tested bacteria, ex-
cept for the lowest Ag concentration against S. aureus that induced at
about 63% reduction).
Finally different coatings (Hydroxyapatite (HAp), Ag2O, SrO or
Ag2O–SrO doped hydroxyapatite) have been deposited on commercial-
ly pure titanium by means of the plasma spray technique [92] and com-
pared; a good adhesion was obtained (16 MPa required for HAp). A
similar Ag release rate in PBS was observed for the Ag– and Ag–Sr-
doped HAp coatings after 7 days of soaking (at about 0.8 ppm at 24 h
and 2 ppm after 7 days), indicating that Sr does not affect silver release.
While some signs of cytotoxicity were noted for Ag-HAp, good
 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
biocompatibity for osteoblasts was instead observed for Ag–Sr-HAp. It
seems that Sr can reduce and balance the toxic effect of Ag on cells.
Both coatings were antibacterial against P. aeruginosa (almost complete
reduction after 24 h).
4.1.8. Alloys
Ti–Ag (Ti–1Ag, Ti–2Ag and Ti–4Ag) [93] and TiNiAg (Ti 49.3, Ni 47.3
and Ag 1.4 at.%) [94] alloys have been developed in order to obtain bulk
antibacterial metals for biomedical applications.
Sand-blasting, by means of large grits, and acid etching of the Ti–Ag
alloys resulted in a micro-rough surface, thus much more Ag was ex-
posed, and the alloy was able to release Ag ions in PBS (at about
1000 ppb after 24 h and 4000 ppb in 7 days for the highest silver con-
centration) and the amount of released silver was consistent with the
Ag content in the alloy. The antibacterial behaviour of the thus treated
alloy was verified against Streptococcus aureus (N 95% reduction for all
the tested concentrations), while biocompatibility was verified on the
osteoblast cells of mice for all the Ag concentrations [93].
Looking at the reported data, the majority of the modified surfaces
present an antibacterial behaviour due to a silver release higher than
the MIC of Ag+ ions for the specified bacterial strain. However, an anti-
bacterial action has been evidenced for AgNPs obtained on Ti substrate
by PIII and IBAD processes, and has been attributed to electrostatic in-
teractions, microgalvanic phenomena and electron storage capability
of AgNPs [53,54,56]. Considering that silver ions release is usually max-
imum in the first day and no author reported ion release longer than
1 month, the opportunity to obtain silver enriched surfaces able to dem-
onstrate an antibacterial action without ion release seems interesting
and needs further investigations.
Another key point, in the development of innovative antibacterial
metallic surfaces for bone contact application, is to guarantee good
bone integration. As reported, there is a sort of “race for the surface”
upon implantation [6,27] between cells and bacteria. Fast and physio-
logical bone integration can consequently reduce the risk of bacterial
colonization. Taking into account these considerations, a surface able
to induce optimal bone integration and, at the same time, exert an anti-
bacterial action is extremely promising for clinical applications. Among
the reported research articles, a bioactive behaviour (material ability to
bond to bone by the formation of an apatite layer) can only be evi-
denced for these materials: Ag-doped hydroxyapatite [57,63,80,88,91,
92], Ag-doped bioactive glass coatings [72,73], Ag enriched NaOH treat-
ed Titanium [70,71] and few TiO2 layers [61,62]. Moreover, in [62] a re-
duction in the bioactivity has been evidenced after silver addition, due
to the reduction of the free Ti–OH groups. In this context, the authors re-
cently published the characterization of an innovative titanium surface
(obtained by means of a controlled oxidation in Ag-enriched hydrogen
peroxide) constituted by titanium oxide embedded with metallic silver
nanoparticles [95]. This surface is bioactive against S. aureus, biocompat-
ible for osteoblasts cells and bioactive (it induces hydroxyapatite pre-
cipitation after 15 day in SBF). No reduction of the bioactive behaviour
has been registered upon silver addition, in this case.
A summary of the strategies utilized in the scientific literature to in-
troduce silver onto titanium surfaces is reported in Table 2. The main
features (Ag release, antibacterial activity and biocompatibility) of the
modified surfaces are also reported.
