Bactericidal behaviour of Ti6Al4V surfaces after exposure to UV-C light
Amparo M. Gallardo-Morenoa,b, Miguel A. Pacha-Olivenzaa,b, María-Coronada Fernández-Calderónb,c,
Ciro Pérez-Giraldob,c, José M. Bruquea,b, María-Luisa González-Martína,b,*
aDepartamento de Física Aplicada, Facultad de Ciencias, Universidad de Extremadura, Avda de Elvas s/n, 06071 Badajoz, Spain
bCentro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina CIBER-BBN, Badajoz, Spain
cDepartamento de Microbiología, Facultad de Medicina, Universidad de Extremadura, Avda de Elvas s/n, 06071 Badajoz, Spain
a r t i c l e i n f o
Received 20 January 2010
Accepted 3 March 2010
Available online 2 April 2010
a b s t r a c t
TiO2-coated biomaterials that have been excited with UV irradiation have demonstrated biocidal prop-
erties in environmental applications, including drinking water decontamination. However, this proce-
dure has not been successfully applied towards the killing of pathogens on medical titanium-based
implants, mainly because of practical concerns related to irradiating the inserted biomaterial in situ.
Previous researchers assumed that the photocatalysis on the TiO2surface during UV application causes
the bactericidal effects. However, we show that a residual post-irradiation bactericidal effect exists on the
surface of Ti6Al4V, not related with photocatalysis. Using a combination of staining, serial dilutions, and
a biofilm assay, we show a significant and time-dependent loss in viability of different bacterial strains of
Staphylococcus epidermidis and Staphylococcus aureus on the post-irradiated surface. Although the
duration of this antimicrobial effect depends on the strains selected, our experiments suggest that the
effect lasts at least 60 min after surface irradiation. The origin of such phenomena is discussed in terms of
the physical properties of the irradiated surfaces, which include the emission of energy and changes in
surfaces charge occurring during electron-hole recombination processes. The method here proposed for
the preparation of antimicrobial titanium surfaces could become especially useful in total implant
surgery for which the antimicrobial challenge is mainly during or shortly after surgery.
? 2010 Elsevier Ltd. All rights reserved.
Ti6Al4V is one of the most commonly used biomaterials in
orthopedic applications. The use of Al and V alloying elements
stabilizes the alphaebeta microstructure, and improves the
mechanical properties in relation to the commercially pure Ti .
An important feature of Ti-based materials, directly related to their
biocompatibility, is their air-induced passivation. This creates
a protective and stable layer of titanium oxide on the surface,
mainly composed of titanium dioxide, with a few nanometers
thickness . This layer minimizes ion release from the implant to
surrounding tissues and hence inflammatory reactions in the body
. Despite these advantages, a post-operative serious and unre-
solved problem leading to the failure of the implant is the
appearance of implant-associated infections. In this sense, both,
anti-adhesive and antibacterial surface modifications are targeted
to prevent the bacterial colonization and subsequent biofilm
formation without compromising the biocompatibility of the
implant [4,5]. It is common to find modified surfaces with inte-
grated antibiotics, antiseptics, metal ions [6e11] most often with
the integration of silver due to its antibacterial properties .
However, biocompatibility of most of these anti-infective surfaces
is still uncertain and needs to be clarified.
In a different scenario that shares this common concern, the
semiconductor properties of TiO2have been successfully applied
with antimicrobial purposes for three decades. Upon excitation
with ultraviolet light, the TiO2 surface generates electron-holes
pairs which induce a series of photocatalytic reactions. Without
exhaustive details, the hole in the valence band can react with H2O
or hydroxide ions adsorbed on the surface to produce hydroxyl
radicals (OH?) and the electron in the conduction band can reduce
O2to produce superoxide ions (O2
short life but are extremely reactive with contacting organic
compounds, leading to the degradation of organic matter. This
novel photocatalytic and bactericidal technology was pioneering
used in the works of Matsunaga et al. in 1985 . Since then it has
been applied to different environmental areas such as those related
to the drinking water, air conditioning filters, food preparation
surfaces and sanitary-ware surfaces [14e19]. In these systems,
?). Both holes and OH?have a very
* Corresponding author. Departamento de Física Aplicada, Facultad de
Ciencias, Universidad de Extremadura, Avda de Elvas s/n, 06071 Badajoz,
Spain. Tel.: þ34924289532; fax: þ34924289651.
E-mail address: firstname.lastname@example.org (M.-L. González-Martín).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/biomaterials
0142-9612/$ e see front matter ? 2010 Elsevier Ltd. All rights reserved.
Biomaterials 31 (2010) 5159e5168
selected strains used for in vitro testing include Escherichia coli
[15, 20], Lactobacillus helveticus , Salmonella choleraesuis, Lis-
teria monocytogenes , Pseudomonas aeruginosa , Bacillus
Recently, a novel application is found in the textile industry.
Here, apatite-coated TiO2particles fixed to cotton textiles, nontoxic
to human dermal fibroblasts, have shown antibacterial activity
upon UV irradiation. This suggests its potential use for reducing the
risk of microorganism transmission in textile applications .
