Accepted Manuscript

Title: Femtosecond laser surface texturing of titanium as a

method to reduce the adhesion of Staphylococcus aureus and
biofilm formation

Author: Alexandre Cunha Anne-Marie-Elie Laurent

Plawinski Ana Paula Serro Ana Maria Botelho do Rego
Amélia Almeida Maria C. Urdaci Marie-Christine Durrieu
Rui Vilar

PII: S0169-4332(15)02521-0
DOI: http://dx.doi.org/doi:10.1016/j.apsusc.2015.10.102
Reference: APSUSC 31583

To appear in: APSUSC

Received date: 30-7-2015

Revised date: 13-10-2015
Accepted date: 16-10-2015

Please cite this article as: <doi>http://dx.doi.org/10.1016/j.apsusc.2015.10.102</doi>

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
*Highlights (for review)

Highlights

 The short-term adhesion of Staphylococcus aureus onto femtosecond laser textured

surfaces of titanium was investigated

 The laser textured surfaces consist of Laser-Induced Periodic Surface Structures (LIPSS)

t
and nanopillars

ip
 The laser treatment enhances the hydrophilicity and the surface free energy of the

cr
material

us
 The laser treatment reduces significantly the adhesion of Staphylococcus aureus and
biofilm formation


an
Femtosecond laser surface texturing of titanium is a simple and promising method for
M
endowing dental and orthopedic implants with antibacterial properties
ed
pt
ce
Ac

Page 1 of 23
*Graphical Abstract (for review)

t
ip
cr
us
an
M
d
p te
ce
Ac

Page 2 of 23
*Manuscript
Click here to view linked References

Femtosecond laser surface texturing of titanium as a method to

reduce the adhesion of Staphylococcus aureus and biofilm
formation
Alexandre Cunhaa,c, Anne-Marie-Elieb, Laurent Plawinskic, Ana Paula Serrod, Ana Maria
Botelho do Regoe, Amélia Almeidaa, Maria C. Urdacib, Marie-Christine Durrieuc, Rui Vilara*

a
Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa,

t
Portugal.

ip
b
Bordeaux University, CBMN UMR 5248, CNRS, Bordeaux Science Agro, 1 Rue du G. de
Gaulle, 33170 Gradignan, France.

cr
c
Bordeaux University, Institute of Chemistry & Biology of Membranes & Nanoobjects
(CBMN UMR 5248, CNRS), European Institute of Chemistry and Biology, 2 Rue Robert

us
Escarpit, 33607 Pessac, France.
d
Instituto Superior Técnico, Universidade de Lisboa, CQE-Centro de Química Estrutural, Av.
Rovisco Pais 1, 1049-001 Lisbon, Portugal.
e an
Instituto Superior Técnico, Universidade de Lisboa, CQFM-Centro de Química-Física
Molecular and Institute of Nanoscience and Nanotechnology (IN), Av. Rovisco Pais 1, 1049-
001 Lisbon, Portugal.
M
*Corresponding author:
ed

Rui Vilar (R. Vilar)

Department of Chemical Engineering, Instituto Superior Técnico, Universidade de Lisboa,
Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal
pt

Tel.:+351 218417259
E-mail address: rui.vilar@tecnico.ulisboa.pt
ce
Ac

Page 3 of 23
Abstract

The aim of the present work was to investigate the possibility of using femtosecond laser
surface texturing as a method to reduce the colonization of Grade 2 Titanium alloy surfaces
by Staphylococcus aureus and the subsequent formation of biofilm. The laser treatments were
carried out with a Yb:KYW chirped-pulse-regenerative amplification laser system with a
central wavelength of 1030 nm and a pulse duration of 500 fs. Two types of surface textures,

t
ip
consisting of laser-induced periodic surface structures (LIPSS) and nanopillars, were
produced. The topography, chemical composition and phase constitution of these surfaces

cr
were investigated by atomic force microscopy, scanning electron microscopy, X-ray
photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction. Surface

us
wettability was assessed by the sessile drop method using water and diiodomethane as testing
liquids. The response of Staphylococcus aureus put into contact with the laser treated

an
surfaces in controlled conditions was investigated by epifluorescence microscopy and
scanning electron microscopy 48 h after cell seeding. The results achieved show that the laser
treatment reduces significantly the bacterial adhesion to the surface as well as biofilm
M
formation as compared to a reference polished surfaces and suggest that femtosecond laser
texturing is a simple and promising method for endowing dental and orthopedic titanium
ed

implants with antibacterial properties, reducing the risk of implant-associated infections

without requiring immobilized antibacterial substances, nanoparticles or coatings.
pt

Keywords: Femtosecond lasers; Laser surface texturing; Dental and orthopedic implants;
Wettability; Bacteria adhesion; Biofilm
ce
Ac

2
Page 4 of 23
1. Introduction

Advancements in metallic biomaterials have been allowing the production of better or longer
lasting dental and orthopedic implants. However, despite the significant progress achieved so
far, implant failure is still a major concern and the improvement of materials with improved
biofunctionality remains a permanent quest [1, 2].

t
Infections are one of the most severe complications leading to implant failure [3, 4]. These

ip
infections are often due to the contamination of the surgical site and of surrounding area
during implantation, and, in particular for bacterial strains presenting high resistance to

cr
antibiotics [5], the treatment of implant-related infections may be difficult and lead to
substantial risk to the patients’ health [6]. The presence of bacteria colonies and biofilms at

us
the implant surface may also prevent proper osseointegration [7]. Consequently it is of utmost
importance to reduce the probability of opportunistic colonization of the implant surface by
an
bacteria, by endowing these surfaces with antibacterial characteristics [8].

