Applied Surface Science 280 (2013) 578–589

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Applied Surface Science

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

Physical vapor deposited titanium thin films for biomedical

applications: Reproducibility of nanoscale surface roughness and
microbial adhesion properties
Claudia Lüdecke a,b,d , Jörg Bossert a,d , Martin Roth b,d , Klaus D. Jandt a,c,d,∗
a
Chair of Materials Science (CMS), Faculty of Physics and Astronomy, Friedrich Schiller University Jena, Löbdergraben 32, D-07743, Jena, Germany
b
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Bio Pilot Plant, Beutenbergstraße 11a, D-07745, Jena,
Germany
c
Jena Center for Soft Mater, Friedrich Schiller University Jena, Jena, Germany
d
Jena School for Microbial Communication (JSMC), Friedrich Schiller University Jena, Jena, Germany

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

Article history: The surface topography is of great importance for the biological performance of titanium based implants
Received 24 July 2012 since it may influence the initial adsorption of proteins, cell response, as well as microbial adhesion.
Received in revised form 6 May 2013 A recently described technique for the preparation of titanium thin films with an adjustable surface
Accepted 10 May 2013
roughness on the nanometer scale is the physical vapor deposition (PVD). The aims of this study were
Available online 17 May 2013
to statistically evaluate the reproducibility of nanorough titanium thin films prepared by PVD using an
atomic force microscopy (AFM) based approach, to test the microbial adhesion in dependence of the
Keywords:
nanoscale surface roughness and to critically discuss the parameters used for the characterization of the
PVD
Titanium thin films
titanium surfaces with respect to AFM microscope settings. No statistically significant differences were
Nanoroughness found between the surface nanoroughnesses of the PVD prepared titanium thin films. With increasing
Escherichia coli surface nanoroughness, the coverage by Escherichia coli decreased and the microbial cells were increas-
AFM scan size ingly patchy distributed. The calculated roughness values significantly increased with increasing AFM
Biomaterials scan size, while image resolution and pixel density had no influence on this effect. Our study shows that
PVD is a suitable tool to reproducibly prepare titanium thin films with a well-defined surface topography
on the nanometer scale. These surfaces are, thus, a suitable 2D model system for studies addressing the
interaction between surface nanoroughness and the biological system. First results show that surface
roughness even on the very low nanometer scale has an influence on bacterial adhesion behavior. These
findings give new momentum to biomaterials research and will support the development of biomaterials
surfaces with anti-infectious surface properties.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction implant surface roughness strongly influences the initial protein

There is a wide range of biomaterials that can be used as implant
materials. Some of the commonly used materials for implants are
metals, such as titanium or titanium alloys [1]. Titanium is a bio-
compatible material, largely due to its inherently stable surface
oxide layer. It is extensively used in dental, orthopedic and cardio-
vascular fields in the form of dental implants, hip-joint replacement
devices or heart-valves and cardiac pacemakers [2]. For the bio-
logical performance of titanium based biomaterials, the texture
and roughness of their surfaces is of great importance [3]. The
∗ Corresponding author at: Chair of Materials Science (CMS), Faculty of Physics
and Astronomy, Friedrich Schiller University Jena, Löbdergraben 32, D-07743, Jena,
Germany. Tel.: +49 3641 947730; fax: +49 3641 947732.
E-mail address: k.jandt@uni-jena.de (K.D. Jandt).
adsorption [4], the cell response [5], the tissue adhesion [6] as well
as the adhesion of microorganisms [7].
Several techniques, such as grinding and polishing, blasting,
plasma spraying or chemical etching can be used for the prepa-
ration of titanium surfaces with different roughnesses [8,9]. Most
of these methods may be accompanied by not only modifying the
surface roughness, but also applying scratches or superordinate
structures and, particularly, changing the surface chemistry [10].
For the investigation of the influence of a variation of the bio-
materials surface roughness on the biological environment, these
preparation techniques might, thus, be disadvantageous.
Previously, we demonstrated that physical vapor deposition
(PVD) is a suitable and straight forward technique to prepare tita-
nium samples with a surface roughness on the nanometer scale
without changing the surface chemistry [11]. These titanium thin
films (TiO2 -TFs) might be suitable surfaces to study the influence