Nevertheless, although there is no doubt on the bactericidal
effect of TiO2coated materials upon UV irradiation, there have been
very few reports about its application on bio-implant-related
infections. In 1988, Cai et al.  were one of the first researchers
who showed a new application of TiO2photocatalyst for medical
applications, by erradicating cancer cells with ultrafine TiO2
powders . Recent in vitro works of Choi et al. [26,27] tested the
antibacterial response of titanium-made orthodontic materials
under UV irradiation with select strains of Lactobacillus acidophilus
and Streptococcus mutans. Riley et al.  used a similar experi-
mental set-up to evaluate the effect of these materials on E. coli
Few details have been published on the TiO2 photocatalytic
activity of clinical isolates, such as S. aureus or S. epidermidis.
Traditional TiO2photocatalysis is effective only upon irradiation of
UV-light at levels that would induce serious damage to human cells
. In addition, taking into account that a direct illumination of
the surface is needed, this methodology cannot be carry out in
implanted biomaterials surrounded by tissues. However, within the
last year different groups have tried to find some ways of using TiO2
photocatalysis in the bio-implant field. Cheng et al.  found that
the new developed carbon-containing TiO2nanoparticles showed
antibacterial properties against S. aureus, Shigella flexneri and Aci-
netobacter baumannii under visible-light illumination. The work of
Cushnie et al.  gave insights on the influence of different vari-
ables on the outcome of TiO2 photocatalysis experiments with
bacteria of clinical interest. Shiraishi et al.  compared the
viability of S. aureus strains against two different TiO2 coated
materials under UV-A exposure.
Nevertheless, at the moment, it is hard to find a direct and
practical application of such as emerging research to the implant
field. Accordingly, this work has been designed to look at a different
direction. We will show that the surface of the biomaterial Ti6Al4V
displays antimicrobial activity after being irradiated with UV light
without compromising the excellent biocompatibility exhibited by
the biomaterial surface previously reported . To this extend, the
aims of this research are twofold. First, we will evaluate the
bacterial viability and biofilm formation on the surface of Ti6Al4V
surface before and after being irradiated with UV-C light. For this
purpose, we will work with two representative strains of the vast
majority of nosocomial infections, S. aureus and S. epidermidis,
while considering also their ability of producing extracellular
polymer substances (EPS). Second, we will give insight the reasons
to the bacterial viability loss testing whether photocatalytic effect
remains on the surface or the bactericidal mode is provoked by
physical phenomena such as energy emission from the irradiated
surface or surface charge excess.
2. Materials and methods
Disks of Ti6Al4V were cut from bars of 25 mm in diameter kindly supplied by
SURGIVAL S.A., Spain. A TIMETAL 6e4 ELI alloy was processed as a hot rolled
annealed bar at 700?C for 2 h, then air cooled. The disks were abraded on succes-
sively finer silicon carbide papers, mechanically polished with diamond paste, and
finished with colloidal silica.
Prior to their use, the Ti6Al4V disks were carefullycleaned with DSF disinfectant
(DERQUIM DSF 11; Panreac Quimica S.A., Spain) at 60?C by vigorously rubbing with
a smooth cotton cloth, then rinsed repeatedly with distilled water and sonicated in
deionized water (Milli-Q system), 70% acetone and finally ethanol for periods of
10 min each. Finally they were dried in an oven at 40?C for 1 h and stored in
a desiccator for no longer than 24 h. Samples used as controls were not subjected to
any further treatment. A second set of samples was exposed to an UV-C source for
15 h. This period was sufficient to guarantee a complete hydrophilization of the
surface, as we have recently shown . G15-T8 UV lamps, emitting predominantly
at a wavelength of 254 nm, were kindly provided by Philips Iberica, Spain. The disks
were positioned at 10 cm from the light source and centered, receiving an intensity
of about 4.2 mW cm?2. The irradiation installation was inside an opaque wood
chamber to prevent interference from the room or daylight, or cause any damage to
2.2. Bacterial strains and culture
S. aureus ATCC29213 (S. aureus), S. epidermidis ATCC35984 (S. epidermidis4), S.
epidermidis HAM892 (S. epidermidis2) and E. coli ATCC25922 (E. coli) were stored at
?80?C in porous beads (Microbank, Pro-Lab Diagnostics, USA). S. epidermidis
HAM892 is a negative extracellular polysaccharide substance producer (EPS-nega-
tive) mutant derived by acriflavine mutagenesis from S. epidermidis ATCC35984
(EPS-positive) and it was kindly provided by L. Baldassarri from the Laboratorio di
Ultrastrutture, Istituto Superiore di Sanita, Rome, Italy. From the frozen stock, blood
agar plates were inoculated and incubated at 37?C to obtain cultures. From these
cultures, tubes of 4 ml of Trypticase Soy Broth (TSB) (BBL, Becton Dickinson, USA)
were inoculated for 10 h at 37?C and then 25 ml of this pre-culture was used again to
inoculate 50 ml flasks of TSB at 37?C for 14 h. This time was sufficient to guarantee
that all strains were at the beginning of the stationary phase of growing (previously
checked with the growing curves for the strains).