Similarly to eukaryotic cells, bacteria are sensitive to surface characteristics such as

M
topography, chemical composition and wettability by the body fluids [9-11]. However, since
they are smaller, more rigid, and less motile than eukaryotic cells, bacteria often react
ed

differently to surface features [12]. Implant surfaces can be provided with bactericidal
properties by direct impregnation with suitable drugs, by coating with thin films of
bactericidal substances, such as copper, silver, titania or silver nitrate or with polymeric or
pt

ceramic-matrix composites films containing antimicrobial agents, such as silver, titania or

drug-functionalized polymeric nanoparticles [2, 13, 14]. However, the antibacterial effect of
ce

these treatments is often accompanied of some risk for the patient, due to the potential
toxicity of the agents used [13]. Thus, surface treatments capable of endowing implants with
Ac

antibacterial characteristics while avoiding harmful effects would be clearly advantageous

and, in this sense, modifying the surface nanotopography of implants appears as a viable
strategy to conventional coating processes. Surface texturing with ultrafast laser pulses allows
creating a wide range of surface micro- and nanotextures on a wide range of materials [15],
including metallic [16, 17], ceramic [18], and polymeric [19] biomaterials. The method
presents unique characteristics of flexibility, simplicity, controllability and reproducibility,
making it a very promising technique for the surface modification of biomaterials. In
addition, laser-based methods offer a negligible risk of surface contamination since they do
not require the use of toxic substances. The influence of surface roughness on microbial

3
Page 5 of 23
retention has been investigated by several authors [20]. The results achieved so far suggest
that bacteria adhere preferentially to polished surfaces [21], and to surfaces with cavities
larger than the bacteria size [12] but the results for nanotextured surface, with surface
features with dimensions in an intermediate range are scarce.. In previous publications the
authors demonstrated that several types of isotropic and anisotropic surface nanotextures with
interesting properties and characteristic dimensions in this intermediate range can be
reproducibly fabricated on titanium alloys using femtosecond pulse duration lasers [16, 22-

t
ip
24]. It was also shown that when human Mesenchymal Stem Cells (hMSCs) are put in
contact with Grade 2 titanium alloy laser treated surfaces, surface nanotextures lead to

cr
reductions of the cell area and of the focal adhesion area as compared to a polished reference
surface but, when the cells were cultured for 4 weeks in an osteogenic medium, they enhance

us
matrix mineralization and bone-like nodule formation, potentially improving hMSC
differentiation into an osteoblastic lineage [25]. In the present work, we investigated the
an
behavior of Staphylococcus aureus (S. aureus), which is one of the most common and
dangerous pathogens causing implant-related infections [26], when they are put in contact
with surface nanotextures produced by laser treatment with femtosecond lasers, aiming at
M
assessing the potential of these laser treatments for producing surfaces with antibacterial
properties, capable of reducing the risk of implant-associated infections.
ed

2. Experimental
pt

2.1. Femtosecond laser treatment

ce

The tests were performed on plates of Grade 2 titanium alloy supplied by Titanium Industries
UK Ltd. Prior to the laser treatment, the specimens surface was ground and polished to
Ac

ensure homogeneity of optical properties, and ultrasonically cleaned to remove residues of

the polishing compounds and of grease.

Surface texturing was carried out with an Yb:KYW chirped-pulse regenerative amplification
laser system (Amplitude Systèmes s-Pulse HP) with a central wavelength of 1030 nm and
pulse duration of 500 fs. The laser treatments were carried out using methods described in
previous publications [16, 22], chosen because they allow preparing surface nanotextures
with characteristic dimensions similar to the bacterium size. They are summarized in Table 1.
The fluence was calculated on the basis of the laser beam radius at e-2 of the maximum

4
Page 6 of 23
 intensity of the Gaussian beam profile, estimated using the D-squared method [27]. The laser
beam was focused by a 100 mm focal length lens on a spot with a diameter of 338  10 m
perpendicularly to the specimens’ surface. The laser treatment was performed in air, by
moving the specimens under the stationary laser beam with a constant scanning speed, by
means of a computer-controlled XYZ stage (PI miCos) while pulsing the laser beam at 1 kHz.
To achieve complete surface coverage, consecutive laser tracks were partially overlapped by
a lateral displacement of about 30 % of the laser track width. The polarization direction was

t
ip
controlled by a half-wave plate introduced in the optical path. Two different types of textures
(LIPSS and nanopillars) were produced using methods and parameter ranges described in

cr
previous publications [16, 22].

us
Table 1. Laser processing parameters used to produce the different types of textures

Repetition Scanning Lateral b

Fluence Linear Number

LIPSS
Texture
[J/cm2]

0.30
rate
[kHz]
1
speed
[mm/s]
5
an displacement
[mm]
0.1
polarization

perpendicular
of pulses

192
M
a perpendicular
Nanopillars 0.30 + 0.10 1 5 + 20 0.1 + 0.1 192 + 45
+ parallel
a
This texture is produced by a two-step treatment.
b
ed

The polarization direction of the laser beam is given in relation to the laser beam scanning direction.