0169-4332/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.apsusc.2013.05.030
 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589
of titanium surface roughness in the nanometer range on the bio-
logical surrounding. Reproducibility of the preparation method is
a mandatory requirement for model surfaces to assure the repro-
ducibility and comparability of the results within a study and
between different studies [12]. The statistical evaluation of the
PVD-TiO2 -TFs for reproducibility was, to our best knowledge, not
part of previous studies.
To characterize surface structures on the nanometer scale,
atomic force microscopy (AFM) is an appropriate and established
method [13]. Investigations of the interaction between a biomate-
rial and its biological environment often require different scan sizes
for AFM measurements, due to differences in the size of proteins,
cells, microorganisms or biofilms [14]. Calculated roughness values
based on AFM measurements can vary with the used AFM scan size
or the image resolution [14–16] which negatively affects the com-
parability of the results between different studies. For uniform and
smooth PVD-TiO2 -TFs with roughnesses in the lower nanometer
range this was not shown before.
The first aim of our study was, therefore, to evaluate the repeat-
ability and reproducibility of the PVD technique for the preparation
of TiO2 -TFs with respect to commonly used surface characteriza-
tion parameters using an AFM based approach. The suitability of the
PVD-TiO2 -TFs as model surfaces for the investigation of the influ-
ence of nanoroughness on the interaction between the biological
system and the biomaterials surface was discussed. In addition, the
influence of an increasing surface nanoroughness on the adhesion
of Escherichia coli was investigated. The second aim of our study was
to critically analyze and discuss the calculated surface parameters
of the TiO2 -TFs with respect to AFM scan size, image resolution and
pixel density.
2. Materials and methods
2.1. Titanium thin film preparation
Glass slides (diameter 15 mm; Borofloat® B33; Jena 4H Engi-
neering GmbH, Jena, Germany) were used as substrates for the
TiO2 -TF deposition. The glass slides were cleaned by different
sonification steps: 30 min in Deconex® 11 Universal Cleaner (1%
v/v solution in double distilled water; Borer Chemie, Zuchwil,
Switzerland) (80 ◦ C), 10 min in double distilled water (20 ◦ C),
10 min in ethanol (>99.9%), 10 min in acetone (20 ◦ C) and 10 min
in double distilled water (20 ◦ C). Subsequently, the glass slides
were dried with filtered, compressed air and immediately used for
the deposition of the TiO2 -TFs. For the preparation of the TiO2 -
TFs, the PVD method was used as described before [11]. With
this technique, the titanium surface roughness can be adjusted
by the deposition rate and the film thickness. For evaluation of
the PVD technique, titanium (purity of 99.99%; Mateck-Material-
Technologie & Kristalle GmbH, Jülich, Germany) was evaporated
with an electron-beam evaporator (Univex 350, Leybold, Germany)
with a deposition rate of 0.5 nm/s and a film thickness of 200 nm
at a vacuum of 6 × 10−6 Torr. The film thickness was monitored
by a quartz balance installed inside the evaporation chamber. The
deposition rate was kept constant and controlled by a deposi-
tion monitor with an error range of 0.01 nm/s. 150 glass slides
were titanium coated in overall 6 deposition runs. From each run,
three coated glass slides were investigated with AFM at three ran-
domly chosen locations on each sample. Due to the contact of the
evaporated titanium thin films with atmospheric oxygen, a tita-
nium dioxide (TiO2 ) layer with an approximate thickness of 5 nm
is immediately formed [11].
For bacterial adhesion tests, in addition, TiO2 -TFs were prepared
with deposition rates and film thicknesses of 0.1 nm/s and 100 nm
(sample group A), 0.5 nm/s and 200 nm (group B), 0.5 nm/s and
500 nm (group C), and 1.0 nm/s and 500 nm (group D), respectively.
579
2.2. Atomic force microscopy (AFM)
An atomic force microscope (Dimension 3100, Digital Instru-
ments, Santa Barbara, CA, USA), equipped with a standard silicon
tip (tip diameter <10 nm) was used to investigate the topography
of the TiO2 -TF surfaces. Prior to the measurements, the accuracy
of the AFM was tested by the measurement of a standard AFM
calibration sample (TGQ1; NT-MDT Co., Moscow, Russia). For
TiO2 -TF surface characterization, the AFM was operated in tapping
mode with a scan rate of 2 Hz, and a scan size of 1 ␮m × 1 ␮m. For
the titanium samples of sample group B, in addition, scan sizes of
25 ␮m × 25 ␮m and 50 ␮m × 50 ␮m were used. All AFM data were
acquired with a resolution of 512 × 512 points. The point-to-point
distance was, thus, 1.95 nm (1 ␮m × 1 ␮m scan size), 48.83 nm
(25 ␮m × 25 ␮m scan size) and 97.66 nm (50 ␮m × 50 ␮m scan
size). Additional images were obtained from three titanium
samples (sample group B) prepared during the fifth deposition
run with a resolution of 128, 256, 512, 1024 and 2048 points
per line with a scan size of 12.5 ␮m × 12.5 ␮m, 25 ␮m × 25 ␮m
and 50 ␮m × 50 ␮m, to keep the pixel density of the AFM images
constant with increasing scan size.
2.3. Bacterial adhesion test and model organism
Bacterial adhesion was investigated on the physical vapor
deposited nanorough titanium surfaces of the sample groups A–D.
E. coli EC081 was used as model organism for the bacterial adhe-
sion tests. EC081 was obtained by transformation of the E. coli K-12
strain RV308 (=ATCC 31608) carrying the plasmid pMK3c2GFP. The
green fluorescent protein (GFP) is constitutively produced in EC081
which ensures a completely fluorescent cell population suitable for
easy monitoring of the cell attachment on the titanium surfaces
with confocal laser scanning microscopy. In addition, the plasmid
contains a kanamycin resistance gene causing post-segregational
killing of plasmid-free cells in the presence of kanamycin.
E. coli EC081 was cultivated as a batch culture in LB (lysogeny
broth, Luria Bertani) medium with kanamycin (50 ␮g/mL) for 3 h, in
an orbital shaker at 200 rpm and 30 ◦ C. In the exponential growth
phase, the culture was diluted with LB to an optical density OD
( = 600 nm) of 0.5, measured with a spectrophotometer (Spekol
1100, Analytic Jena, Jena, Germany). 3 mL of the diluted culture
were used to inoculate each titanium sample (group A–D, n = 3,
sterilized) placed in a standard 12-well culture plate. Samples were
taken after 6 h of incubation at room temperature.
2.4. Confocal laser scanning microscopy and scanning electron
microscopy
Titanium samples with adhered bacteria were fixed with
phosphate-buffered formaldehyde solution (4%, Roti® -Histofix,
Carl Roth GmbH, Karlsruhe, Germany) for 12 h at 4 ◦ C and air-dried
at room temperature in the dark, afterwards. During preparation,
samples were handled with special care to prevent a detachment
and loss of adhered bacteria e.g. due to shear forces during rins-
ing. Dried samples were mounted on microscope slides, embedded
using the ProLong® Antifade Kit (P7481, Molecular Probes® , Darm-
stadt, Germany) according to the manufacturer’s instruction and
covered with cover slips to prevent photobleaching effects dur-
ing fluorescence microscopy and to enable long-time storage (for
months) of the samples at −20 ◦ C.
A confocal laser scanning microscope (CLSM; Zeiss LSM 510
Meta, Carl Zeiss MicroImaging, Jena, Germany) equipped with an
Argon/2 laser unit (488 nm) and a 63× NA 1.3 oil immersion lens
objective (Zeiss PLANAPOCHROMAT® ) was used for fluorescence
imaging of the bacteria. With a 1.5-fold digital zoom, the basic field
of view for each image was 71.4 ␮m × 71.4 ␮m. Images were taken
580 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589
at five different randomly chosen points on the sample groups
A–C and at 10 different randomly chosen points on the samples of
group D.
For scanning electron microscopy (SEM), biofilm samples were
fixed with glutardialdehyde solution (2.5% in PBS) at room tem-
perature for 12 h. After fixation, samples were washed for 10 min
in PBS buffer solution and twice in distilled water. Subsequently,
samples were dehydrated using an ascending ethanol series from
10 to >99.9% with 10 min incubation per step and two times 30 min
for the last step. The dehydrated samples were critical point dried
(EMITECH K850, Quorum Technologies Ltd., East Grinstead, UK) and
gold sputter coated (∼5 nm) (S150B, Edwards Ltd., Crawley, UK).
For SEM imaging, an AURIGA 60 CrossBeam® FIB-SEM scanning
electron microscope (Carl Zeiss AG, Oberkochen, Germany) was
used at a magnification of ∼6000× operated at 5 kV and a working
distance of about 4 mm.
2.5. Propagation of uncertainty
The AFM measurements of the TiO2 -TFs and respective calcula-
tions of the surface characterizing values were carried out on three
different randomly chosen points on each sample with overall n = 3
titanium samples, thus, giving 9 different locations, overall. CLSM
images were acquired at 5 (sample group A–C) or 10 (group D)
different randomly chosen points on each titanium sample with
overall n = 3 samples. For all mathematical operations which were
performed on the measured quantities, a propagation of uncer-
tainty was applied according to [17] since the quantity of interest
for statistical analysis was not obtained by measuring this quantity
directly.
2.6. Accuracy and precision
The experimental error was determined by its accuracy and its
precision [12]. The accuracy describes how close a measured value
is to the true or accepted value. The accuracy of the AFM measure-
ments was determined as:
|E − A|
%error =
A
where E (experimental value) is the measured average surface
profile height of a standard sample for AFM calibration and A
(true/accepted value) is the height of the calibration sample given
by the manufacturer (see Section 2.2).
For the calculated surface roughnesses of the TiO2 -TFs, the pre-
cision was determined. The precision describes how closely two
or more measurements agree with each other and is in this study
referred to as repeatability and reproducibility [12]. Repeatability is
the degree of agreement of measurements on replicate specimens
by the same researcher in the same laboratory. The repeatability
was tested by the statistical comparison (analysis of variance, see
below at Section 2.7) of the mean roughness values and according
to standard deviations of the titanium surfaces produced during
the 6 different deposition runs.
Reproducibility is the degree of agreement between measure-
ments on replicate specimens in different locations by different
people and at different time points. The reproducibility was, there-
fore, determined by the comparison of the experimental results
of the current study with the results of the study from Cai et al.
[11]. The mean roughness values of the TiO2 -TFs produced during
the 6 deposition runs calculated for the scan size 50 ␮m × 50 ␮m
were statistically compared to the surface roughness values of the
TiO2 -TFs investigated in Cai’s study.
2.7. Statistical analysis
Using the ANOVA analysis and a Scheffé t-test (95% confi-
dence interval, p ≤ 0.05), the deposition runs and the roughness
values were statistically analyzed with Statgraphics Centurion XV
(StatPoint Inc., Warrenton, USA). Results are given as p-values and
F-ratio. The F-ratio calculated by the software is the quotient of
the variances of two groups of samples. The critical value for the
F-ratio is 4.43 based on the F-distribution: F21 (p-value), whereas
1 and 2 are the degrees of freedom from the sample groups, and
p is 0.05. If the F-ratio is >4.43, the variances of the sample groups
are statistically significantly different. Using the ANOVA analysis
and a Scheffé t-test (95% confidence interval, p ≤ 0.05) the bacterial
adhesion on the titanium samples (estimated surface coverages)
was statistically analyzed.
2.8. Image processing and analysis
The AFM image processing and the calculation of the surface
parameters were performed with Gwyddion 2.25 free SPM data
analysis software (Brno, Czech Republic, http://gwyddion.net/).
The AFM image processing was applied based on the guidelines
for raw AFM micrographs [18]. A plane leveling was used, whereas
the plane was computed from all the image points and was sub-
tracted from the data [19]. In addition, a line by line fit was applied
and scanning errors parallel to the scanning axis were removed.
Roughness parameters were calculated according to the following
equations (Standard: ISO 4287-1997) with the number of points N
(intersections of the surface profile with the mean line), rj the height
of the profile at point j and Rq as the RMS roughness parameter [19]:
Arithmetic average roughness (Ra )
1
N
Ra = |rj | (1)
N
j=1
Root mean square roughness (RMS)