The bacteria were then harvested by centrifugation for 5 min at 1000 g (Sorvall
TC6, Dulont, USA) and washed three times with 0.15 M phosphate buffered saline
(PBS, pH 7.2) pre-conditioned at 37?C. Then the bacteria were re-suspended in PBS
at a concentration of 3 ?108bacteriaml?1for the adhesion experiments.
2.3. Adhesion experiments
Adhesion experiments were carried out with the help of a sterile reusable sili-
cone chamber (flexiPERM, Greiner bio-one, Germany). Due to the highly adhesive
underside of the chamber, it was fixed to the Ti6Al4V surface by applying a little
pressure to its top part. Then 2 ml of the bacterial suspension was added to the
chamber well and the contact with the Ti6Al4V surface was allowed for different
times, ranging from 10 to 60 min. During these times samples were introduced in an
environmental chamber at 37?C and were subjected to slight orbital shaking of
20 rpm (Heidolph Rotamax 120, Heidolph Electro GmbH and Co, Germany) to
minimize sedimentation effects. After the adhesion time, the silicone chamber was
removed and the samples were carefully immersed twice in a volume of 25 ml of
freshly prepared PBS to remove loosely bound bacteria. To this extend, rinsing forces
were carefully applied trying to exert always the same shear strength to the surface
and guarantee the comparison between samples The use of the silicone chamber
permitted a very precise adhesion area on the metal surface.
2.4. Viability tests on adhered bacteria
After the adhesion process, different viability tests were carried out to show out
the viability of adhered bacteria to the Ti6Al4V surface before and after being
exposed to the UV irradiation.
2.4.1. Staining-based methods
For most of the experiments the kit Live/Dead Baclight L-7012 (Invitrogen SA,
Spain) was used to stain live and dead bacterial cells according to the manufacturer.
Concretely, 2 ml of dimethyl sulfoxide were mixed with 10 ml of SYTO 9 and propi-
dium iodide and 15 ml of this mixturewere put in contact with thesample surface for
15 min avoiding exposure to room light. Then, stained bacteria were observed at
randomly chosenlocations under
membranes, fluorescence green while dead bacteria with damaged membranes
fluoresce red. For comparisons, some samples were also stained with acridine
orange, 6-Acridinediamine N,N,N0,N0-tetramethyl-monohydrochloride (Molecular
Probes, Inc., USA) (lex¼500 nm, lem¼526 nm). The stain is a DNA binding dye in
which the acridin orange fluorocrome interacts with DNA and RNA by intercalation.
Live bacteria, withintact
2.4.2. Serial dilution method
After the adhesion process, the samples were immersed in 25 ml of sterile PBS
and sonicated for 15 min in an ultrasonic bath at 110 W (Ultrasons,J.P. Selecta, Spain)
which ensures the detachment of the adhered bacteria. Then, viable bacteria were
evaluated with the serial dilution method by using agar plates. Statistical analysis
were made with paired samples, it means, control and irradiated surface using the
same bacterial suspension.
A.M. Gallardo-Moreno et al. / Biomaterials 31 (2010) 5159e5168
2.4.3. Direct transfer of bacteria to solid agar
In this qualitative test, the Ti6Al4V surface with adhered bacteria was used to
directly inoculate the solid agar of a series of petri dishes by carefully rubbing the
Ti6Al4V surface with the solid agar of the plates. Each sample was rubbed on agar
plates, trying to detach the highest number of adhered bacteria. This method,
avoiding sonication, allows the establishment of an intimate relationship with the
serial dilution method since in both cases the colony forming units (CFU) are
determined. Statistical analysis was also made with paired samples.
2.5. Biofilm formation on the Ti6Al4V surface
Bacteria was inoculated and incubated in blood agar plates at 37?C for 24 h to
obtain cultures, and then transferred to Trypticase Soy Broth medium (TSB) (BD,
Becton Dickinson and Company, Sparks, USA) for overnight culture. The microor-
ganisms were transferred to fresh TSB until a turbidity equivalent to a 0.5 McFarland
standard and then diluted 1/100 to obtain an inoculum of approximately 106cfu/ml.
Then, 500 ml of the bacterial suspension was cultivated on the metal surface using
a sterile silicone culture chamber in a similar way to the bacterial adhesion exper-
iments for different periods of time, 8 h and 24 h. Afterthose times thecultures were
carefully washed three times with sterile PBS in order to eliminate the non-adherent
bacteria. Later, 1 ml of PBS was added per well for removing the biofilm from the
titanium alloy surface and the suspension was homogenized by sonication in
a ultrasonic bath (Ultrasons; J.P. Selecta, Spain) for 15 min.
These bacteria included in biofilm were quantified using the BacTiter-Glo?
Microbial Cell Viability Assay (Promega Corporation, USA) according to the manu-
facturer. This is a homogeneous method for determining the number of viable
microbial cells in biofilm culture based on the quantification of ATP present. ATP is
an indicator of metabolically active cells and, by consequence, this method has been
frequently used for adhesion and biofilm development in vitro [35,36]. The lumi-
nescent signal, in relative light units, in the ATP-bioluminiscence assay is propor-
tional to the amount of ATP present in the biofilm, which, in turn, is directly
proportional to the number of viable cells in the biofilm.