4.2. Copper introduction onto titanium surfaces
Although an extensive amount of literature exists on the enrichment
of titanium surfaces with silver, very few research papers have consid-
ered the application of copper on titanium, as an inorganic antibacterial
agent. This is unexpected because lower cytotoxicity is reported for cop-
per than for silver [96], so copper is a very interesting antibacterial
agent. The research works have reported various techniques (ion im-
plantation, alloys, sputtering, sol–gel and chemical vapour deposition)
971
that are used to introduce copper onto titanium substrates, in metallic
or oxide form.
4.2.1. Ion implantation
As mentioned before, ion implantation has been employed to
introduce different antibacterial ions (Ag, Cu) onto metallic substrates
[51,97]. As far as copper is concerned, an increased antibacterial activity
against S. aureus was observed, as the copper content on the surface in-
creased (0.5–4 1017 ions/cm2, concentrations comparable with the ones
previously described for silver). Cu ion implantation induced a slight
improvement in the wear resistance of the metallic substrate. On the
other hand, and contrary to what has been observed for silver implanted
surfaces, described in the previous paragraph, a reduction in corrosion
resistance was recorded for Cu-enriched samples.
4.2.2. Ti–Cu alloys
Ti–Cu alloys with different copper contents (1% and 5%, [96] and
10 wt.% [98]) have been prepared. Ti2Cu and Cu rich phases have been
detected on the 10% Ti–Cu alloy [98]. A significant increase in the
micro-hardness, yield strength and compressive strength was observed,
as well as a reduction in ductility, for the Ti–Cu alloy, compared to bare
Ti [98]. Unlike what was reported for Cu ion implanted surfaces [97], an
increase in the corrosion resistance was observed [98]. No inhibition
halo was observed for the 10% Ti–Cu alloy, but a significant reduction
in bacteria adhered onto the surface was observed for both the
10 wt.% Ti–Cu alloy [98] and for the 1% and 5% ones [96]. In [98], a low
Cu release (0.05 mg/l after 72 h in a physiological solution) indicated
that bacteria can only be killed when they are directly in contact with
the surface. In [96], it was shown that antibacterial behaviour against
S. aureus and E. coli increased as the copper content in the alloy in-
creased, as well as the cytotoxic effect on fibroblast cells; a 1% Cu con-
tent was shown to be the most promising one to limit bacterial
infection and guarantee tissue integration. No cellular tests were report-
ed in [98].
4.2.3. Sputtering
Magnetron sputtering has been employed for the deposition of a
copper containing film on Ti6Al4V [99] and steel [100] substrates. In
the former case, different techniques were considered (direct current
magnetron sputtering, dual magnetron sputtering and dual high
power impulse magnetron sputtering), and different chemical composi-
tions were obtained for the deposited films [99]. Generally, these films
were constituted by metallic Ti and Cu, but their oxides were also de-
tected. Cu release was on occasion verified in DMEM (up to 6 mmol/l
in 24 h), depending on the chemical characteristics of the sample; an in-
crease in antibacterial behaviour (up to the complete reduction of
planktonic bacteria and biofilm), as well as in cytotoxicity, was evi-
denced as the amount of released copper increased. In the latter case,
TiN and Cu were deposited [100]. A reduction in the friction coefficient
was observed for the coated samples. Unlike what has been reported for
ion implanted copper [51,97], and according the results reported for Ti–
Cu alloys [98], an improvement in corrosion resistance was observed for
these materials [100], probably because of the deposition of a TiN layer
on the top of the surface. Antibacterial activity was demonstrated
against E. coli (80–90% bacterial reduction after 24 h, depending on
the coating characteristics).
A plasma surface alloying technique has also been employed to
obtain a Cu–Ni layer on commercially pure titanium [101]. Composi-
tional analyses evidenced a higher surface enrichment in Ni than in
Cu, despite of the known cytotoxicity of Ni no biocompatibility stud-
ies have been reported in [101]. The 10 μm coating conferred anti-
bacterial properties (almost complete reduction of bacterial
viability by direct counting of the colonies on the sample surface)
against E. coli and S. aureus. Antibacterial activity has been attributed
to copper release, but no measurements are reported in [101]. An
 972
Table 2
Techniques for the preparation of silver enriched titanium substrates and the related properties.

Technique Chemical state of Ag Release Tested bacteria Biocompatibility References

Ion implantation Ag ionic metallic/nanoparticles Not reported S. aureus Osteoblasts (if Ag b 1e17 ions cm−2, 20 keV) [51] [52]
(IBAD)

S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978

Plasma Immersion Ion Implantation Ag nanoparticles No (b10 ppb in 60 days) S. aureus, E. coli Osteoblasts [53] [54]
(PIII)
Ion beam assisted deposition (IBAD) Metallic/Ag nanoparticles No (b10 ppb cm2 in 70 days) for Ad doped S. aureus, E. coli Osteoblasts [55,56,57]
titanium.