2.2. Surface characterization

pt

The surface topography of the laser treated materials was analyzed by atomic force
ce

microscopy (AFM) and scanning electron microscopy (SEM). AFM observations were
performed with a Veeco DI CP-II atomic force microscope. Images of 20 x 20 m2 surface
Ac

regions were acquired in contact mode using standard silicon tips. At least two images were
taken at different locations for each specimen. Quantitative assessment of the surface
roughness was carried out by computer analysis of stereoscopic pairs of SEM micrographs
recorded at 0 and 10 tilting angle with Alicona-MeX software. The SEM observations
were performed with a JEOL JSM-7001F field emission gun scanning electron microscope
(FEG-SEM). The stereoscopic pairs were analyzed The surface roughness was expressed by
the parameters Ra (arithmetic mean height) and Rz (maximum height), according to the ISO
4287 standard calculated from at least ten profiles taken from each stereoscopic pair, in a
direction perpendicular to the laser beam scanning direction.

5
Page 7 of 23
The chemical composition of the laser treated surfaces was investigated by X-ray
photoelectron spectroscopy (XPS), while the phase constitution was assessed by micro-
Raman spectroscopy and glancing incidence X-ray diffraction (GI-XRD). The XPS
spectrometer used was a XSAM800 KRATOS operated in the fixed analyzer transmission
(FAT) mode. The specimens were irradiated with a non-monochromatized Al K radiation
(h = 1486.6 eV) in ultra-high vacuum ( 10-7 Pa) at room temperature. Peak fitting was
performed with Gaussian-Lorentzian products using the XPS Peak 4.1 freeware, after

t
ip
subtraction of a Shirley background. The sensitivity factors Ti 2p: 1.75, O 1s: 0.66, and C 1s:
0.25 were used for the calculation of the atomic concentrations. The peak components were

cr
assigned on the basis of the NIST X-ray Photoelectron Spectroscopy Database of the
National Institute of Standards and Technology. The micro-Raman analyses were performed

us
with a Horiba LabRAM HR800 Evolution (Jobin-Yvon) spectrometer provided with a He-Ne
laser (laser =633 nm). Spectra were recorded in the range 100-1200 cm-1 with a resolution of
an
2 cm-1, acquisition time of 10 s and 10 accumulations. X-ray diffraction experiments were
performed with a Siemens D 500 diffractometer in glancing incidence configuration. The
M
diffractograms were recorded in the 2 range 20-90, with an incidence angle of 1,
increment of 0.04, and integration time of 8 s. The diffraction peaks were indexed on the
basis of the XRD database of the International Centre for Diffraction Data (ICDD).
ed

2.3. Assessment of wettability

pt

Wettability was measured by the sessile drop method, using distilled-deionized water and
diiodomethane (Merck) as testing liquids. These liquids were selected because they present
ce

very distinct polarities. Droplets of about 4 µL of the liquids were deposited with a
micrometric syringe on the surface of 20 x 20 x 1 mm3 specimens placed inside a closed
Ac

chamber (Ramé-Hart) saturated with the vapor of the respective liquid at room temperature.
Images of the droplets were acquired at regular time lapses, between the droplet deposition
instant until cessation of droplet spreading or at least 600 s, using a video camera (jAI CV-
A50) mounted on a microscope (Wild M3Z, Leica). All images were acquired in a direction
perpendicular to the laser beam scanning direction. The contact angles were determined from
the droplet profile analysis using the Axisymmetric Drop Shape Analysis Profile (ADSA-P)
method [28]. At least 10 measurements were taken for each type of surface and liquid.

6
Page 8 of 23
The surface free energy ( ) and its dispersive ( ) and polar ( )
components were estimated according to Owens and Wendt geometric mean approach [29],
by applying equation (1):

, (1)

Where, is the surface tension of the testing liquid, and its dispersive and polar

t
ip
components, respectively, and the contact angle. The values of the surface tension and its
dispersive and polar components for water and diiodomethane are given in Table 2.

cr
Table 2. Surface tension ( ) and its dispersive ( ) and polar ( ) components for water

us
and diiodomethane [30]

Testing media
Water
[mN/m]
72.0
an
[mN/m]
21.3
[mN/m]
50.7
Polarity
Polar
Diiodomethane 49.4 49.0 0.4 Nonpolar
M

2.4. Bacterial cell culture

ed

Prior to cell seeding, the specimens were ultrasonically cleaned in absolute ethanol (Sigma
Aldrich) for 15 minutes. They were then put on 12-well culture dishes, sterilized in 70 %
pt

ethanol for 10 minutes, and washed five times with phosphate buffered saline (PBS 1x;
ce

GIBCO) solution. S. aureus was used in the experiments because it is the commonest cause
of metal implant-associated infections and a biofilm-forming bacteria [26]. S. aureus
(American Type Culture Collection; ATCC 25923) was precultured in Tryptic Soy Broth
Ac

(TSB; Sigma Aldrich) medium for 24 h at 37 C. The optical density (OD = 0.6) of the
precultured bacteria at a wavelength of 600 nm was measured using a spectrophotometer
(Genesis 20, Thermo Scientific). Bacteria were seeded on each specimen at a concentration
of about 7.3 x 108 Colony-Forming Unit (CFU)/mL and incubated for 48 h at 37 C, under
static conditions. At least three specimens per each type of texture were tested. Polished
titanium surfaces were used as reference.