Specifically, 1 ml of BacTiter-Glo? Reagent was added directly to bacterial cells
suspension and transfer to wells of sterile 96-well white polystyrene flat-bottomed
microtiter plates (Greiner bio-one, Germany). The light emission (luciferin-luciferase
reaction) was measured in a Microplate Fluorescence Reader (FLx800; Bio-Tek
Instruments, Inc. USA). Positive controls (control surface with bacteria) and negative
controls (control surface without bacteria) were considered. Each assay was per-
formed in duplicate and repeated at least three times in order to confirm
Although the method was applied for the three strains studied, only S. epi-
dermidis ATCC35984 induced the production of biofilm in enough quantity to be
Similar to the adhesion experiments, results were analyzed with paired samples
and independent experiments, up to fourteen, were done.
2.6. Evaluation of the changes on the Ti6Al4V surface after irradiation
A series of experiments were carried out in order to elucidate the changes on the
irradiated surface in respect to the control, responsible for the changes in the
viability of adhered bacteria. All the experiments were repeated at least three times
to confirm data.
2.6.1. Photocatalysis on the irradiated surface
Different testing solutions were used to check whether photocatalytic reactions
remain on the irradiated surface after turning off the UV lamp. The degradation of
dyes species is tested intensively with regard to their photodynamic treatment.
Brilliant green (BG, SigmaeAldrich, USA) and the complex (TPTZ)2Feþþ(TPTZ: 2,4,6-
tripyridyl-1,3,5-triazine, Fluka) were used by means of colorimetric methods eval-
uating the optical absorbance with the help of a spectrophotometer (Spectronic 601,
Milton Roy, USA) before contact and after contact with the control and irradiated
surface. In the case of BG the protocol proposed by Chen et al.  was employed.
Briefly, stock solutions containing 1 gL?1of BG dye in water were prepared, pro-
tected from light and stored at 4?C. For the complex (TPTZ)2Feþþ, without
exhaustive details, based on the work of Pilch et al. , the TPTZ was diluted in
glacial acetic acid and sodium acetate 1 M. This solution was mixed with the
compound Fe(NH4)2$6H2O diluted in deionized water for obtaining an optical
density of about 0.8e0.9 at 593 nm. In all the experiments the ratio between the
initial (before contact) and final (after contact with the sample) optical density was
evaluated. Different assays were made changing the initial optical density ranging
from 0.15 to 0.9. Further experiments were also carried out by using the reactive
specie t,t-muconic acid (E,E-2,4-hexadienedioic acid, SigmaeAldrich, USA). This
compound degrades in the presence of hydroxyl radicals through double C-C bonds
attack . Its concentration in solution was determined before and after being in
contact with the control (dark) and the irradiated surfaces by HPLC (1100 Hewlett-
Packard, USA). The mobile phase was a mixture acetonitrileewater (15/85 v/v) with
0.1% phosphoric acid (flow rate: 1 mlmin?1). A C18 Kromasil 15 cm long, 0.4 cm
diametercolumnwas used. Detectionwas made at 264 nm. The contact betweenthe
probe chemicals and the samples were established with the help of silicone wells
and contact time was set as 60 min.
2.6.2. Duration time of the post-radiation effect
Bacteria were let to adhere onto the irradiated surface of Ti6Al4V not immedi-
ately after turning off the UV lamp but after different time intervals: 5,15, 30, 60, 90
and 120 min. For any time, bacteria were let in contact with the irradiated surface
during 60 min. We refer to those times as post-radiation waiting times and the Live/
Dead test was used to verify the viability of adhered bacteria.
2.6.3. Radiation emission from the irradiated surface
Three different probe non-conducting thin films (0.2 mm thickness), quartz,
microscopic slide top glass (glass film) and black plastic film, were selected to test
whether the Ti6Al4V surface was able to release energy in a radiation form after
being irradiated. The films were placed over the Ti6Al4V surface immediately after
irradiation (control samples were studied in the same way but without irradiation).
Then, a 250 ml bacterial suspension droplet was deposited on their surface, allowing
the adhesion of microorganisms to the film surface for 60 min at 37?C. At that time,
the bacterial suspensionwas removed and the film surface was slightly washed with
PBS to remove loosely bound bacteria. Adhered bacteria were stained with the Live/
Dead viability kit and the results were analyzed in terms of viable microorganisms.
2.6.4. Electrical interaction potential at the irradiated surface
Zeta potential of control and irradiated surfaces was obtained from the
streaming potential  by using an electrokinetic Analyzer (EKA, Anton Paar,
Austria). The measurements were made with the electrolyte 0.001 M KCl setting
a ramp pressure of 600 mb. The pressure gradient (DP) between both ends of the
clamping cell provoked the movement of the electrolyte inside the electrokinetic
channel, which, in turn, was reflected in a potential difference between both ends
(DV). Both magnitudes were related to provide the value of z,
z ¼DV ? h ? k0:1M? R0:1M
DP ? 3 ? R0:001M
with R0.001Mbeing the resistivity of the 0.001 M KCl electrolytic solution and k0.1Mand
R0.1Mthe electrical conductivity and resistivity of a 0.1 M solution of a KCl needed for
correcting the surface conductivity of the sample.