Up to 15 days in water for Ag-np doped HAp
(max in the first hours 0.4–0.8 ppm in 24 h,
0.78–1.70 ppm in 172 h)
Plasma Electrolytic Oxidation (PEO) Ag-nanoparticles Not reported S. aureus Not reported [58,59,61]
Anodic spark deposition Ag nanoparticles In PBS max at 24 h (1.2 ng ∗ g−1 ∗ mm−2) and S. epidermidis Not reported [60]
constant up to 15 days (0.4 ng ∗ g−1 ∗ mm−2)
Electrophoresis TiO2–Ag nanoparticles Not reported Not reported Not reported [62]
Plasma Electrolytic Process (PEP) Ag-enriched HAp Not reported E. coli Not reported [63]
In situ synthesis of Ag-nps on TiO2 Ag nanoparticles In PBS maximum in the first days (0.15–0.45 ppm S. aureus, E. coli Moderate cytotoxicity for osteoblasts [64] [65,66,67]
by UV reduction of AgNO3 after 24 h) and slow down till 14 days
Ion exchange Ag ions/Ag nanoparticles 300–510 ppb in physiological saline solution, S. aureus, Meticillin Resistant Osteoblasts for Ag-doped bioactive glass [70,71,72,73]
320–440 pp. in PBS and 64,000–82,000 ppb in FBS, Staphylococcus aureus (MRSA) coatings
after 24 h for Ag-doped sodium titanate
Up to 4 μg/mm2 in SBF for Ag-doped bioactive glass
coatings
Sol–gel Ag nanoparticles Maximum after 1 h (0.4 ppm) in water for Ag E. coli, S. aureus Osteoblasts (0.8 wt.% Ag in HAp) [74,75] [76] [77]
nanoparticles containing TiO2 coatings [78] [79] [81]
Ag-doped HAp E. coli, S. albus [80]
Sputtering Ag-nanoparticles Artificial saliva, PBS max at 24 h S. aureus, Fibroblasts [82] [83] [84] [85]
A. actinomycetemcomitans [89]
Ag-nanorods HNO3 1 M, up to 20 days, maximum at 8 days E. coli Not reported [87]
(550 nM/ml) reduced for capped nanostructures
Plasma-spray Metallic/oxide in Ag–TiO2 coatings In water up to 28 days E. coli Osteoblasts [90]
(max at 24 h, 0.11 μg/(ml ∗ cm2)).
Ag-HAp on plasma spray Ti Max at 24 in SBF (up to 0.4 ppm) E. coli, P. aeruginosa, S. aureus Not reported [91]
AgO/SrO doped HAp In PBS up to 7 days (2 ppm) P. aeruginosa Osteoblasts (ok for Ag–Sr-HAp) [92]
Alloys Ti–Ag In PBS up to 7 days (4000 ppb) S. aureus Osteoblasts [93]
Controlled oxidation in Ag–H2O2 Ag nanoparticles In water 0.08–0.2 μg/ml in 24 h S. aureus Osteoblasts [95]
 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
improved wear resistance was observed on the coated surfaces, com-
pared to pure titanium.
4.2.4. Sol–gel
Copper doped TiO2 mono- and multi-layer coatings on Ti6Al4V sub-
strates [46] and CuO/TiO2 composite nanofibres [102,34] have been ob-
tained by means of the sol–gel technique. Titanium isopropoxide [102]
and tetrabutoxytitanate [46] were used as TiO2 precursors, while copper
acetate [46,102], copper nitrate [34] or copper nanoparticles [102] were
used as Cu ones. As far as coatings are concerned [46], the dip-coating
technique has been used to deposit the copper onto substrates and it
was observed that the antibacterial action against S. aureus increased
with the number of Cu-containing layers (up to 6log10 for 4 layers),
but some signs of cellular suffering were observed, when the layer num-
ber was increased [46]. As far as fibres are concerned, electrospinning
was employed for [102,34] and a deleterious effect on the fibre structure
was observed for copper acetate, while good results were obtained with
Cu nanoparticles and copper nitrate. The TiO2 fibres were characterized
by anatase, while a rutile signal appeared for the Cu doped ones. A CuO
signal clearly appeared for fibres obtained using Cu nanoparticles [102],
while it was not present for those obtained using copper nitrate [34]. In
the latter formulation, Cu rich particles were observed by means of
FESEM and microanalysis techniques. Antibacterial activity was tested
against Klebsiella pneumonia for fibres obtained using Cu nanoparticles
[102], and against S. aureus and E. coli for fibres obtained using Cu nitrate
[34]. Increased antibacterial action was observed in the former case
[102], when the copper content was increased, together with photocat-
alytic behaviour. Cell wall damage and DNA condensed deposits were
visible for bacteria treated with Cu enriched fibres and with Cu nitrate
[34], which can be explained considering the mechanism of copper
against micro-organisms.