 N
1
RMS =  r2 j
(2)
N
j=1
(i) Skewness (Rsk )
1 
N
Rsk = rj3 (3)
NRq3
j=1
Kurtosis (Rku )
1 
N
Rku = rj4 (4)
NRq4
j=1
In addition, the measured surface area (MSA) and the fractal
dimension (FD) were calculated. The FD characterizes the rough-
ness of a self-similar surface and varies from 2 for a totally smooth
surface to 3 for an infinitely rough surface. For the fractal analysis,
the cube counting method was used [19]. The algorithm is based
on the following steps: a cubic lattice with a lattice constant h is
superimposed on the z-expanded surface. Initially, h is set at X = 2
(where X is the length of the edge of the surface), resulting in a
lattice of 2 × 2 × 2 = 8 cubes, with N(h) being the total number of
cubes. The lattice constant h is then reduced stepwise by a factor of
2 and the process is repeated until h equals the distance between
two adjacent pixels. The slope of a plot of log N(h) versus log (1/h)
gives the fractal dimension directly [19].
For the calculation of the average peak-to-peak distances of
the nanorough titanium surfaces, the coordinates of the local
 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589 581

Table 1
Roughness values, fractal dimension, measured surface area (MSA) and the deviation of MSA from the scanned surface area (SSA) of the titanium thin films (deposition rate
0.5 nm/s, film thickness 200 nm) dependent on scan size and deposition run (mean ± standard deviation; with n = 3 for the titanium thin films (with 3 images/sample and
per scan size) and n = 1 for industrial purchased Borofloat glass slides.

Scan size Runa Ra (nm) RMS (nm) Skewness Kurtosis Fractal dimension MSA (␮m2 ) Deviation MSA
from SSA (%)

1 ␮m × 1 ␮m 1 2.41 ± 0.46 3.00 ± 0.54 −0.13 ± 0.12 −0.14 ± 0.22 2.42 ± 0.03 1.10 ± 0.04 8.74 ± 4.37
2 2.07 ± 0.35 2.58 ± 0.44 0.13 ± 0.09 −0.09 ± 0.14 2.41 ± 0.04 1.10 ± 0.07 8.42 ± 5.69
3 2.15 ± 0.09 2.70 ± 0.11 0.21 ± 0.10 −0.01 ± 0.08 2.43 ± 0.01 1.10 ± 0.02 8.84 ± 1.35
4 2.15 ± 0.17 2.70 ± 0.23 0.35 ± 0.05 0.20 ± 0.49 2.45 ± 0.02 1.18 ± 0.02 9.16 ± 1.12
5 1.89 ± 0.12 2.37 ± 0.14 0.40 ± 0.06 0.06 ± 0.14 2.42 ± 0.02 1.07 ± 0.01 6.28 ± 1.13
6 1.98 ± 0.15 2.47 ± 0.19 0.31 ± 0.05 −0.11 ± 0.06 2.42 ± 0.02 1.07 ± 0.03 6.37 ± 2.18
Glass 0.21 0.27 0.38 0.98 2.37 1.002 0.179

25 ␮m × 25 ␮m 1 2.80 ± 0.24 3.46 ± 0.31 0.29 ± 0.09 0.07 ± 0.33 2.58 ± 0.05 626.84 ± 0.81 0.29 ± 0.13
2 2.62 ± 0.61 3.29 ± 0.77 0.27 ± 0.13 0.35 ± 0.41 2.61 ± 0.04 628.09 ± 1.80 0.49 ± 0.28
3 2.73 ± 0.08 3.40 ± 0.10 0.27 ± 0.06 0.01 ± 0.13 2.61 ± 0.02 627.51 ± 0.24 0.40 ± 0.04
4 2.11 ± 0.16 2.45 ± 0.48 0.25 ± 0.04 −0.02 ± 0.31 2.61 ± 0.04 626.20 ± 0.31 0.19 ± 0.05
5 1.86 ± 0.44 2.41 ± 0.74 0.47 ± 0.23 −0.24 ± 0.10 2.55 ± 0.05 625.46 ± 0.07 0.07 ± 0.01
6 2.81 ± 0.51 3.50 ± 0.64 0.31 ± 0.05 0.04 ± 0.16 2.64 ± 0.03 629.12 ± 1.81 0.65 ± 0.28
Glass 2.52 2.96 0.67 0.01 2.17 625.04 0.006