2.6.5. Surface charge excess on the irradiated surface
A thin opaque conducting film (0.2 mm thickness) was placed over the irradi-
ated surface immediately after turning off the UV lamp (parallel experiments were
performed with control samples). Then, a 250 ml bacterial suspension droplet was
deposited on their surface, allowing the adhesion of microorganisms to the film
surface for 60 min at 37?C. At that time, the bacterial suspension was removed and
the film surface was slightly washed with PBS to remove loosely bound bacteria.
Adhered bacteria were stained with the live/dead viability kit and the results were
analyzed in terms of viable microorganisms.
2.7. Statistical analyses
Different statistical tests were employed to confirm differences between
samples. Depending on the number of data and its distribution, it was selected
parametric paired t-test or non parametric Wilcoxon matched-pairs signed-ranks
test, using the statistical software SPSS for Windows v12 (Chicago, Illinois, USA). The
level of significance was set at P values <0.05. Data are reported as mean?SD. In
this sense, special attention must be paid when comparing paired data (experiments
with control and irradiated samples handled in parallel), since differences may not
be shown graphically when plotting mean and SD.
3.1. Quantification of adhered bacteria to the control and irradiated
The total number of adhered bacteria on the surface, no matter
their viability, at the different contact times is plotted in Fig. 1.
Those samples not exposed to UV light (control) were maintained
in contact with the bacterial suspension for the maximum time of
60 min. There is a progressive increase in the number of adhered
bacteria to the surface with time. The adhesion kinetics is the
highest for S. epidermidis4 for mostly all the adhesion times tested,
while S. aureus and S. epidermidis2 show similar behaviors, S. epi-
dermidis2 being the strain with the lowest mean adhesion at any
time. In the case of S. epidermidis4, it is also worth to mention that,
due to the particularities of its bacterial surface, the exact number
A.M. Gallardo-Moreno et al. / Biomaterials 31 (2010) 5159e5168
of adhered bacteria was very difficult to quantify since the aggre-
gation of cells in big 3-Dcumulus lead tothe underestimation of the
total number of adhered bacteria. This fact became morerelevant at
long adhesion times since bacteria could even promote their
aggregation in suspension, for this reason, it is very likely that the
real number of bacteria on the surface at 60 min of adhesion is the
highest for this biofilm-producing strain. Another interesting
phenomenon is that the surface coating at 60 min is similar in the
control than in the irradiated surface (not statistically significant
3.2. Evaluation of the viability of adhered bacteria to the control
and irradiated surface
Focusing on the viability of adhered bacteria, Fig. 2a presents
various images of typical areas on the Ti6Al4V after 15 h of irradi-
ation for the different contact times,10, 20, 40 and 60 min. Control-
viability imagesarealso shownin the last column for the maximum
time of contact. At first glance, the most interesting observation is
that all bacteria deposited on the non-irradiated surface are 100%
viable (green), while bacteria attached to the irradiated surface
showa progressivelyloss inviability. This phenomenonseems to be
a function of the contact time between bacteria and surface:
bacteria become more damaged as the time of contact between
0 10 2030 405060
n (bacteria cm-2) x 10-4
Fig. 1. Total number of adhered bacteria (viable and non-viable) on the Ti6Al4V irra-
diated surface as a function of contact time and on the control surface after 60 min of
20 min 40 min 60 min 60 min
10 2040 60
Live bacteria (%)
Fig. 2. (a) Qualitative images of the cells viability loss as a function of the contact time between the three strains and the irradiated surface after turning off the UV lamp. Control
images refer to the maximum time of contact between bacteria and nonirradiated Ti6Al4V surface. (b) Quantitative analysis of the percentage of live bacteria adhered to the control
and irradiated surfaces obtained by using the kit Live/Dead kit. Error bars represent standard deviations of the average.
A.M. Gallardo-Moreno et al. / Biomaterials 31 (2010) 5159e5168
bacteria and the irradiated surface increases. For 10 and 20 min
there are a number of bacteria stained in yellow-like and orange-
like color mixed with green spots, which indicates the beginning of
the bacterial damage, but the color of cells becomes completely red
at 60 min, which means, based on the description of the test, that
mostly all bacteria are compromised or dead.
A quantitative analysis of these results has been performed and
presented in Fig. 2b. Remarkably, a complete loss of cell viability is
observed after 60 min of contact. It is important to note that the
largest decrease in cell viability (w75%) occurs after the first 10 min
viability decreases by w2.5% consecutively. From this time on all
bacteria on the surface appeared red. This figure also shows that,
within experimental error, the viability loss is independent on the
strain selected, it seems that the post-radiation effect is not depen-
dent on the strain selected but on the exposure time to the surface.
Although the surface of the biomaterial analyzed is mainly
colonized by gram-positive staphylococci, mostly represented by
both staphylococcal strains selected, we performed similar exper-
iments with a gram-negative E. coli strain, vastly used in UV-light
related systems [15,20,41]. Interestingly, bacteria also turned into
red after 60 min of contact with the irradiated surface, showing the
post-radiation efficacy with bacteria with completely different cell
The study of viability of microorganisms with one specific dye is
sometimes contrasted with other staining techniques [42,43] to
avoid problems with viability detection by differentiation between
viable and non-viable cells. For this reason some experiments were
carried out using acridine orange and the results obtained
confirmed those observed with the live/dead kit.