Moreover TiO2–Cu composite nanoparticles (10 nm) have been ob-
tained by means of the UV photocatalytic reduction of copper chloride
containing TiO2 sol [103]. Anatase, ionic and metallic copper were iden-
tified as the particle constituents. Significant antibacterial activity, in the
absence of light, against E. coli (complete inhibition with 21.4 μg/ml
nanoparticles) and S. aureus (complete inhibition with 14.3 μg/ml nano-
particles) was observed for the composite particles. The indicated
values are lower than the ones reported in the literature for pure titani-
um and copper/copper oxide nanoparticles. It has been suggested that
negatively charged TiO2 particles could attract Cu ions and act as car-
riers, but no release tests are reported.
4.2.5. Chemical vapour deposition
Copper containing TiO2 coatings have been prepared on steel, silicon
and glass by means of pulsed Direct Liquid Injection Chemical Vapour
Deposition (DLI-CVD) using copper bis(2,2,6,6-tetramethyl-3,5-
heptadionate) and titanium tetra-iso-propoxide as Cu and TiO2 precur-
sors [104]. Anatase and metallic copper were individuated as the main
constituents of the coating; an early crystallization of rutile was also
detected for the highest Cu content, suggesting the ability of copper to
Table 3
Techniques for the preparation of copper enriched titanium substrates and the related properties.
Technique Chemical state of Cu Release
Ion implantation Ionic Not reported
Alloys Ti–Cu Up to 72 h in physiological
solution (0.05 mg/l)
Magnetron sputtering Metallic/ionic Up to 10 days in DMEM
(up to 6 mmol/l in 24 h)
Plasma surface alloying Cu-Ni alloy Not reported
Sol–gel Oxide Not reported
UV reduction CuCl2 TiO2–Cu nanoparticles Not reported
DLI-CVD Metallic Not reported
CVD Oxide Not reported
973
favour rutile crystallization, and confirming other results already re-
ported for silver containing titanium oxide films and previously
discussed [74,84,83]. A decrease in the film growth rate and in the
mean crystal size was observed, while a decrease in the grain growth
was also observed for silver containing titanium oxide [79]. Finally the
antibacterial behaviour of Cu-containing coatings was demonstrated
against S. aureus (100% of bacterial reduction for a Cu content higher
than 1.5 mol%), as was the maintenance of coating properties for up to
5 months of storage in ambient conditions with a 30% decrease for lon-
ger times [104].
The CVD technique has also been employed for the deposition of
multi-layer titanium and copper oxide coatings [105]. A significant anti-
bacterial activity against E. coli was noted under UV irradiation (N5log
bacterial reduction after 20–40 min), and a significant effect of surface
roughness on antibacterial behaviour was observed. In particular, the
effect of nanostructures on the improvement of the surface area and
Cu release was underlined. Cu release in distilled water was measured
in the range 3.3–5.5 ppm after 80 min immersion, depending on the
coating type. Moreover, the ability of these coatings to self-regenerate
under UV irradiation was reported.
As far as copper enriched surfaces are concerned, the antibacterial
activity, analogously to silver enriched surfaces, is often attributed to
the release of Cu2+ ions, except for Ti–Cu alloys [96,98] for which an ex-
tremely low release was registered and antibacterial action attributed to
contact killing. Compared to silver, few data on copper release are
reported.
Moreover, no bioactivity data have been reported for Cu-enriched
surfaces.
A summary of the strategies utilized in the scientific literature for the
introduction of copper onto titanium surfaces is reported in Table 3. The
main features (Cu release, antibacterial activity and biocompatibility) of
the modified surfaces are also reported.