7
Page 9 of 23
2.5. Assessment of bacterial adhesion and biofilm formation

48 h after bacterial cell seeding, the specimens were washed five times with sterile PBS
solution to remove non-adherent and loosely adherent bacteria. The adherent bacteria were
fixed with 4 % (w/v) solution of paraformaldehyde (PFA; Electron Microscopy Sciences) in
PBS for 30 minutes at 4 C, and fluorescently labeled with 0.3 % carboxyfluorescein
succinimidyl ester (CFSE; Difco) in PBS solution for 30 minutes at 37 C, protected from

t
ip
ambient light.

The specimen fluorescence after adding the fluorescent dye was used to quantify the biofilm

cr
bacteria. The experiments were carried out using a LEICA DM5500B epifluorescence
microscope with a 40X oil immersion objective and a FITC filter (480/500 nm,

us
excitation/emission wavelengths). In parallel with imaging and photographing with the Leica
microscope camera, the fluorescence signal was monitored with the Q-LUT function of the
an
microscope image processing software Qwin (Leica Microsystems), as indicated by the
supplier. TIFF images were acquired with a FITC filter (green, 480 nm). To quantify the
M
extension of fluorescent areas in microscope images ImageJ freeware [31] was used. The
surface covered by bacteria was measured using the threshold function of ImageJ by
determining the area occupied by the 480 nm bacterial signal. The bacterial fluorescence
ed

intensity was also measured using ImageJ software. At least 20 images of randomly selected
areas were acquired for each type of surface in order to ensure that the data are statistically
pt

significant.

The bacterial biofilm was observed in the secondary electron imaging mode using a JEOL
ce

JSM-7001F field emission gun electron microscope (FEG-SEM). Prior to SEM observation,
the specimens were fixed for 30 minutes at 4 C and dehydrated in a graded ethanol series
Ac

(70, 80, 90, and 100 %) for 30 minutes each at room temperature. To avoid charging, the
specimens were coated with a carbon thin film by DC sputtering (Q150T ES, Quorum
Technologies). At least 10 micrographs at different magnifications were taken for each
specimen.

2.6. Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey’s
test for multiple comparisons using OriginPro 8 software (OriginLab Corporation, USA).

8
Page 10 of 23
Differences were considered statistically significant if the p-value was lower than 0.05
(confidence of 95 %).

3. Results

3.1. Topography of the laser treated surfaces

t
SEM micrographs and AFM images of the textures produced are presented in Fig. 1. The

ip
texture depicted in Fig.1 a-b consists of low spatial frequency laser-induced periodic surface

cr
structures (LIPSS) and forms for fluences slightly larger than the material ablation threshold
[32, 33]. Their average period and maximum peak-to-valley distance are 710  60 and

us
250  80 nm, respectively. The ripples are perpendicular to the laser beam polarization,
which was perpendicular to the beam scanning direction in these experiments. The Ra and Rz
of these surfaces are 0.3  0.1 and 1.5  0.2 m, respectively. The second type of texture,
an
consisting of nanopillars (Fig. 1 c-d), forms when the surface is submitted to a two-step
treatment with laser processing parameters in the LIPSS formation range and mutually
M
perpendicular polarization directions [16], the second laser treatment leading to the
fragmentation of the LIPSS formed in the first treatment. Their bottom diameter and
ed

maximum height are 750  130 and 175  40 nm, respectively. The Ra and Rz of these
surfaces are 0.3  0.2 and 1.6  0.8 m, respectively. The mechanisms of formation of these
surface textures were discussed in previous publications [16, 22, 24].
pt
ce
Ac

9
Page 11 of 23
 t
ip
cr
us
an
M
Fig. 1.
ed

3.2. Chemical composition and phase constitution of the surfaces

pt

Fig. 2 presents detailed XPS spectra of Ti 2p and O 1s peaks for polished and laser treated
ce

surfaces. The Ti 2p peak can be fitted with three doublets with a spin-orbit split of
5.7  0.1 eV (Fig. 2 a and c). The main component is centered at 458.3  0.1 eV, and can be
assigned to Ti+4 in TiO2. A less intense component, centered at 455.6  0.2 eV, can be
Ac

attributed to Ti+3 in Ti2O3. For polished surfaces and surfaces textured with nanopillars a
component centered at 453.8  0.2 eV is observed, which can be associated to metallic
titanium. The O 1s peak can be fitted with three sub-peaks situated at 529.9  0.2,
531.5  0.1, and 532.7  0.3 eV (Fig. 2 b and d), which can be attributed to O-2, a mixture of
hydroxyl (OH) and carbonyl groups (C = O), and oxygen singly bound to carbon (C – O),
respectively. The presence of C – O and C = O groups is also confirmed by the analysis of
the C 1s peak (not shown). The relatively large carbon peak may be attributed to surface
contamination by carbon-containing species, which occurs frequently on surfaces exposed to