Despite the viability loss shown by staining techniques, Huang
et al.  have recently pointed out that in some cases damages to
the cell wall can be repaired during the subculture of cells onto agar
plates for the viability study. For this reason, and due to the valu-
able results found in the present study, we considered extremely
interesting to contrast this finding with other methodology, able to
tell us whether red-like adhered bacteria can proliferate when
finding a nutrient-rich media.
Experiments were carried out under the situation that gave the
highest number of red-stained bacteria and in the control state,
both referred to as 60 min of bacteria-surface contact. Viability of
bacteria in nutrient-rich containing media was evaluated and
contrasted with two different protocols.
In the first one, bacteria were detached from the surface by
sonication, as explained in the experimental section, and the serial
dilution method with agar plates counting was performed to detect
the colonies present in each surface. For all the cases studied, there
was a significant reduction in the number of CFU in the irradiated
sample. This decrease was similar for the three strains, although for
S. aureus and S. epidermidis4 the reduction was about 40 % and for
S. epidermidis2 was near 35 %.
Statistical comparisons were made assuming a Gaussian distri-
bution and considering that data are randomly distributed. The
paired t-test gave a P value of 0.0012 and the Wilcoxon test of
0.0039, both significant.
The second protocol was carried out by carefully rubbing the
control and the irradiated surface directly against a series of agar
plates, avoiding sonication. This method gave similar relationships
than the previous one, i.e., there was always a reduction in the
number of CFU in the irradiated surface with respect to the control
one. In this case the maximum reduction values found were of only
20 % (P¼0.0377) likely due to the impossibility of detaching all
adhered bacteria with the rubbing, which was confirmed with the
microscopic observation of the surface alloy after rubbing.
3.3. Assessment of bacterial biofilm growth on the control and
A linear relationship was found between the detected amount of
bacterial ATP and the number of viable cells (CFU/ml) using
S. epidermis4 with a high correlation factor (r2¼0.975, data not
shown). Then, experiments were carried out with this biofilm
producer strain, as S. epidermidis4 exhibited a behavior with regard
to the UV-irradiated surfaces similar to the other strains and has
shown reductions in viable cells of the same order of magnitude
than the others.
After both times selected for the growth of biofilms there was
reduction in microbial biofilm formation on the irradiated
surfaces versus the control. It is worth to mention that after 8 h, 14
% of the experiments performed showed no reduction, while 86 %
of them showed a markedly reduction in the ATP bioluminiscence
signal. These data are summarized in Fig. 3. It is interesting to
note that half of the total number of experiments presented
a biofilm reduction comprised between 45 % - 75 %, and for the
other cases the reduction was distributed equally in the other
three ranges considered. The two-tailed P value was 0.0085, i.e.,
After 24 h of culture, the changes in biofilm formation between
the control and the irradiated samples were lower. Though reduc-
tions were observed in all the cases, the percentage in biofilm
decrease was never higher than 16%. The small changes gave a two-
tailed P value very close to the significance level limit (P¼0.0577),
which implied not quite significant differences.
3.4. Insights into the post-radiation antimicrobial mechanisms
3.4.1. Photocatalysis on the irradiated surface
None of the reactive species employed were degraded by the
control or the irradiated surface (data not shown), in any of the
several repetitions carried out, which indicates that photocatalysis
is not the reason for the antimicrobial activity manifested by the
surfaces after irradiation.
3.4.2. Duration time of the post-radiation effect
A summary of the results concerning the post-radiation waiting
times for S. epidermidis4 is presented in Fig. 4 (similar images were
obtained for the other strains). In all cases bacteria were in contact
with the surface for 60 min, the time of maximum damage
observed for bacteria in contact with the irradiated surfaces.
In these images it can be appreciated that microorganisms
become more viable as the post-radiation waiting time increases.
After awaiting time of 30 min bacteria appear stained in red, and as
< 15 %
15 % - 45 %
Ranges of biofilm reduction (%)
45 % - 75 %
> 75 %
Number of experiments
Fig. 3. Biofilm reduction (in range) associated with the number of experiments that
presented visible reductions for the strain S. epidermidis4.
A.M. Gallardo-Moreno et al. / Biomaterials 31 (2010) 5159e5168
this waiting time increases a higher number of green spots are
visible. Finally, when this time goes up to 120 min, the viability of
adhered bacteria is about 100 %. In the case of S. epidermidis2 and
S. aureus this final state is reached at the 90 minwaiting time, while
in the case of S. aureus a very high proportion of green bacteria is
observed at 60 min.
3.4.3. Radiation emission
Fig. 5 shows representative images for S. epidermidis2, adhered
to the surface of the different non-conducting film surfaces inves-
tigated. Control surfaces always showed complete viability while
irradiated surfaces were able to turn into yellow-like or orange-red
color those bacteria adhered to quartz and glass film. However,
when a black film was used, with the same thickness than quartz
and glass film, adhered bacteria were 100% viable.
The characterization of the three films employed was carried
out by obtaining the transmission curves for different wave-
lengths, ranging from 254 nm to 490 nm and results are presented
in Fig. 6. It can be observed that quartz has nearly complete
transmission in the UV range selected, while glass film shows high
transmission from 305 nm. No transmission is observed for the
black plastic film.