50 ␮m × 50 ␮m 1 6.42 ± 0.39 7.71 ± 0.56 0.56 ± 0.07 −0.10 ± 0.63 2.41 ± 0.08 2501.95 ± 2.31 0.08 ± 0.09
2 4.98 ± 0.96 6.08 ± 1.11 0.49 ± 0.09 0.01 ± 0.37 2.55 ± 0.04 2504.97 ± 0.62 0.20 ± 0.04
3 5.66 ± 0.43 6.72 ± 0.49 0.41 ± 0.07 −0.49 ± 0.22 2.45 ± 0.04 2501.83 ± 0.40 0.07 ± 0.02
4 4.66 ± 0.77 5.52 ± 0.89 0.41 ± 0.06 −0.45 ± 0.59 2.49 ± 0.01 2500.91 ± 0.43 0.04 ± 0.02
5 4.41 ± 1.19 5.20 ± 1.30 0.35 ± 0.29 −0.19 ± 0.89 2.40 ± 0.05 2500.31 ± 0.08 0.01 ± 0.00
6 4.95 ± 0.79 6.12 ± 1.02 0.59 ± 0.08 0.03 ± 0.33 2.63 ± 0.05 2509.05 ± 4.41 0.36 ± 0.18
Glass 5.22 6.19 0.78 −0.23 2.15 2500.05 0.002

AFM measurements: images were obtained in tapping mode in air with a resolution of 512 × 512 pixel; scan speed, scan angle, gains and setpoint were kept constant. AFM
image processing: raw images were subjected to a plane leveling, a 1st order line-by-line fit and a scar removal parallel to the scanning axis.
a
Number of deposition run during sample preparation of the titanium thin films; deposition rate 0.5 nm/s; film thickness 200 nm; in comparison roughness values, fractal
dimension, MSA, and deviation of MSA from SSA are given for the uncoated Borofloat® B33 glass slides.

maxima of the AFM height images were estimated using ImageJ
(National Institutes of Health NIH, Bethesda, Maryland, USA). Based
on a Delaunay triangulation algorithm the distances between
the neighboring maxima were calculated using MATLAB (Math-
Works, Natick, Massachusetts, USA). Surface coverages of the CLSM
images were calculated using the free software bioImage L v.2.1
[20]. For image analysis a factor of 0.03 for noise reduction was
applied.
3. Results
3.1. Evaluation of titanium thin films prepared with PVD
The accuracy of the AFM was tested by measuring an AFM
calibration standard sample and revealed an experimental value
of 21.18 ± 0.86 nm height compared to the true, respectively,
accepted value of 20.5 ± 1.5 nm given by the manufacturer. There-
fore, the accuracy (%error) of the AFM measurements calculated
according to Eq. (1) was 0.0332 which corresponds to a deviation
of 3.32% from the measured value to the true value. Furthermore,
the calculated mean value and the true value given by the company
are in the range of each other which is shown by their overlapping
standard deviations. Thus, no further calibration of the AFM was
necessary.
Table 1 summarizes the roughness values, the FD, the measured
surface area (MSA) and the deviation of the MSA and the scanned
surface area (SSA) depending on the deposition run (run 1–6) and
the used scan sizes for the TiO2 -TFs prepared with a deposition
rate of 5 nm/s and a film thickness of 200 nm. Table 2 shows the
results of the statistical analysis (Analysis of Variance, ANOVA) of
the different deposition runs for the scan size 1 ␮m × 1 ␮m. There
were no statistically significant differences between the roughness
values, the FD and the MSA of the six deposition runs (p ≥ 0.05, F-
ratio <4.43). For skewness (p ≤ 0.05, F-ratio >4.43), we found the
titanium deposition run 1 statistically significantly different from
the other five deposition runs.
To test the reproducibility of the surface roughness of the TiO2 -
TFs and, thus, the reproducibility of the PVD technique for the
preparation of these surfaces, the calculated RMS mean values
of the titanium samples (deposition rate 5 nm/s, film thickness
200 nm, AFM scan size 50 ␮m) produced during the 6 deposition
runs were compared to the surface roughness values of the TiO2 -
TFs (prepared with the same parameters) investigated by Cai et al.
[11]. The statistical analysis (Scheffé t-test) showed that there was
no statistically significant difference between the roughness values

Table 2
Results of the statistical analysis (Analysis of Variance) of the surface roughness values, fractal dimension and measured surface area (MSA) of the titanium thin films
(deposition rate 0.5 nm/s, film thickness 200 nm) for scan size 1 ␮m × 1 ␮m dependent on the deposition run (n = 9 per run); confidence interval 95% with p ≤ 0.05, critical
value for F-ratio is 4.43.

Ra RMS Skewness Kurtosis Fractal dimension MSA

Run 1 × × × × × ×
2 × × × × × × ×
3 × × × × × × ×
4 × × × × × ×
5 × × × × × ×
6 × × × × × ×
p-Value 0.18 0.18 0.00 0.08 0.25 0.20
F-ratio 1.82 1.82 8.13 2.58 1.55 1.76