4.3. Zinc introduction onto titanium surfaces
Only a few works can be found on the introduction of zinc to impart
antibacterial properties to titanium substrates. Similar techniques to the
ones described for silver and copper doping, such as electrochemical
techniques, ion implantation and plasma spraying, have been used for
the introduction of zinc onto titanium substrates. Biocompatible and an-
tibacterial surfaces have been obtained using these techniques.
Zn-doped titanium oxide coatings (5–10 μm thickness, well adhered
to the substrate) have been obtained on commercially pure titanium by
means of plasma electrolytic oxidation, introducing zinc acetate into the
electrolyte solution [106]. Final concentration comprised between 4.6
and 9.3 at.% was obtained. XPS revealed that Zn entered the coating as
ZnO. Anatase and small amounts of rutile have been detected on Zn con-
taining coatings while only anatase was present onto Zn-free ones, thus
suggesting a tendency to facilitate rutile crystallization for zinc, as al-
ready observed for silver and copper [74,82,83,104]. A significant in-
crease in surface wettability was observed upon oxidation, but no
Tested bacteria Biocompatibility References
S. aureus Not reported [51] [97]
S. aureus, E. coli Fibroblasts (ok 1% Cu) [96] [98]
S. epidermidis, S. aureus, E. coli Some cytotoxic effect on osteoblasts [99] [100]
E. coli, S. aureus Not reported [101]
S. aureus, K. pneumonia, E. coli Fibroblasts, signs of cytotoxicity for [46,102] [32]
high Cu content
E. coli, S. aureus Not reported [103]
S. aureus Not reported [104]
E. coli Not reported [105]
974 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
alteration was introduced upon Zn addition. Zinc containing coatings
are completely biocompatible for rat bone marrow stromal cells,
which also present an increased proliferation and ALP production
[106]. A significant reduction in the viability of E. coli (40–100% reduc-
tion) and S. aureus (96–100% reduction) for Zn containing coatings,
which increased with the Zn content, was reported. A Zn release in a
Tris–HCl solution, proportional to the Zn content in the coating, was
observed for up to 14 days (up to 3.6 ppm in 14 days); the values
were all below the cytotoxicity limits for Zn ions (at about 10 ppm).
Moreover, using a similar electrochemical technique (anodic spark de-
position), Huo et al. [107] obtained titanium oxide nanotubes (with differ-
ent diameters of 30 and 80 nm) on commercially pure titanium, and they
then enriched them with different amounts of Zn (up to 60.2 μg/cm2),
through a hydrothermal process. ZnTiO3 was observed on the Zn-
enriched surfaces, and a small increase in the nanotube wall thickness
was observed after the introduction of Zn. The Zn release in PBS increased
with the Zn content; it reached a maximum after 1 day (up to 0.06 ppm
after 24 h immersion), then it decreased with time, and was almost ab-
sent after 1 month. The nanotubes increased protein absorption on the
surface; this phenomenon depended to a great extent on the surface
nano-roughness, and the highest Zn containing surfaces (which are
slightly smoother) therefore induced less protein absorption. The modi-
fied surfaces were antibacterial against S. aureus, both adhered bacteria
and planktonic ones were significantly reduced (57–97% reduction of
planktonic bacteria and 77–100% reduction of adhered ones at day 1).
The antibacterial behaviour increased with the Zn content and decreased
with time (45–78% reduction of planktonic bacteria and 43–87% reduc-
tion of adhered ones after 7 days, according to the release tests). A more
rapid decrease in antibacterial activity was observed in planktonic bacte-
ria, compared to the surface adhered ones, suggesting a double bactericid-
al mechanism of doped surfaces: Zn release and ROS production induced
by surface bonded zinc. Differently from the observation on Ag-enriched
nanotubes [65], no cytotoxic effects were observed for Zn doped surfaces,
while an increase in cellular spreading and mineral nodule formation was
observed for the Zn containing materials.
Plasma Immersion Ion Implantation deposition (PIIID) has been also
applied in order to enrich a c. p. Ti gr4 surface with zinc [108]. Varying
the deposition time, different Zn concentrations were obtained (1.1–
2.1 at.%). Increasing antibacterial activity against Steptococcus mutans,
one of the main constituents of dental plaque, (69–96% reduction of ad-
hered bacteria) was observed, as the Zn content was increased.