3.4.4. Electrical interaction potential at the irradiated surface
Zeta potential at the same pH of adhesion experiments (pH 7)
did not change after irradiating the surface. The values obtained
were ?31?2 mV and ?30?2 mV for control and irradiated
surfaces, respectively, which would denote a similar negatively
charged surface in both cases.
3.4.5. Surface charge excess on the irradiated surface
Fig. 7 presents the viability of bacteria adhered to the con-
ducting film deposited over the control and irradiated surface after
turning off the UV lamp. It is clearly appreciated that bacteria are
degraded in the case of the irradiated surface, while control surface
exhibits 100% viability.
4.1. The antimicrobial effect
Irradiation of Ti6Al4V surfaces with UV-C light provokes
a residual post-radiation effect which directlyaffects the viability of
adhered bacteria. Staining-based methods have shown that
bacteria attached to the irradiated surfaces present a progressively
Fig. 4. Post-radiation waiting times experiments. Bacteria are allowed to contact with the irradiated surface for 60 min not immediately after turning off the UV lamp, but after
waiting different times. Images refer to those waiting times. The strain used was S. epidermidis4.
A.M. Gallardo-Moreno et al. / Biomaterials 31 (2010) 5159e5168
loss in viability, reaching its maximum damage after 60 min of
contact (Fig. 2). These results have been contrasted with different
methodologies based on the evaluating of the CFU on the control
and irradiated surfaces. Although experiments support the
hypothesis that not all red-stained bacteria correspond to dead
cells, as some of them are able of survive when finding a nutrient
rich media, bacteria adhered to irradiated surfaces always show
a significant reduction in their viability after turning off the UV
lamp. This reduction was also reflected by the different ways in
which the remaining live bacteria begin to build a biofilm over
irradiated vs. nonirradiated Ti6Al4V surfaces (Fig. 3). The decrease
in biofilm production on the irradiated surfaces during the first
hours of its development represents avaluable finding in relation to
minimize the infectious incidences. The initial time after the
implantation is crucial for avoiding the proliferation of microbial
biofilms,the competitive process
eukaryotic cells and other biological molecules yields tothe success
or failure of the implantation.
Results have also revealed that the duration time of the post-
radiation effect is a time-dependent action (Fig. 4). Regardless of
the strain selected, the antimicrobial activation of the surface is
guarantee for at least 60 min after turning off the UV lamp, then it
begins to attenuate. Consequently, the method here proposed for
the preparation of antimicrobial titanium surfaces could become
Fig. 5. Images of adhered bacteria (S. epidermidis2) to different film surfaces placed over the Ti6Al4V surface before irradiation and immediately after irradiation.
black plastic film
Fig. 6. Emission graphs for quartz, glass film and black plastic film in the UVevisible
A.M. Gallardo-Moreno et al. / Biomaterials 31 (2010) 5159e5168
especially useful in totally internal implant surgery, for which
the antimicrobial challenge is mainly during or shortly after
4.2. Mechanisms underlying the antimicrobial effect
Reactive oxygen species are known as the basis of the photo-
catalytic disinfection system, hydroxyl radicals being frequently
assumed to be the major factor responsible for the antimicrobial
activity observed in the TiO2photocatalysis . Therefore, we
firstly hypothesized that surface free radicals could be the reasons
for the antimicrobial activity shown by the irradiated surface.
In bibliography, oxidant analysis with testing solutions is
commonly employed to reveal the presence of such radicals in TiO2
systems under UV light [37,39,46]. Hereby based on such studies,
different probe chemicals, with different methodology, have been
used to check the presence of oxygen reactive species in the irra-
diated surface. Neither the applied tests based on the degradation
of dyes nor the accurate experiments based on chromatography
have demonstrated that reactive species are generated on the
irradiated surface after turning off the UV lamp.
Viability results when films made from dielectric materials are
deposited on the Ti6Al4V surface (Fig. 5) have provided useful
information about the causes underlying the antimicrobial effect.
Bacteria can be receiving an external energy from the irradiated
surface that potentially damages the cell membranes even though
they are not in direct-intimate contact with the surface. It is very
likely that, after irradiation, the excited Ti6Al4V surface returns the
absorbed energy in a radiation form. Electron-hole pairs induced in
the semiconductor energy bands of TiO2upon irradiation do not
recombine instantly. In ordinary semiconductors, after activation,
the electron-hole pairs are recombined very fast, however, in the
titanium dioxide this process is relatively slow and by this reason,
after the photoactivation, the TiO2surface continues stable, this
aspect being decisive for the vastly use of TiO2as photocatalytic
support . In our research it is shown a direct consequence of
such a slow recombination, implying the emission of radiation
while recombining. From the transmission curves of the three
selected films presented in Fig. 6, we have observed the nearly
complete transmission of quartz in the UV range selected and the
high transmission of the glass film from about 305 nm. Considering
that, according tothe manufacturer, our short-wave UV light source
generates radiation almost exclusively at 254 nm (www.philips.
com/uvpurification) it is very likely that emission might be
carried out at similar and longer wavelengths since both quartz and
glass film showloss in bacterialviability. This is also consistent with
theless intense colorof bacteria adhered tothe glass film in relation
to the quartz surface (Fig. 5).