Homogeneous groups are displayed by crosses (×); if the crosses are beneath each other, there is no statistical significance of difference. If the crosses are moved towards
the right, there is a statistically significant difference.
582 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589
Table 3
Characterization of the PVD-TiO2 -TF surfaces prepared with different deposition rates and film thicknesses (sample group A–D) used for the test of the bacterial adhesion:
surface roughness values Ra and RMS, the maximum vertical peak-to-valley distance and the average horizontal peak-to-peak distance. In addition, bacterial surface coverage
after 6 h of incubation is given. AFM scan size 1 ␮m × 1 ␮m, resolution 512 × 512 pixel; n = 3 for AFM images and n = 3 for CLSM images used for analysis.
Sample group Rate (nm/s)/thickness (nm) Ra ± SD (nm) RMS ± SD (nm)
A 1/100 1.58 ± 0.10 2.00 ± 0.12
B 5/200 1.81 ± 0.01 2.28 ± 0.01
C 5/500 2.35 ± 0.14 2.96 ± 0.19
D 10/500 4.82 ± 0.18 6.13 ± 0.19
a
Coordinates of the local maxima of the AFM height images were estimated and based on a Delaunay triangulation algorithm the distances between the neighboring
maxima were calculated.
of the TiO2 -TFs prepared in this study compared to that prepared
in the study of Cai et al. (data not shown). The calculated mean
surface roughness (RMS) of all TiO2 -TFs produced during the six
deposition runs was 6.2 ± 1.6 nm. The mean RMS value of the TiO2 -
TFs prepared and investigated by Cai et al. was 4.9 ± 0.2 nm. Both
values were within the deviation range of each other.
3.2. Bacterial adhesion on nanorough titanium thin films
TiO2 -TFs were prepared with different deposition rates and film
thicknesses resulting in different surface nanoroughnesses (sample
groups A–D; Table 3, Fig. 1). The calculated average surfaces rough-
ness (Ra ) and root mean square roughness (RMS) of the TiO2 -TFs
based on AFM images with a scan size of 1 ␮m × 1 ␮m increased
from 1.58 ± 0.10 nm, respectively, 2.00 ± 0.12 nm for the samples
of group A to 4.82 ± 0.18 nm, respectively, 6.13 ± 0.19 nm for the
samples of group D. The maximum peak-to-valley distance (verti-
cal) and the average peak-to-peak distance (horizontal) increased
from 16.94 ± 1.72 nm and 13.12 ± 6.43 nm to 45.17 ± 2.84 nm and
42.17 ± 23.20 nm.
The bacterial coverage decreased from 23.12 ± 1.60% to
19.11 ± 7.09% with increasing surface roughness (Table 3, Fig. 2).
Although this decrease is an almost one fifth reduction of the sur-
face coverage, there were no statistically significant differences
between the average coverage of the TiO2 -TFs of groups A and D,
since the standard deviation of group D is notably high. The bacte-
rial adhesion on the nanorough titanium surfaces after 6 hours as
well as histograms of the distribution of the data are shown in Fig. 3.
It has been observed that the distribution of the bacteria attached to
the thin films of group A was very homogeneous resulting in a low
standard deviation of the mean coverage value. On the contrary,
the distribution of the bacteria attached to the thin films of group
D was inhomogeneous and patchy, as shown by the CLSM image
Fig. 3 and visualized by the according histogram, which results in
a relatively high standard deviation of the bacterial coverage on
the titanium surfaces of sample group D. A beginning production
of extracellular polymeric substances (EPS) after 6 h of incubation
of the bacteria on the surfaces was observed (Fig. 4).
3.3. Titanium surface characterizing parameters
AFM micrographs of a titanium surface prepared with the PVD
technique (deposition rate 5 nm/s, film thickness 200 nm) obtained
with different scan sizes are shown in Fig. 5b–d. The TiO2 -TFs were
deposited as uniform layers on the glass substrates. Fig. 5a shows
the uncoated borofloat® glass substrate.
The calculated values of Ra , RMS and skewness statistically sig-
nificantly increased with increasing AFM scan size, whereas the
values of kurtosis and FD did not statistically significantly increase
with increasing scan size (Table 1). With increasing scan size the
calculated Ra mean value increased from ∼1.9 nm to ∼6.4 nm, the
calculated RMS mean value from ∼2.4 nm to ∼7.7 nm and the calcu-
lated mean value of skewness from ∼−0.1 to ∼0.6. Kurtosis values
varied between −0.5 and 0.3 and the FD between 2.4 and 2.6. The
Peak-to-valley ± SD (nm) Peak-to-peak ± SD (nm)a Bacterial surface
coverage ± SD (%)
16.94 ± 1.72 13.12 ± 6.43 23.12 ± 1.60
19.93 ± 1.21 17.87 ± 8.05 22.78 ± 5.67
24.66 ± 6.49 26.40 ± 11.33 20.54 ± 3.28
45.17 ± 2.84 42.17 ± 23.20 19.11 ± 7.09
MSA proportionally decreased with increasing scan size with up
to 12% deviation from the measured surface area to the scanned
surface area for 1 ␮m × 1 ␮m scan size and below 0.4% deviation
from the MSA to the SSA for 50 ␮m × 50 ␮m scan size.
The effect of increasing calculated Ra and RMS values with
increasing scan size used for AFM measurements was described
in the literature before but no comprehensive explanation for this
was found in these studies [14,15,21]. This effect was, therefore, for
the most often used roughness values investigated in more detail.
The roughness values Ra and RMS were calculated with respect
to pixel resolution and scan size (Fig. 6a) and in relation to scan
size and pixel density (Fig. 6b). Our results show that an increas-
ing resolution (256, 512 and 1024 pixel/line) had no influence on
the measured Ra and RMS values, nevertheless, with increasing
scan size the calculated roughness values increased. The effect
of increasing measured roughness with increasing scan size was
identical for all measured pixel resolutions (Fig. 6a) and pixel den-
sities (Fig. 6b), respectively. Measured Ra and RMS values were, as
expected for the uniform titanium layers, not statistically signif-
icantly different from each other within the same scan size, but
they became significantly higher with increasing AFM scan size
independent of resolution or pixel density of the AFM image.
As additional or alternative parameter for the surface character-
ization of the TiO2 -TFs next to the roughness, the fractal dimension
was calculated with respect to the deposition run and the scan size
with a constant resolution of 512 × 512 pixel per image (Table 1)
and with respect to different resolutions between 256 and 1024
pixel/line for the three scan sizes. Fig. 7 shows that the FD, given by
the slope of the linear fitting, was the same for the different reso-
lutions (a–c) and differed only slightly between the different scan
sizes (d).
4. Discussion
4.1. Evaluation of the PVD technique
The PVD technique allows for the preparation of TiO2 -TFs with
desired surface properties of structure and composition [11]. By
varying the deposition rate and the film thickness it is easily and
controllably possible to prepare titanium samples with various
surface roughnesses in the nanometer range between 1 nm and
21 nm [11,22]. Since surface roughness is one of the most impor-
tant factors influencing the biological performance of an implant,
decisively affecting the adsorption of proteins and biomolecules
[23], the attachment of cells [3] or the adhesion of microorganisms
[24], the PVD technique provides a powerful tool to significantly
influence the interaction of an implant surface with its surrounding
environment. For the qualitative and quantitative characterization
of biomaterials surfaces and interfaces in general and rough tita-
nium surfaces in particular, atomic force microscopy is a suitable
and commonly used tool which provides excellent results on the
nanometer scale [13] and was, therefore, the method of choice to
be used in our study.
 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589
Fig. 1. AFM 3D height images of the PVD-TiO2 -TF surfaces prepared with different
deposition rates and film thicknesses used to test of the bacterial adhesion: 0.1 nm/s
and 100 nm (a), 0.5 nm/s and 200 nm (b), 0.5 nm/s and 500 nm (c) and 1 nm/s and
500 nm (d); scan size 1 ␮m × 1 ␮m.
To evaluate the PVD method, the repeatability and reproducibil-
ity of this technique was investigated. Repeatability is the degree
of agreement of measurements on replicate specimens by the same
researcher in the same laboratory [12]. Six independent titanium
deposition runs were carried out. Several surface characterizing
parameters were calculated and analyzed with respect to scan size
and deposition runs (Table 1). To test statistically for variations
between the runs, thus, for the repeatability of the method, an anal-
ysis of variance (ANOVA) was performed on the results of the scan
583
Fig. 2. Surface coverage of Escherichia coli cells (plotted as empty bars with standard
deviation; n = 3 with 5 measured points on each sample) adhered to PVD-TiO2 -TF
prepared with different deposition rates and film thicknesses resulting in differ-
ent surface nanoroughnesses (plotted as filled bars; n = 3): 0.1 nm/s and 100 nm,
2.00 ± 0.12 nm (sample group A), 0.5 nm/s and 200 nm, 2.28 ± 0.01 nm (sample
group B), 0.5 nm/s and 500 nm, 2.96 ± 0.19 nm (sample group C), and 1 nm/s and
500 nm, 6.13 ± 0.19 nm (sample group D).
size 1 ␮m × 1 ␮m. The statistical analysis (Table 2) revealed that
there were no significant differences between the calculated rough-
ness parameters Ra , RMS and kurtosis of the TiO2 -TFs produced
during the 6 deposition runs. PVD is, therefore, a suitable method
to repeatably prepare TiO2 -TFs with a surface roughness on the
nanometer scale. The mean arithmetic average surface roughness
Ra calculated for 1 ␮m × 1 ␮m scan size of all deposition runs was
2.10 nm with a standard deviation of 0.30 nm. This result showed
that within an error range of 14.3%, including the error of tita-
nium sample preparation and AFM measurements, it is possible
with the PVD technique to prepare TiO2 -TFs with desired surface
roughnesses. Nevertheless, since the surface roughnesses are on
the nanometer scale, this error range means a deviation of much
less than 1 nm. The fractal analysis of the surfaces and the calcula-
tion of the measured surface area and the deviation between the
measured and the scanned surface area supported these findings.
However, it remains unclear why the mean skewness value of the
first run statistically differs from the mean values calculated for the
titanium surfaces prepared during the other five runs.
Reproducibility is the degree of agreement between measure-
ments on replicate specimens in different locations by different
people and at different time points [12]. The reproducibility was,
therefore, determined by the comparison of the experimental
results of the current study with the results of the study from Cai
et al. [11] which was performed at a different location, by a dif-
ferent researcher and at a different time point. It was shown that
there was no statistically significant difference between the rough-
ness values of the TiO2 -TFs prepared in this study compared to that
prepared in the study of Cai et al. Furthermore, the mean surface
roughness (RMS) for the TiO2 -TFs investigated in the current study
and in the study of Cai et al. were within the standard deviation
range of each other. Thus, the PVD method provides next to repeat-
able also reproducible results since both compared measurements
agree well with each other. Accordingly, the PVD technique can
be described as a precise method referring to the definition given
584 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589