Furthermore TiO2, Ti(Zn)O2 and ZnO have been deposited on com-
mercially pure titanium (c. p. Ti) by cathodic arc deposition [109]. A po-
rous layer was obtained for Ti(Zn)O2 (with a surface zinc content of
7.6 at.%), and a fibrous one for ZnO (with a surface zinc content of
45.7 at.%). An increase in the water contact angle was observed for the
coated samples (at about 84°, 85° and 89° for TiO2, Ti(Zn)O2 and ZnO,
respectively) compared to c. p. Ti (at about 78°). A reduction in
S. aureus adhesion and viability was observed for the Zn coating (78%
for Ti(Zn)O2 and 87% for ZnO). Completely biocompatible behaviour
was instead verified for the Ti(Zn)O2 coatings, while a certain cytotoxic
effect on osteoblasts was observed on the ZnO ones.
Plasma spray coatings of Zn-enriched calcium silicate (Ca2ZnSi2O7),
obtained by means of the sol–gel method, have also been considered
on Ti6Al4V substrates [110,35]. Zn addition reduced calcium silicate
Table 4
Techniques for the preparation of zinc enriched titanium substrates and the related properties.
Technique Chemical State of Zn Release
Plasma electrolytic oxidation Oxide Up to 14 days in TRIS–HCl (max 3.6 ppm)
Anodic spark deposition ZnTiO3 In PBS max at 24 h, up to 0.06 ppm, complete
after 1 month
PIIID Ionic Not reported
Cathodic arc deposition Ti(Zn)O2, ZnO Not reported
Plasma spray Ca2ZnSi2O7 Tris–HCl up to 14 days (4 ppm)
dissolution in a Tris–HCl buffer, but the coating ability to induce hy-
droxyapatite precipitation in a simulated body fluid was maintained
after 28 days of soaking. Zn release in Tris–HCl buffer was observed up
to 14 days (at about 1.5 ppm after 1 day and 4 ppm after 14 days). Zn
showed significant antibacterial behaviour against S. aureus (93% reduc-
tion) [110] and E. coli (93% reduction) [35]. A detailed study on the zinc
mechanism of action against E. coli has been performed, which revealed
that Zn ions induced significant damage of the cell wall and membrane,
as well as DNA condensation and a loss of the internal cell organization
[35]. As widely discussed for Ag+ and Cu2+ ions, Zn2+ ions can easily at-
tach to bacterial cells, thanks to their positive charge (the opposite of
the bacterial one) and to their ability to tightly bond S-containing mol-
ecules [35].
According to the data reported for silver and copper, the antibacteri-
al action of zinc enriched surfaces is attributed to the release of Zn2+
ions, while no other mechanism has been suggested for these surfaces.
Bioactivity has been described only for Zn-enriched calcium silicate
coatings [110,35].
A summary of the strategies utilized in the scientific literature for the
introduction of zinc onto titanium surfaces is reported in Table 4. The
main features (Zn release, antibacterial activity and biocompatibility)
of the modified surfaces are also reported.
4.4. Other strategies adopted to impart antibacterial properties to titanium
surfaces
The addition of fluorine [111,112], iodine [113,114], iron [115],
nitrogen [116] and cobalt [117] has also been considered to impart
antibacterial properties to titanium surfaces.
The ion implantation of F induces antibacterial activity against
P. gingivalis and A. actinomycetemicomitans, even though no fluorine re-
lease has been observed in a physiological saline solution after 7 days
[111]. The activity was attributed to the formation of a metal-fluoride
complex on the surface. The F-modified samples resulted biocompatible
for fibroblast cells. Unlike almost all of the studies in this field, the Ag
and Zn enriched surfaces with Ion Beam Mixing and N-ones doped
with TiN coating or N implantation did not show the ability to kill the
tested bacteria. Anodic oxidation in a fluorine-containing electrolyte
has also been considered in order to introduce F onto titanium surfaces
[112]. The oxidation conditions were optimized to obtain a compact
layer and to separate the chemical effect from the topographical one.
The ability of F-enriched surfaces to inhibit 6 clinical strains of
S. aureus and S. epidermidis was demonstrated. This property was attrib-
uted to enzymatic activity inhibition by TiF4 complexes.
