Supplementary material
Figure S1. Schematic sketching of the femtosecond ablation system.
Figure S2. Representative SEM images of S. aureus CIP 65.8 adhesion on polished and lotus-like Ti after incubation for 0 min,
10 min, 30 min and 60 min.
Table S1. Number of attached cells and biovolume of produced EPS on as-received and lotus-like Ti surfaces.
Bacterial strains
Retained cells
a
6 10
5
[number of cells mm
72
]
Biovolume of EPS production
(mm)
As-received Lotus-like As-received Lotus-like
S. aureus CIP 65.8
T
0.47 + 0.06 11.09 + 1.51 2.33 + 0.49 8.69 + 1.96
S. aureus ATCC 25923 1.58 + 0.80 2.44 + 0.56 3.00 + 0.60 6.30 + 2.80
S. epidermidis ATCC 14990
T
1.72 + 0.46 8.73 + 2.70 4.50 + 1.00 7.00 + 1.47
Planococcus maritimus KMM 3738 5dl
a
0.53 + 0.13 5dl 0.20 + 0.09
a
Below detection limit.
Ti surface topography and wettability
The surface topography at both micro- and nano-metric scale
is evidenced to place an influence upon the adhesive
behaviours of bacteria (Anselme et al. 2010; Ploux et al.
2010; Rizzello et al. 2011; Webb et al. 2011). In order to
understand surface topography at the micro/nano-scale, three
dimensional topography of as-received and lotus-like Ti over
10 mm 6 10 mm is shown in Figure S2 and a comprehensive
description of surface topography is evaluated by several
surface parameters in Table S2. After femtosecond laser
ablation, there was a significant change in the surface
topography of the as-received Ti surfaces. As shown in
Figure S2, lotus-like Ti surfaces exhibited deep valleys with a
depth of 3.4 mm and large plateaux with the width of 10 mmto
20 mm. On the top of these plateaux, there is a second tier of
nanotopography with undulations of 200 nm. This two-tier
topography was formed spontaneously under femtosecond
laser irradiation. This ‘self-organisation’ effect was reported
previously after femtosecond laser ablation (Vorobyev et al.
2007; Fadeeva et al. 2011). In Table S1, the S
a
, S
q
and S
max
of
lotus-like Ti surfaces are considerably greater than those of
as-received Ti on both the 10 mm 6 10 mm and 5 mm 6 5 mm
scanning areas. The developed surface area ratio (ie the ratio
of surface area to projected area) of lotus-like Ti surfaces
(S
dr
¼ 42.70% and 50.09% respectively for 10 mm 6 10 mm
and 5 mm 6 5 mm scanning areas) is significantly higher than
as-received Ti (S
dr
¼ 0.13% and 0.24% respectively for
10 mm 6 10 mm and 5 mm 6 5 mm scanning areas). To
provide sufficient information on the surface architecture,
skewness (S
sk
) and kurtosis (S
ku
) were also utilised to
describe the distribution of heights. On both the
10 mm 6 10 mm and 5 mm 6 5 mm scanning areas, as-
received Ti surfaces had S
sk
values of 8.63 and 10.99
respectively, and S
ku
values of 137.13 and 211.05 over the
same scanning areas, indicative of a highly uneven
distribution of peaks and valleys and a complicated
surface architecture. In contrast, lotus-like Ti surfaces
have S
sk
values close to 0 and S
ku
values close to 3,
highlighting the relatively symmetric distribution of peaks
and valleys across the analysed surfaces.
The wettability of lotus-like Ti surfaces was examined in
a previous study in which the water contact angle was 1668
(Fadeeva et al. 2011). It can be explained by the Cassie-
Baxter model of wettability, in which the air component
entrapped between micro/nano-structures enhances the
natural surface wettability, according to:
cos y ¼ f
1
ðcos y
1
þ 1Þ1
where y is the composite contact angle of the heterogeneous
surface, f
1
is the area fraction of surface component of Ti and
y
1
is the contact angle on the projected surface of Ti (Cassie
and Baxter 1944).
References
Anselme K, Davidson P, Popa AM, Giazzon M, Liley M,
Ploux L. 2010. The interaction of cells and bacteria with
surfaces structured at the nanometre scale. Acta Bioma-
terial 6:3824–3846.
Cassie ABD, Baxter S. 1944. Wettability of porous surfaces.
Trans Faraday Soc 40:546–551.
Fadeeva E, Truong VK, Stiesch M, Chichkov BN, Crawford
RJ, Wang J, Ivanova EP. 2011. Bacterial retention on
superhydrophobic titanium surfaces fabricated by fem-
tosecond laser ablation. Langmuir. 27:3012–3019.
Ploux L, Ponche A, Anselme K. 2010. Bacteria/material
interfaces: role of the material and cell wall properties. J
Adhes Sci Technol 24:2165–2201.
Rizzello L, Sorce B, Sabella S, Vecchio G, Galeone A,
Brunetti V, Cingolani R, Pompa PP. 2011. Impact of
nanoscale topography on genomics and proteomics of
adherent bacteria. ACS Nano 5:1865–1876.
Vorobyev AY, Makin VS, Guo C. 2007. Periodic ordering of
random surface nanostructures induced by femtosecond
laser pulses on metals. J Appl Phys 101:no. 034903.
Webb HK, Hasan J, Truong VK, Crawford RJ, Ivanova EP.
2011. Nature inspired structured surfaces for biomedical
applications. Curr Med Chem 18:3367–3375.
Table S2. Extended set of surface roughness parameters of control and lotus-like Ti surfaces over scanning areas of
10 mm 6 10 mm and 5 mm 6 5 mm.
Surfaces S
a
(nm) S
q
(nm) S
max
(nm) S
sk
S
ku
S
dr
(%)
10 mm 6
10 mm
As-received Ti 2.12 + 0.34 4.68 + 0.76 120.60 + 19.58 8.63 + 1.40 137.13 + 22.26 0.13 + 0.02
Lotus–like Ti 257.74 + 41.84 328.74 + 53.37 2392.60 + 388.42 70.43 + 0.07 3.03 + 0.49 42.70 + 6.93
5 mm 6
5 mm
As-received Ti 1.01 + 0.16 2.26 + 0.37 71.68 + 11.64 10.99 + 1.78 211.05 + 34.26 0.24 + 0.02
Lotus–like Ti 96.38 + 15.65 121.58 + 19.74 945.37 + 153.47 70.07 + 0.01 3.00 + 0.49 50.09 + 8.13
Table S3. Adhesion kinetics of the polished and lotus-mimicked superhydrophobic Ti surfaces by S. aureus CIP 65.8.
Incubation time
Retained cells
a
6 10
4
[number of cells mm
72
] Biovolume of EPS production (mm)
As-received Lotus-like As-received Lotus-like
10 min 5dl
b
0.3 + 0.07 5dl 1.1 + 0.2
30 min 5dl 9.1 + 0.9 5dl 1.4 + 0.8
60 min 5dl 12.7 + 4.3 5dl 1.5 + 0.6
a
Cell densities have estimated errors of *15–20% due to local variability in the surface coverage;
b
below detection limit.