10
Page 12 of 23
the atmosphere [34]. The results of the Ti 2p and O 1s peaks analysis and the XPS atomic
ratios, summarized in Table 3, show that the thickness of the surface oxide film is larger in
the laser treated specimens with LIPSS texture as compared to the reference, the metallic
titanium peak disappearing after the laser treatment, but this does not occur for the
nanopillars textures. On the other hand, the increase of the Ti+4/Titotal ratio and the slight
decrease of the Ti+3/Titotal ratio indicate that the laser treatment leads to an increase of the
proportion of TiO2 and a decrease of the proportion of Ti2O3 in the oxide film.

t
ip
cr
us
an
M
ed
pt
ce

Fig. 2.
Ac

Table 3. XPS atomic ratios

Ratios
Surfaces
x100 [%] x100 [%] x100 [%]
Polished 7.2 5.2 87.6
LIPSS - 4.2 95.8
Nanopillars 3.3 5.1 91.6

11
Page 13 of 23
The micro-Raman spectra also present bands corresponding to TiO2 polymorphs (Fig. 3). The
bands around 144-147 and 198 cm-1 may be attributed to anatase, while those around 248,
448, 612, and 827 cm-1 are due to rutile [35]. The rutile band at 827 cm-1 is not observed for
the laser treated surfaces. The Raman bands are broad and slightly shifted, indicating the
presence of short-range order and/or oxygen vacancies in TiO2 [36]. The X-ray
diffractograms for all the specimens are presented in Fig. 4. Most of the diffraction peaks can
be indexed to Ti -phase hcp crystal structure planes. A diffraction peak corresponding to the

t
ip
plane (111) of rutile is observed at 2  41.2 for the laser treated surfaces.

cr
us
an
M
ed

Fig. 3.
pt
ce
Ac

Fig. 4.

12
Page 14 of 23
3.3. Wettability

The values of the contact angles of both liquids on polished and laser treated surfaces are
presented in Fig. 5. The laser treatment enhances significantly the hydrophilicity of the
specimens. The contact angle values of water are 48.2  3.1, 12.6  3.1, and 32.1  9.0 for
polished, LIPSS and nanopillars textured surfaces, respectively. Diiodomethane presents a
similar evolution, except for surfaces textured with nanopillars.

t
ip
cr
us
an
M
ed

Fig. 5.

The values of the contact angle for this liquid are 43.7  0.7, 15.5  3.5, and 54.3  1.6 for
pt

polished, LIPSS and nanopillars textured surfaces, respectively. The values of the surface
free energy ( ) and its dispersive ( ) and polar ( ) components for the different
ce

specimens are given in Table 4. The laser treatment increases , in agreement with the
evolution observed for the hydrophilicity.
Ac

Table 4. Surface free energy ( ) and its dispersive ( ) and polar ( ) components for
the titanium surfaces

Surfaces [mN/m] [mN/m] [mN/m]

Polished 54.7  2.1 32.1  0.6 22.6  2.4
LIPSS 74.5  1.0 41.3  0.4 33.2  1.2
Nanopillars 62.3  5.2 25.6  1.2 36.6  6.1

13
Page 15 of 23
3.4. Bacterial adhesion and biofilm formation

Fluorescence microscopy images of S. aureus 48 h after bacterial cell seeding are depicted in
Fig. 6.

t
ip
cr
us
an
M
ed
pt
ce

Fig. 6.
Ac

Bacteria adhere both to the laser treated specimens and the reference polished specimen, but
much less so to the laser treated surfaces. Moreover, the size of S. aureus bacterial deposits is
smaller on laser treated surfaces. The average fraction of the surface area covered by bacteria
is  7 % for the laser treated surfaces as compared to  25 % for the polished specimen (Fig.
7a) and these results are consistent with the measurements of fluorescence intensity (Fig. 7b).
Bacteria adhesion is similar for the two types of laser treated surfaces within the limits of
experimental error. SEM micrographs of bacteria colonized surfaces 48 h after seeding are
depicted in Fig. 8. An extracellular polymeric substance is observed on all the surfaces,

14
Page 16 of 23
confirming the production of biofilm. However, while for polished surfaces the film is
continuous and presents embedded bacteria (Fig.8 a-b), for the laser treated surfaces the film
is discontinuous and the number and size of bacteria aggregates are much smaller (Fig. 8 c
and e). Fig. 8 d and f show that S. aureus does not penetrate significantly in the texture
depressions and, as a result, the biofilm tends to adhere only to the LIPSS crests.

t
ip
cr
us
an
M
Fig. 7.
ed
pt
ce
Ac

15
Page 17 of 23
 t
ip
cr
us
an
M
ed

Fig. 8.
pt

4. Discussion
ce

Preventing infections is of upmost importance to ensure the long-term service of dental and
orthopedic implants [13]. In the present work, we analyzed the possibility of using
Ac

femtosecond laser surface nanotexturing as a method to prevent the colonization of titanium

surfaces by bacteria and subsequent formation of biofilm.