The introduction of iodine onto titanium surfaces by means of anodic
oxidation in an I-containing electrolyte has also been performed [113,
114]. This process resulted in effective antibacterial activity against
S. aureus and E. coli, without in vitro (fibroblast) or in vivo (rabbit) cyto-
toxicity [113]. Moreover, these I-enriched implants reduced inflamma-
tion and infection and improved bone integration. Clinical trials on
humans [114] have demonstrated good antibacterial activity, without
cytotoxic effects or thyroid abnormalities. After 1 year of implantation,
20–30% of the introduced iodine was still present on the material sur-
face [114]. A broad spectrum of iodine activity was reported, and the
Tested bacteria Biocompatibility References
E. coli, S. aureus Rat bone marrow stromal cells [106]
S. aureus Mesenchymal stem cells [107]
S. mutans Not reported [108]
S. aureus Osteoblasts (ok Ti(Zn)O2, some [109]
cytotoxicity for ZnO
S. aureus, E. coli Osteoblasts [110] [35]
 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
mechanism was attributed to an oxidizing action, related to protein de-
naturation, and to consequent micro-organism destruction [114].
Moreover Fe containing titanium oxide films have been produced on
a glass substrate, by means of the sol–gel dip coating technique [115].
Fe3+ ions entered the TiO2 crystal lattice, where they substituted the
Ti4 + ions, thanks to the similarity between the ionic radii (Fe3 + =
0.063 nm, Ti4+ = 0.068 nm). The Fe-doped TiO2 showed antibacterial
activity against E. coli under fluorescent irradiation.
N-doped TiO2 films have also been obtained on stainless steel by
means of oxidation of a TiN layer [116]. These films demonstrated anti-
bacterial activity against S. aureus, under a fluorescent lamp. Increased
corrosion resistance was observed for the modified samples.
Finally Co-enriched titanium oxide nanofibres (350–450 nm) have
been obtained by means of the electrospinning technique [117]. Co
was observed as cobalt oxide, and it induced antibacterial activity
against S. aureus and Salmonella typhimurium. The proposed Co antibac-
terial action mechanism is analogous to the Ag, Cu and Zn one: the pos-
itive ion binds to the negatively charged bacterium, causing bacterial
cell walls to rupture and enzyme inactivation. On the other hand no
cellular tests were performed on these materials to verify their
biocompatibility.
Surface functionalization with organic molecules has also been con-
sidered to obtain antibacterial titanium surfaces.
In addition to the systemic administration of antibiotics, their
coupling with biomaterials has been considered in order to obtain
local delivery. The most common example of biomaterial-antibiotic
coupling is that of antibiotic loaded bone cements [118].
As far as titanium implants are concerned, the possibility of intro-
ducing the drug directly onto a metal surface has also been considered.
Antoci et al. [119,120] covalently grafted vancomycin onto silanized
titanium surfaces. The specificity of the antibiotic for gram positive bac-
teria was demonstrated after the grafting. The covalently bounded mol-
ecule was not released in solution, but remained stable for up to 3 weeks
in serum.
Vancomycin has also been incorporated in a sol–gel silica coating on
a Ti6Al4V alloy [121]. The antibiotic maintained its structure upon load-
ing and did not alter the silica structure. However, the coating degrada-
tion rate increased with the vancomycin content. An antibiotic release
of up to 7 days was obtained with this technique.
Moreover vancomycin or tobramycin loading in hyaluronic acid/
polylactic acid hydrogel has been performed for the coating of titanium
[122]. The coated samples were resistant to autoclave sterilization and
biocompatible for human dermal fibroblasts.
Furthermore polymeric nanoparticles have been functionalized with
gentamicin sulphate through pH-sensitive bonding [123]. This strategy
allows an antibiotic to only be released in the typical acidic pH of infec-
tions. Functionalized nanoparticles were coupled with silanized titani-
um by means of covalent bonding.
Gentamicin has also been introduced onto titanium surfaces by
means of gentamicin loaded bioresorbable PDLLA coatings [124]. The
coatings were antibacterial against Bacillus subtilis and biocompatible
for osteoblasts. Antibiotic release was very quick (60% in the first min-
ute), both in vitro and in vivo.
Finally a patented spray technique has been employed for the depo-
sition of gentamicin on Ti6Al4V blasted substrates [125]. A PLGA coating
was also deposited to protect the antibiotic during implantation proce-
dures. The polymeric coating degraded, thus allowing gentamicin re-
lease. An effective antibacterial action was demonstrated against 30
clinically isolated bacterial strains.