Fig. 3. CLSM images of Escherichia coli cells adhered to PVD-TiO2 -TF prepared with different deposition rates and film thicknesses resulting in different surface nanorough-
nesses (RMS): 0.1 nm/s and 100 nm, 2.00 ± 0.12 nm (Group A), 0.5 nm/s and 200 nm, 2.28 ± 0.01 nm (Group B), 0.5 nm/s and 500 nm, 2.96 ± 0.19 nm (Group C) and 1 nm/s
and 500 nm, 6.13 ± 0.19 nm (Group D). In addition, the histograms of the estimated surface coverages of E. coli cells on the different nanorough titanium surfaces are shown,
giving the distribution of the data.

above. The higher mean and the relatively high standard deviation and low costs [25]. Our results showed that with the PVD technique
of the surface roughness in our study could be explained by the it is possible to prepare titanium surfaces with controlled surface
lower number of measured replicates but could be also due to the nanotopographies which match these requirements, as the thin
different software used for the calculations or was even caused by a films provide uniform and reproducible two-dimensional surfaces
higher order fit of the measured values during the data processing with well-defined nanoroughnesses. The thin films were deposited
in the study of Cai et al. which could have led to a loss of height on glass surfaces with an average surface roughness of 0.21 nm
data and correspondingly decreasing roughness values [18].
We demonstrated that the PVD technique provides a powerful
tool for the repeatable and reproducible preparation of TiO2 -
TFs with a well-defined nanoscale surface topography. The PVD
method, thus, expands the pool of available techniques for the
preparation of nanorough titanium surfaces.

4.2. PVD deposited titanium thin films as 2D nanorough model

surfaces

The surface topography of biomaterials is well known as one of

the most important factors influencing biological reaction [3] and,
thus, contributing to the biocompatibility of the material. But, much
more work is necessary to further advance a detailed understand-
ing of the very complex interactions between biological systems
and nanostructured biomaterials surfaces. For this, first, simple
two-dimensional (2D) model systems are required with well-
characterized surfaces and carefully designed nanotopographies
[3]. To fabricate 2D surfaces with well-defined nanotopographies
for cellular and microbial studies or protein adsorption, it is impor- Fig. 4. Scanning electron micrograph of the model organism Escherichia coli EC081
tant to focus on preparation techniques which produce highly used in this study. The image shows microbial cells which are adhered to a PVD-
TiO2 -TF (sample group B; deposition rate 5 nm/s, film thickness 200 nm) after 6 h
reproducible nanoscale features, uniform and coherent structures
of incubation and produced extracellular polymeric substances (EPS) as matrix for
over large areas in the range of cm2 and which are quick with an biofilm formation. Samples were critical-point dried and gold sputter coated before
appropriate number of produced replicas for large scale screenings imaging (average gold thickness of ∼5 nm).
 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589 585

Fig. 5. AFM height images of the uncoated Borofloat® B33 glass substrate (a) and the titanium thin films (deposition rate 0.5 nm/s, film thickness 200 nm) with a scan size
of 1 ␮m × 1 ␮m, 25 ␮m × 25 ␮m and 50 ␮m × 50 ␮m (b–d).