It is widely established that radiation at wavelengths below
320 nm causes disinfection, with the optimum effect occurring
around 260 nm [48,49]. In this sense, the inactivation of microor-
ganisms under UV light is understood by means of DNA alterations,
basically, adjacent thymine bases form chemical bond that creates
dimmers preventing DNA replication [50,51]. However, by the time
the UV illumination is ceased, some microorganisms, particularly
bacteria, are known to be capable of repairing their damage DNA in
the presence or absence of visible light by mechanisms commonly
referred to as photoreactivation and dark repair, respectively .
The basis of such activating mechanisms relies on what is also
known as SOS responses. The term SOS response has been applied
to the cellular response to DNA damage produced by UV as well as
other agents such as pH, high pressure stresses or subinhibitory
doses of antibiotics [52e55]. A previous work of Crowley and
Courcelle  has shown that E. coli is able to challenge the DNA
lesions caused upon irradiation with near ultraviolet (254 nm) by
upregulating the expression of several genes which function to
repair the DNA lesions, maintaining the integrity of the DNA
replication fork and preventing premature cell division. Also, in
a recent review of Erill et al.  the SOS response is considered as
a moderately infrequent event, triggered mainly by UV irradiation.
Photoreactivation, dark repair or SOS response must be acting on
the adhered bacteria once the energy emission from the irradiated
surface finishes, explaining the observed viability increase in those
experiments carried out with nutrient-rich media.
The relativelyslowrecombination of electron-holes pairs of TiO2
after irradiation can also provoke a surface charge excess on the
Ti6Al4V surface which could directly affect the viability of sessile
bacteria. It has been already described that the bacterial adhesion
state to metallic surfaces can be altered by the application of little
currents . Although it has not been observed changes in the
zeta potential between the control and irradiated surfaces, in
a previous work of our group  we have shown that irradiated
surface displayed a huge increase in the electronedonor parameter
of the surface tension, as consequence of the electron promotion
from the valence to the conduction band after irradiation. Probably,
this excess of surface charge is not detected by streaming potential
because of the particularities of the technique: time needed for
conditioning and filling the measuring streaming channel without
air bubbles is long (it can take about 30 min) and it can be crucial
for detecting any electrical change between both surfaces. Never-
theless, the viability tests applied on the adhered bacteria to a thin
opaque conducting film (with the same thickness than previous
dielectric films) placed over the irradiated surface immediately
Fig. 7. Viability of adhered bacteria (S. epidermidis2) to a thin opaque conducting film placed over the control and Ti6Al4V irradiated surface after turning off the UV lamp.
A.M. Gallardo-Moreno et al. / Biomaterials 31 (2010) 5159e5168
after turning off the UV lamp (Fig. 7), confirmed the degradation of
bacteria in such conditions. Bacteria turned into complete green
when repeating the experiments but delaying the deposition of
bacterial suspension to 30 min. Surface charge changes during
recombination process would induce small potential gradients,
which, in turn, can be directly related to small surface electrical
currents. Both effects, the surface charge excess on the Ti6Al4V
surface after irradiation and the energy emission while the elec-
tron-hole recombinations must be present at the initial steps of
bacterial adhesion as shown by the enormous reduction (z95%) in
the bacterial viability during the first 20 min of adhesion experi-
ments (Fig. 2b). Present experiments in our group are devoted to
measure such as electrical surface currents by using more specific
The surface of the biomaterial Ti6Al4V shows bactericidal effect
after being exposed to UV-C for both gram-positive and gram-
negative bacteria. This effect is a time-dependent action but,
regardless the strain selected, it is guaranteed at least 60 min after
turning off the UV lamp before it begins to attenuate. Deepening in
the observed phenomena, radiation emission from the irradiated
surfaces and changes in the surfaces charge taking place during the
electronehole recombination process are proposed as the mecha-
nisms responsible for the post-radiation effect. This research can
bring great benefits in the implant field. The first reason relies on
the fact that irradiation of the prosthesis before implantation can
diminishes the infectious incidences in totally internal implants
through preventing the bacterial proliferation on the Ti6Al4V
surface. The second reason is built on the low costs and easiness in
the application of the methodology proposed.
This work was supported by Grants MAT2006-12948-C04-(03-
04) and MAT2009-14695-C04-(01-03) from the “Ministerio de
Ciencia e Innovación”, Grant PRI08A124 from the “Consejería de
Economía, Comercio e Innovación” of the “Junta de Extremadura”.
The authors thank the contribution of E.M. Rodríguez from the
Departmentof Chemical Engineering and Physical-Chemistryof the
UEx for the HPLC measurements. Thanks also to V. Vadillo-Rodrí-
guez from the Department of Applied Physics of the UEx and T.A.
Camesano from the Department of Chemical Engineering of the
Worcester Polytechnique Institute (MA, USA) for the useful correc-
tions-comments in the elaboration of this manuscript.
Figure with essential colour discrimination. Most of the figures
in this article have parts that are difficult to interpret in black and
white. The full color images can be found in the on-line version, at
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