The influence of surface roughness on microbial retention has been widely investigated [20].
Bacteria adhesion to surfaces depends on several factors, namely the topography and
chemical composition of the surface and its wettability and surface free energy, but the
surface topography seems to be the most influential factor [20]. Bacteria seem to adhere
preferentially to surfaces with surface roughness in specific ranges, which bear a relationship
with the bacteria size. For example Truong et al. [21] showed that S. aureus adheres

16
Page 18 of 23
preferentially to titanium surfaces with Ra in the range 1-4 nm a conclusion confirmed by
Ivanova et al. [37] and by Wu et al. [12] for Staphylococcus epidermidi. Braem et al. [38]
also found a strong correlation between the roughness of porous Ti coatings and the behavior
of S. aureus and S. epidermidis, the formation of biofilm being more intense on specimens
with Ra in the range 5-8 μm than on polished (Ra = 30 nm) or machined (Ra = 0.5 μm)
surfaces. These studies suggest that bacteria adhere preferentially to low roughness polished
surfaces, with Ra in the nanometer range, but also adhere to surfaces that present topographic

t
ip
features larger than the bacteria size (typically 1-2 μm). Our results, which concern an
intermediate surface roughness show that bacterial adhesion is much lower for laser treated

cr
surfaces, with Ra ≈ 0.3 μm, slightly smaller than the bacteria size, and dense features, which
inhibit bacteria to contact the surface and, contrarily to micron-sized cavities, limits their

us
agglomeration. The main conclusion of the present work is that the presence of a surface
relief with characteristic dimensions smaller that the bacterium size and very dense features
an
reduces the contact area between bacteria and the surface, inhibiting bacterial colonization
and reducing bacteria retention. Vasudevan et al [39] found that Enterobacter cloacae
bacteria in contact with a textured polydimethylsiloxane elastomer (PDMS) surfaces colonize
M
preferentially the recessed regions of pillars and pits with dimensions in the range 2-23 m
while Fadeeva et al. [40] and Truong et al. [21] concluded that S. aureus bacteria locate
ed

preferentially in the depressions between 10-20 m-sized pillars created in titanium by

femtosecond laser treatment and just a few bacteria adhered to the top of the pillars, in
pt

agreement with our results. Taking into account all these observations, it is possible to
conclude that concave microscale regions facilitate bacteria adhesion by providing a large
ce

contact area between the cell and the surface, while LIPSS and nanopillars inhibit bacterial
colonization because the S. aureus cells cannot penetrate into the depressions to establish s
stable bond to the surface. In this situation, in the laser-induced nanotextures the bacteria can
Ac

only attach to individual features’ apices, with much smaller contact area and, consequently,
much reduced adhesion strength as compared to the reference surfaces.

Surface chemical composition also plays a role in bacterial adhesion. Del Curto et al. [41]
and Giordano et al. [42] showed that the presence of a significant proportion of anatase in the
surface oxide film decreases the adhesion of S. aureus and S. epidermidis [41] and similar
observations were made for Streptococcus mutans, S. salivarius, and S. sanguis. [42]. Since
the laser treatment was carried out in air, the changes in surface topography were
accompanied by an increase of the thickness of the oxide film, as shown by the XPS analysis

17
Page 19 of 23
results. In addition, the micro-Raman analyses indicate that the proportion of anatase in the
oxide film increased after the laser treatment. According to the results quoted, the presence of
anatase may also contribute to the antibacterial properties of the laser treated surfaces.
However, the native oxide film in the reference specimens also contains an important
proportion of anatase, and a significant adhesion of bacteria is observed for these surfaces.
With that in mind, one may conclude that the chemical constitution of the oxide film plays a
minor role in the bacterial behavior observed in this work.

t
ip
Regarding wettability, previous studies showed that hydrophilic surfaces encourage bacterial
adhesion [40] but this is not the case for laser nanotextured surfaces. The laser treatment

cr
increases the hydrophilicity of the materials (contact angles in the range 13-32) but bacterial

us
adhesion decreases. Similarly to the chemical constitution, wettability does not seem to play a
crucial role in the bacterial adhesion.

5. Conclusions an
The present work shows that the laser treatment reduces significantly the adhesion of S.
M
aureus and biofilm formation as compared to the reference polished specimens. The
nanotopography of the laser-induced textures reduces bacteria adhesion because the size of
ed

individual features and the average distance between them is such that the penetration of
bacteria is inhibited, reducing the area of the contact interface between individual bacterium
and the metal. On the other hand, bacteria agglomeration is reduced, decreasing the tendency
pt

to form biofilms. The present work shows that femtosecond laser surface texturing is
promising method for endowing dental and orthopedic titanium implants with antibacterial
ce

properties, reducing the risk of implant-associated infections without using immobilized

antibacterial substances, nanoparticles or coatings.
Ac

Acknowledgements

Alexandre Cunha acknowledges the Fundação para a Ciência e a Tecnologia (FCT) for the
doctoral grant SFRH/BD/61002/2009. The authors also acknowledge the Programme
d’Actions Universitaires Intégrées Luso-Françaises (PAUILF) for financial support in the
scope of the International Doctorate School in Functional Materials (IDS-FunMat). A special
thanks to Daniel Pomiel de Jesus, Bruno Nunes, Liliana Cangueiro, and Annie-Marie Elie for

18
Page 20 of 23
their assistance in the preparation of the metallographic specimens, AFM and micro-Raman
analyses, and in vitro experiments.