Fig. 6. Surface roughness values of the titanium thin films (n = 3; deposition rate 0.5 nm/s, film thickness 200 nm) depending on resolution and scan size (a) and depending
on scan size and pixel density (b); results of the statistical analysis (multi-way ANOVA) are given with a confidence interval of 95% and p ≤ 0.05; *denotes a statistically
significant difference between the values (a) or the groups (b), respectively.
586 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589
Fig. 7. Fractal dimension FD (calculated with cube counting method) of the titanium thin films (deposition rate 0.5 nm/s, film thickness 200 nm) depending on resolution (pixel
per line) for the different scan sizes: 1 ␮m × 1 ␮m (a), 25 ␮m × 25 ␮m (b) and 50 ␮m × 50 ␮m (c) and depending on the scan size with a constant resolution of 512 pixel/line
(d); FD is given by the slope of the linear fitting.
(Table 1) in comparison to that of the thin films between 1.58 and
2.82 nm (Table 3). Thus, there is no influence of the underlying sub-
strate surface topography on the topography of the titanium thin
films and no surface structures on the micrometer scale are present
on the sample surfaces. In addition, titanium samples in a number
of tenth to some hundreds can be fabricated within relatively short
time and with moderate costs.
As any differences, even small, in the surface chemistry may
influence cellular, microbial or protein adhesion behavior and
can, thus, mask the effect of topography, model substrates with
an absolutely uniform surface chemistry even while varying the
topography are required [25]. It has been shown that in most
cases it is difficult to decouple topographical from chemical effects
[26] since topographical variations of the materials surfaces due to
e.g. grinding, polishing or blasting are most often accompanied by
chemical heterogeneities [9,22,27]. Cai et al. observed no statisti-
cally significant changes in the chemical surface properties of the
PVD-TiO2 -TFs during the variation of the surface nanoroughness.
Qualitative and quantitative X-ray photoelectron spectroscopy as
well as X-ray diffraction measurements showed no differences in
the chemical composition or the crystal structure of the TiO2 -TFs
prepared with different deposition parameters.
In summary, the findings of Cai et al. and our statistical evalu-
ation of the PVD method and the deposited titanium thin films in
the current study showed that these surfaces are due to their high
repeatability and reproducibility and constant chemical composi-
tion during the variation of the surface nanoroughness an excellent
simple 2D model system required for studies addressing the influ-
ence of titanium surface roughness on the nanometer scale on the
biological performance of biomaterials. The use of these model
surfaces for relevant studies, thus, provides a high comparability
within one study and also between different studies.
4.3. The interaction between nanorough surfaces and the
biological system
On the macro- and microscale it has been shown that sur-
face topography has strong effects on human cells [28], since it
can influence cell adhesion, morphology, migration and orienta-
tion as well as cytoskeleton development and cell differentiation
[25]. Much less is known about how cells react to nanoscale struc-
tures. Cell dimensions are about 10–20 ␮m [29], however, some
functional domains present on the cell membrane have dimensions
on the nanoscale. In addition, in vivo cells live inside an extracel-
lular matrix containing nanoscale collagen fibrils, thus, cells are
likely to be able to respond not only to surface topography on
the micrometer range but also on the nanometer range [3]. Cell
filopodia, for instance, have a tip diameter of 50–100 nm, while
integrin-mediated adhesion is in the order of 15 nm, hence, are of
sufficient resolution to determine changes in the substrate nanoto-
pography. Nevertheless, the mechanisms by which cells respond
to nanoscale surface topography and the subsequent signal trans-
duction pathways have to date not been resolved in detail [25]. The
PVD-TiO2 -TFs as 2D model surfaces provide a suitable option for
the investigation of these interactions, which should be addressed
in further studies.
It is well established that biomaterials are immediately coated
with biomolecules and proteins from the blood or interstitial
 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589
fluid when they are implanted or inserted into the human body.
Therefore, cells may be in some cases essentially controlled by
these surface adsorbed biomolecules and proteins rather than by
the nanoscale surface topography [30]. It is, thus, of fundamen-
tal interest to investigate how nanostructured materials influence
the adhesion behavior, structure and stability of biomolecules
and proteins to understand the actual contribution of the sur-
face topography of a biomaterial to its biocompatibility. Recently,
we investigated the adsorption of bovine serum albumin and
human plasma fibrinogen on PVD-TiO2 -TFs [22]. A significantly
larger amount of adsorbed fibrinogen has been observed on the
nanorough titanium surfaces compared to albumin. No significant
differences have been found regarding the different surface nanor-
oughnesses of the TiO2 -TFs. In contrast, Singh et al. [31] found a
significant increase in the amount of adsorbed proteins originated
from fetal bovine serum on nanorough titanium surfaces prepared
with supersonic cluster beam deposition. The conflicting results of
these two studies show that much more work has to be done to fully
understand the mechanisms behind protein adsorption in general
and the influence of surface nanoroughness on protein adsorption
in particular.
Biomaterials-related infections are most frequently associated
with microbial adhesion and biofilm formation on the indwelling
implants. Far less is known about the mechanisms of surface
sensing by microorganisms than by eukaryotic cells and the influ-
ence of surface topography on the nanometer scale on the microbial
adhesion [3]. In the current study, the bacterial adhesion of E. coli
on TiO2 -TFs with different nanoroughnesses was investigated. The
bacterial coverage decreased from approximately 23% to 19% with
increasing surface nanoroughness from ∼2 nm (sample group A)
to ∼6 nm (sample group D) (Fig. 2). The influence of surface struc-
tures size on the microbial attachment is controversially discussed
in the literature. Some studies consider that microorganisms pre-
fer the attachment to surfaces which provide surface features, such
as roughness, corresponding in size to the size of the bacterial
cells [32]. It was hypothesized that the microorganisms are in that
way protected against shear stress and abrasion. Other studies
showed that microorganisms attach less to surfaces with features
corresponding in size to the size of the cells [3]. It is believed
that these structures cause mechanical stress in the cytoskele-
ton of the microorganisms which make them uncomfortable in
this sessile state. Since the maximum peak-to-valley distance and
the average peak-to-peak distance for the roughest titanium sur-
faces (group D) were approximately 45 nm and 42 nm, respectively,
these effects can be excluded as influencing factors on the bacte-
rial adhesion on the nanorough TiO2 -TFs. Moreover, the average
size of the thin film surface structures of the sample groups A
and B (in the range of ∼15 nm) which were correlated with the
highest measured bacterial surface coverages, corresponds to some
extent to the size of the EPS (mediate the bacterial adhesion to sur-
faces and provide the mechanical stability of biofilms) produced
by the bacteria. Scanning electron micrographs of the adhered
bacteria revealed a diameter of an EPS strand of 5–10 nm (Fig. 4).
This size roughly corresponds to the structure size of the tita-
nium surfaces of the samples of group A and B and might, thus,
promote bacterial adhesion on the one hand and increase mechan-
ical stability of the adhered bacteria against abrasion on the other
hand.
The distribution of the bacteria attached to the thin films became
increasingly inhomogeneous and patchy with increasing surface
nanoroughness (Fig. 3). This observed effect might be due to bacte-
rial co-adhesion [33] and correlates with the generally decreasing
bacterial surface coverage. On surfaces less attractive for bacteria,
they might prefer to attach to areas on the materials surface, where
other bacteria have previously attached and already produced EPS.
This EPS may then promote bacterial adhesion.
587
A more detailed investigation and discussion of the bacterial
adhesion on the nanorough PVD-TiO2 -TFs goes beyond the scope
of this study and will be published elsewhere.
4.4. Critical analysis of the titanium surface characterizing
parameters
One of the most commonly used parameters for the quantita-
tive characterization of biomaterials surfaces is the roughness [34].
Comparing the results of the different scan sizes to each other (for
TiO2 -TFs sample group B, deposition rate of 0.5 nm/s, film thick-
ness of 200 nm), it was shown that the calculated roughnesses
values Ra , RMS and skewness statistically significantly increased
with increasing scan size (Table 1). For Ra and RMS of grinded and
polished titanium surfaces, this effect was described in the liter-
ature before but no comprehensive explanation for this has been
found in these studies [14,15,21]. To our knowledge, for skewness
this effect was not shown before.
The surface roughness depends on the lateral and vertical
dimensions of the surface features and only if all these features
are included in the scanned and analyzed image frame, the rough-
ness is unaffected of the scanning area [15]. With decreasing AFM