Chitosan [126] and chitosan, coupled with hyaluronic acid, [127,128]
have also been explored as antibacterial molecules. Their effective
action against S. aureus has been demonstrated [127,128], and it has in
particular been observed that chitosan presents an antibacterial action,
while hyaluronic acid can only reduce bacterial adhesion [127]. Since
a certain reduction in osteoblast adhesion has been recorded for chito-
san and hyaluronic acid modified titanium [127,128], a further
975
functionalization with VEGF [127] or RGD [128] has been performed
to improve the material bone integration. Carboxymethylchitosan
(CMCS) has been bonded to titanium via several strategies (e.g. silane,
dopamine, polydopamine) [129]. Its resistance to several sterilization
and ageing conditions was investigated, and it was demonstrated that
covalent bonding improved molecule resistance.
Chitosan coating on TiO2 nanotubes loaded with selenium has been
considered in order to couple the anticancer activity of Se to the antibac-
terial activities of chitosan [130].
Antimicrobial peptides (AMPs) have also been considered, and re-
cently reviewed [131], thanks to their wide spectrum of activity, high ef-
ficacy at low concentrations, specificity, synergistic action with
conventional antibiotics and low resistance development. AMPs are
small (15–20 amino acids) and positively charged and can therefore
bind to a bacterial surface, which is generally negatively charged. The
antibacterial activity of AMPs, after grafting onto solid supports, is cur-
rently under investigation; the necessity of a proper spacer has been
suggested [131].
Cecropin B peptide has been grafted onto a polished titanium surface
via an intermediate polydopamine layer [132] in order to induce anti-
bacterial activity against S. aureus, B. subtilis, P. aeruginosa and E. coli.
Biocompatibility was verified for osteoblast cells, together with an
anti-inflammatory effect on macrophages.
5. Conclusions and future outlooks
The surface modification of titanium substrates seems a challenging
strategy to prevent the development of prosthetic infections. The
grafting of an antimicrobial agent directly onto an implant surface pre-
vents bacterial colonization and allows the localized delivery of a thera-
peutic agent to help prevent or treat an infection. The presence of the
therapeutic principle directly at the implant site can improve the effica-
cy of the treatment in case of biofilm development on implant surface.
Moreover this way of application can reduce the side effects of systemic
application by reducing the amount of the antibacterial agent and by its
local delivery (only were needed).
Numerous solutions have been proposed in the scientific literature,
and have been reviewed in the present paper. The considered solutions
are mainly based on inorganic antibacterial agents (such as silver, cop-
per or zinc), which have been introduced in order to overcome the
growing problem of bacterial resistance to antibiotics.
The main open problems related to this strategy are the time limited
release of the antibacterial agent (generally reaching a maximum in
1 day and then rapidly decreasing over the next 10–30 days), the anti-
bacterial effect that increases with the concentration of the antibacterial
agent, coupled to cytotoxic behaviour at the highest concentration and
the limited resistance (e.g. chemical, mechanical) of the modified layer.
Actually no titanium based implant with antibacterial properties is
available on the medical devices market, probably due to the aforemen-
tioned open problems. Moreover the presence of an active principle that
can be released from the material surface can complicate the classifica-
tion procedure of the related medical device increasing time and costs of
commercialization. However, the problem of prosthetic infection is a
critical issue for dental and orthopaedic implantology. In fact, prolonged
antibiotic therapies, the consequent development of antibiotic resistant
bacterial strains, the sometimes consequent need to remove and to sub-
stitute the implant and increasing costs for the healthcare system are
the main consequences of prosthetic infections.
In this context the development of titanium based surfaces with
effective and prolonged antibacterial action, a good bone integration
ability and stability in the physiological environment can represent a
promising solution to the problem.
The future research in the biomaterial field should be directed to the
development of innovative metallic surfaces able to improve and fasten
tissue integration and offer a prolonged antibacterial action. The devel-
opment of surface modifications able to guarantee a prolonged release
976 S. Ferraris, S. Spriano / Materials Science and Engineering C 61 (2016) 965–978
of the active principle in sufficient amount for bacterial killing but in the
biocompatible range represent and extremely challenging topic. On the
other hand the opportunity to develop surfaces able to reduce bacterial
adhesion and exert a local contact killing action for bacteria, without the
release of any substance, represent a promising strategy in order to
overcome classification and toxicity concerns.
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