Competing interests disclosure

The authors have no other relevant affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict with the subject matter or materials

t
discussed in the present manuscript apart from those disclosed.

ip
cr
References

us
[1] M. Elder, Advanced Orthopedic Technologies, Implants and Regenerative Products,
Report n. HLC052B, BBC Research LLC, Wellesley, MA, USA, 2011.
[2] A. Simchi, E. Tamjid, F. Pishbin, A.R. Boccaccini, Recent progress in inorganic and

[3]
an
composite coatings with bactericidal capability for orthopaedic applications,
Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 22-39.
M.E. Török, N.P.J. Day, Staphylococcal and streptococcal infections, Medicine 42
(2014) 1-7.
M
[4] P. Astagneau, C. Rioux, F. Golliot, G. Brucker, Morbidity and mortality associated
with surgical site infections: results from the 1997-1999 INCISO surveillance, J. Hosp.
Infect. 48 (2001) 267-274.
[5] T.R. Garrett, M. Bhakoo, Z. Zhang, Bacterial adhesion and biofilms on surfaces, Progr.
ed

Nat. Sci. 18 (2008) 1049-1056.

[6] D. Campoccia, L. Montanaro, C.R. Arciola, The significance of infection related to
orthopedic devices and issues of antibiotic resistance, Biomater. 27 (2006) 2331-2339.
pt

[7] G. Subbiahdoss, R. Kuijer, D.W. Grijpma, H.C. van der Mei, H.J. Busscher, Microbial
biofilm growth vs. tissue integration: "the race for the surface" experimentally studied,
Acta Biomater. 5 (2009) 1399-1404.
ce

[8] J.A. Wright, S.P. Nair, Interaction of staphylococci with bone, Int. J. Med. Microbiol.
300 (2010) 193-204.
[9] R.J. Crawford, H.K. Webb, V.K. Truong, J. Hasan, E.P. Ivanova, Surface topographical
factors influencing bacterial attachment, Adv. Colloid. Interf. Sci. 179-182 (2012) 142-
Ac

149.
[10] K. Anselme, P. Davidson, A.M. Popa, M. Giazzon, M. Liley, L. Ploux, The interaction
of cells and bacteria with surfaces structured at the nanometre scale, Acta Biomater. 6
(2010) 3824-3846.
[11] L. Rizzello, B. Sorce, S. Sabella, G. Vecchio, A. Galeone, V. Brunetti, R. Cingolani,
P.P. Pompa, Impact of nanoscale topography on genomics and proteomics of adherent
bacteria, ACS Nano 5 (2011) 1865-1876.
[12] Y. Wu, J.P. Zitelli, K.S. TenHuisen, X. Yu, M.R. Libera, Differential response of
Staphylococci and osteoblasts to varying titanium surface roughness, Biomater. 32
(2011) 951-960.
[13] D. Campoccia, L. Montanaro, C.R. Arciola, A review of the biomaterials technologies
for infection-resistant surfaces, Biomater. 34 (2013) 8533-8554.

19
Page 21 of 23
[14] L. Pichavant, G. Amador, C. Jacqueline, B. Brouillaud, V. Heroguez, M.C. Durrieu,
pH-controlled delivery of gentamicin sulfate from orthopedic devices preventing
nosocomial infections, J. Controlled Release 162 (2012) 373-381.
[15] A.Y. Vorobyev, C. Guo, Direct femtosecond laser surface nano/microstructuring and its
applications, Laser & Photon. Rev. 7 (2013) 385-407.
[16] V. Oliveira, S. Ausset, R. Vilar, Surface micro/nanostructuring of titanium under
stationary and non-stationary femtosecond laser irradiation, Appl. Surf. Sci. 255 (2009)
7556-7560.
[17] M. Erdoǧan, B. Öktem, H. Kalaycıoǧlu, S. Yavaş, P.K. Mukhopadhyay, K. Eken, K.
Özgören, Y. Aykaç, U.H. Tazebay, F.Ö. Ilday, Texturing of titanium (Ti6Al4V)

t
medical implant surfaces with MHz-repetition-rate femtosecond and picosecond Yb-

ip
doped fiber lasers, Optics Express 19 (2011) 10986-10996.
[18] R.A. Delgado-Ruíz, J.L. Calvo-Guirado, P. Moreno, J. Guardia, G. Gomez-Moreno,

cr
J.E. Mate-Sánchez, P. Ramirez-Fernández, F. Chiva, Femtosecond laser
microstructuring of zirconia dental implants, J. Biomed. Mater. Res. B 96 (2011) 91-
100.

us
[19] C.A. Aguilar, Y. Lu, S. Mao, S. Chen, Direct micro-patterning of biodegradable
polymers using ultraviolet and femtosecond lasers, Biomater. 26 (2005) 7642-7649.
[20] L.G. Harris, R.G. Richards, Staphylococci and implant surfaces: a review, Injury 37
(2006) S3-14.
an
[21] V.K. Truong, R. Lapovok, Y.S. Estrin, S. Rundell, J.Y. Wang, C.J. Fluke, R.J.
Crawford, E.P. Ivanova, The influence of nano-scale surface roughness on bacterial
adhesion to ultrafine-grained titanium, Biomater. 31 (2010) 3674-3683.
M
[22] V. Oliveira, A. Cunha, R. Vilar, Multi-scaled femtosecond laser structuring of
stationary titanium surfaces, J. Optoelectron. Adv. Mater. 12 (2010) 654-658.
[23] V. Oliveira, N.I. Polushkin, O. Conde, R. Vilar, Laser surface patterning using a
Michelson interferometer and femtosecond laser radiation, Opt. Laser Techn. 44 (2012)
ed