scan size, some surface features, e.g., some height information may
get lost and the calculated roughness becomes smaller. Vice versa,
with increasing scan sizes the calculated surface roughness might
increase [15,35]. Since the TiO2 -TFs investigated in our study had
a very coherent and uniform structure with surface features in the
nanometer range [11], we rule out this effect for our samples.
The resolution of the AFM image, more precisely the number of
measured points per line, might be another possible reason hypoth-
esized in the literature for an increased measured, respectively,
calculated surface roughness with increasing scan size [11,15,16].
In the studies mentioned above, the resolution was not increased
proportionally with increasing scan size, thus, the point-to-point
distance increased and, accordingly, the pixel density of the images
decreased. Using a mathematically constructed surface profile, it
has been shown previously that with an increasing number of
points per line the measured roughness decreased until a critical
value of approximately 105 points per line and roughness values
remained constant above this value [15], although this value is only
a theoretical one since in reality with AFM it is just possible to
measure with resolutions some order of magnitudes below. To test
these reported findings for the TiO2 -TFs investigated in our study,
we calculated Ra and RMS in dependence of the image resolution
and the scan size (Fig. 6a) and in relation to scan size and pixel den-
sity (Fig. 6b). The effect of the increasing measured roughness with
increasing scan size occurred independently from the resolution or
the pixel density. Our results, therefore, demonstrate that the res-
olution as well the pixel density had no influence on the calculated
roughness parameters of the PVD-TiO2 -TFs.
All images were obtained and processed by a plane leveling, a 1st
order line-by-line fit and a scar removal parallel to the scanning axis
to ensure that the calculated roughness parameters for the TiO2 -
TFs were valid and comparable. However, one more reason for the
effect of the increasing measured roughness with increasing scan
size that we found may be a still insufficient image processing. A
common appearance in AFM images is the scanner bow. It is caused
by a swinging motion of the free end of the scanner. This leads to
a curve in the image plane especially with increasing scan sizes.
Possibly, this effect was still present in the AFM images. Neverthe-
less, it has to be considered that the way of image processing can
directly affect the calculated roughness values, since a high order
image processing, which can be useful to erase the AFM scanner
bow, may lead to the loss of height data, thus, the roughness val-
ues decrease with a progressive image processing [18]. AFM image
processing should be applied without the elimination of important
588 C. Lüdecke et al. / Applied Surface Science 280 (2013) 578–589
superordinate structures, such as waviness or structuring features.
Thus, the processing of AFM images is mostly a balance act between
a sufficient image processing and height data loss.
The percentage increase of a projected surface area relative to
the scanned image area gives considerable information about the
topography of a surface, too, and was used in numerous studies
for the investigation of a variety of materials surfaces and inter-
faces [14]. This percentage increase is a suitable parameter in
particular for biomedical applications since the deviation of the
measured surface area (MSA) from the scanned surface area (SSA)
is positively correlated with the specific surface area available for
the initial adsorption of proteins or biomolecules, tissue adhesion
or the attachment of microorganisms. The surface area that can
participate in any exchange between an implant surface with its
surrounding environment is, thus, much bigger for surfaces with a
higher deviation of the MSA from the SSA.
Our results show that the deviation of the measured from
the scanned surface area was, contrary to the roughness values
discussed above, negatively correlated with the scan size. With
increasing scan size, the percentage deviation between the mea-
sured and the scanned area decreased significantly. Furthermore,
we found that the deviations mean value (of run 1–6, Table 1) for
the scan length of 50 ␮m (0.12 ± 0.14%) represented approximately
1/50 of the mean value of the 1 ␮m scan length (9.17 ± 2.01%) and
1/2 of the mean value of the 25 ␮m scan length (0.32 ± 0.16%). Thus,
the differences between the deviations mean values of the differ-
ent scan sizes directly depended on the magnifications used for the
AFM images. This observed negative correlation between the devi-
ation of the measured from the scanned surface area and the AFM
scan size seems to be a mathematical effect since with increasing
scan size the AFM image resolution remained constant and the den-
sity of measured points per line decreased accordingly. This led to
the relative decrease of the measured surface area with increas-
ing scan size since surface features, especially for such nano rough
surfaces, were missed due to the increasing distance between the
measured points. Thus, for the characterization of surface prop-
erties in the low nanometer range, higher AFM scan sizes, such
as 25 ␮m or 50 ␮m accompanied by a relatively low resolution of
512 × 512 pixels are not sufficient to display the real surface topog-
raphy since the pixel size is bigger than the smallest surface feature.
Nevertheless, some surface characterization parameters used in
our study were not affected in the way described above. The calcu-
lated values for fractal dimension and kurtosis have not increased
or decreased with increasing scan size. The fractal dimension (FD) is
a frequently used parameter for the characterization and compari-
son of rough surfaces in natural sciences, e.g. biology [36], medicine
[37] or geo sciences [38]. The FD characterizes the roughness of a
self-similar surface and varies from 2 for a smooth surface to 3
for an infinitely rough surface. From the literature it is known that
the fractal dimension is not influenced by an underlying surface
topography [39] which was confirmed by the results of our study.
The small difference of the FD values between the scan length of
1 ␮m and 50 ␮m and the scan length of 25 ␮m remains unclear
and should be addressed in further studies. However, the fractal
dimension as well as the kurtosis seem to be suitable parameters
for the characterization of rough surfaces in general since they are
not influenced by an increasing scan size, by the image resolution or
the pixel density. Furthermore, these surface characterizing param-
eters seem not to be influenced by the commonly occurring AFM
scanner bow. Thus, it is not absolutely necessary to apply a high
order processing to the AFM raw images which may be accompa-
nied by a possible height data loss.
For AFM based investigations, an identical scan size and image
processing are particularly important as well as to use the smallest
possible scan sizes which include all important surface features.
Nevertheless, sometimes it may also be required to use different
scan sizes, e.g. for the investigation of the interaction of biomateri-
als surfaces with their biological environment. The used AFM scan
size might be larger for the investigation of the interaction of the
biomaterial with tissues, cells or biofilms and might be smaller for
the investigation of the interaction of the biomaterials surface with
microorganisms, proteins or other biomolecules. Then, the effects
discussed above have to be taken into account during data analysis,
interpretation and discussion. As additional or alternative parame-
ter for the characterization of rough surfaces next to Ra and RMS, the
fractal dimension seems to be well suited, since it is not influenced
by the AFM scan size, pixel density or image resolution.
5. Summary and conclusions
PVD is a suitable method to reproducibly prepare TiO2 -TFs with
a surface roughness on the lower nanometer scale. Within an error
range of 14.3% which corresponds to a deviation of much less than
1 nm it is possible to prepare TiO2 -TFs with desired surface rough-
nesses.
We demonstrated that surface roughnesses even on the low
nanometer scale influence bacterial adhesion behavior. With
increasing surface roughness bacterial surface coverage decreased
and became increasingly inhomogeneous.
PVD-TiO2 -TFs are an excellent 2D model system for studies
addressing the influence of surface roughness on the nanometer
scale on the biological performance of biomaterials. Our findings
give, thus, new momentum to biomaterials research and will sup-
port the development of biomaterials surfaces with anti-infectious
surface properties.
The critical analysis of the surface characterizing parameters
used in our study showed that the calculated values of Ra , RMS and
skewness were significantly influenced by the AFM scan size. This
effect has, in general, to be taken into account for studies using dif-
ferent scan sizes or for comparing results between different studies.
Acknowledgements
The authors thank M.M.L. Arras for help with ImageJ and MAT-
LAB, M. Beyer for introduction to the statistical analysis of the data,
T. Keller and J. Reichert for helpful discussions and the Graduate
School “Jena School for Microbial Communication” (JSMC MikroIn-
ter supported by the TMBWK) which is funded by the German
Excellence Initiative for financial support. Furthermore, KDJ grate-
fully acknowledge the partial financial support of the Deutsche
Forschungsgemeinschaft (DFG), grant reference INST 275/241-1
FUGG, and the TMBWK, grant reference 62-4264 925/1/10/1/01.
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