2072-2075.
[24] A. Cunha, A.P. Serro, V. Oliveira, A. Almeida, R. Vilar, M.-C. Durrieu, Wetting
behaviour of femtosecond laser textured Ti–6Al–4V surfaces, Appl. Surf. Sci. 265
(2013) 688-696.
pt

[25] A. Cunha, O.F. Zouani, L. Plawinski, A.M. Botelho do Rego, A. Almeida, R. Vilar,
M.C. Durrieu, Human mesenchymal stem cell behavior on femtosecond laser-textured
ce

Ti-6Al-4V surfaces, Nanomedicine: Nanotechnology, Biology and Medicine 10 (2015)

725-739.
[26] H. Humphreys, Staphylococcus aureus: The enduring pathogen in surgery, The Surgeon
10 (2012) 357–360.
Ac

[27] J.M. Liu, Simple technique for measurements of pulsed Gaussian-beam spot sizes, Opt.
Lett. 7 (1982) 196-198.
[28] P. Cheng, D. Li, L. Boruvka, Y. Rotenberg, A.W. Neumann, Automation of
axisymmetric drop shape analysis for measurements of interfacial tensions and contact
angles, Colloid. Surf. 43 (1990) 151-167.
[29] D.K. Owens, R.C. Wendt, Estimation of the surface free energy of polymers, J. Appl.
Polym. Sci. 13 (1969) 1741-1747.
[30] A.P. Serro, Biomineralização de materiais de implante: estudos de molhabilidade, PhD
thesis, Instituto Superior Tecnico, Universidade Técnica de Lisboa, Lisbon, Portugal,
2001.
[31] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image
analysis, Nat. Methods 9 (2012) 671-675.

20
Page 22 of 23
[32] J. Eichstädt, G.R.B.E. Römer, A.J. Huis in ‘t Veld, Determination of irradiation
parameters for laser-induced periodic surface structures, Appl. Surf. Sci. 264 (2013) 79-
87.
[33] J.Z.P. Skolski, G.R.B.E. Römer, J.V. Obona, V. Ocelik, A.J. Huis in 't Veld, J.T.M. De
Hosson, Laser-induced periodic surface structures: Fingerprints of light localization,
Phys. Rev. B 85 (2012) 075320.
[34] F. Variola, J.H. Yi, L. Richert, J.D. Wuest, F. Rosei, A. Nanci, Tailoring the surface
properties of Ti6Al4V by controlled chemical oxidation, Biomater. 29 (2008) 1285-
1298.
[35] C. Anandan, K.S. Rajam, MicroRaman study of the effect of oxide layer on nitriding of

t
Ti–6Al–4V, Appl. Surf. Sci. 254 (2008) 2783-2789.

ip
[36] V.V. Yakovlev, G. Scarel, C.R. Aita, S. Mochizuki, Short-range order in ultrathin film
titanium dioxide studied by Raman spectroscopy, Appl. Phys. Lett. 76 (2000) 1107.

cr
[37] E.P. Ivanova, V.K. Truong, J.Y. Wang, C.C. Berndt, R.T. Jones, I.I. Yusuf, I. Peake,
H.W. Schmidt, C. Fluke, D. Barnes, R.J. Crawford, Impact of nanoscale roughness of
titanium thin film surfaces on bacterial retention, Langmuir 26 (2010) 1973-1982.

us
[38] A. Braem, L. Van Mellaert, T. Mattheys, D. Hofmans, E. De Waelheyns, L. Geris, J.
Anne, J. Schrooten, J. Vleugels, Staphylococcal biofilm growth on smooth and porous
titanium coatings for biomedical applications, J. Biomed. Mater. Res. Part A 102
(2014) 215-224.
an
[39] R. Vasudevan, A.J. Kennedy, M. Merritt, F.H. Crocker, R.H. Baney, Microscale
patterned surfaces reduce bacterial fouling-microscopic and theoretical analysis,
Colloids Surf. B: Biointerf. 117 (2014) 225-232.
M
[40] E. Fadeeva, V.K. Truong, M. Stiesch, B.N. Chichkov, R.J. Crawford, J. Wang, E.P.
Ivanova, Bacterial Retention on Superhydrophobic Titanium Surfaces Fabricated by
Femtosecond Laser Ablation, Langmuir 27 (2011) 3012-3019.
[41] B. Del Curto, M.F. Brunella, C. Giordano, M.P. Pedeferri, V. Valtulina, L. Visai, A.
ed

Cigada, Decreased bacterial adhesion to surface-treated titanium, Int. J. Artificial

Organs 28 (2005) 718-730.
[42] C. Giordano, E. Saino, L. Rimondini, M.P. Pedeferri, L. Visai, A. Cigada, R. Chiesa,
Electrochemically induced anatase inhibits bacterial colonization on Titanium Grade 2
pt

and Ti6Al4V alloy for dental and orthopedic devices, Colloids Surf. B: Biointerf. 88
(2011) 648-655.
ce
Ac

21
Page 23 of 23