caracterizaciÓn y respuesta antibacteriana de la
TRANSCRIPT
CARACTERIZACIÓN Y RESPUESTA ANTIBACTERIANA DE LA
SUPERFICIE DEL BIOMATERIAL TI6AL4V SOMETIDO A
DIFERENTES MODIFICACIONES FÍSICAS
Memoria presentada por D. Miguel Ángel Pacha Olivenza para optar al Título de
Doctor por la Universidad de Extremadura
Tesis Doctoral realizada bajo la dirección de los Doctores Dña Amparo María Gallardo
Moreno, Dña María Luisa González Martín y D. Ciro Pérez Giraldo
Departamento de Física Aplicada
Universidad de Extremadura
ÍNDICE
Capítulo 1: Introducción ............................................................................................ 1
1.1. Los implantes y el problema asociado a las infecciones....................................... 3
1.2. La aleación Ti6Al4V .......................................................................................... 9
1.2.1. Pasivado superficial del Ti6Al4V ............................................................... 10
1.3. La superficie y su relevancia en la adhesión microbiana .................................... 11
1.3.1. Propiedades superficiales ........................................................................... 13
1.3.1.1. Hidrofobicidad .................................................................................... 13
1.3.1.2. Energía de Gibbs superficial................................................................ 14
1.3.1.3. Potencial eléctrico de interacción. Potencial zeta ................................. 17
1.3.1.4. Caracterización electroquímica de la superficie ................................... 19
1.3.1.4.1. Polarización lineal ....................................................................... 21
1.3.1.4.2. Espectroscopía de Impedancia Electroquímica (EIS) ................... 23
1.3.1.4.2.1. Interpretación del sistema en base a un circuito eléctrico
equivalente ........................................................................................... 26
1.3.1.4.3. Propiedades electrónicas de las vacantes anódicas y catódicas.
Ensayos Mott-Schottky ............................................................................... 29
1.4. Métodos de evaluación de la adhesión microbiana ............................................ 30
1.4.1. Modelos teóricos de adhesión microbiana .................................................. 30
1.4.1.1. Determinación de la energía de Gibbs de interacción ........................... 33
1.4.2. Metodología empírica: Adhesión estática y dinámica ................................. 34
1.5. Objetivo ............................................................................................................ 37
1.6. Bibliografía....................................................................................................... 37
Modificación superficial basada en implantación con silicio................................... 47
Capítulo 2: Si+ ion implantation reduces the bacterial accumulation on the
Ti6Al4V surface ....................................................................................................... 48
2.1. Introduction ...................................................................................................... 49
2.2. Materials and methods ..................................................................................... 50
2.2.1. Ti6Al4V surface modification .................................................................... 50
2.2.2. Surface characterization ............................................................................. 50
2.2.3. Bacterial strains .......................................................................................... 50
2.2.4. Adhesion experiments ................................................................................ 50
2.3. Results and discussion ...................................................................................... 51
2.4. References ........................................................................................................ 53
Modificación superficial basada en irradiación con luz UV-C ................................ 55
Capítulo 3: Effect of UV irradiation on the surface Gibbs energy of Ti6Al4V and
thermally oxidized Ti6Al4V ...................................................................................... 56
3.1. Introduction ...................................................................................................... 57
3.2. Materials and methods ..................................................................................... 59
3.2.1. Sample characterization ............................................................................. 59
3.2.2. Experimental procedures ............................................................................ 60
3.3. Results and discussion ...................................................................................... 61
3.4. Conclusions ...................................................................................................... 72
3.5. References ........................................................................................................ 72
Capítulo 4: Influence of slight microstructural gradients on the surface properties
of Ti6Al4V irradiated by UV .................................................................................... 76
4.1. Introduction ...................................................................................................... 77
4.2. Materials and methods ..................................................................................... 79
4.2.1. Material...................................................................................................... 79
4.2.2. Microstructural characterization ................................................................. 79
4.2.3. Surface wettability characterization ............................................................ 80
4.3. Results and discussion ...................................................................................... 82
4.4. Conclusions ...................................................................................................... 91
4.5. References ........................................................................................................ 91
Capítulo 5: In vitro biocompatibility and bacterial adhesion of physico-chemically
modified Ti6Al4V surface by means of UV irradiation ........................................... 95
5.1. Introduction ...................................................................................................... 96
5.2. Materials and methods ..................................................................................... 97
5.2.1. Ti6Al4V ..................................................................................................... 97
5.2.2. Cell culture ................................................................................................ 98
5.2.3. Cell spreading assays ................................................................................. 99
5.2.4. Bacterial strains and culture........................................................................ 99
5.2.5. Bacterial adhesion-retention experiments ................................................. 100
5.2.6. Physico-chemical surface characterization ................................................ 101
5.2.6.1. Contact angle measurements ............................................................. 101
5.2.6.2. Zeta potential measurements ............................................................. 101
5.2.7. X-DLVO analysis .................................................................................... 102
5.2.8. Statistical analysis .................................................................................... 103
5.3. Results and discussion .................................................................................... 103
5.3.1. Effect of UV treatment on cell behaviour ................................................ 104
5.3.2. Effect of UV treatment on bacterial adhesion ........................................... 107
5.3.3. Effect of ultra-violet treatment on bacterial retention ................................ 111
5.3.4. Physico-chemical approach to adhesion and retention .............................. 114
5.4. Conclusions .................................................................................................... 117
5.5. References ...................................................................................................... 118
Capítulo 6: Bactericidal behavior of Ti6Al4V surfaces after exposure to UV-C light ................................................................................................................................. 123
6.1. Introduction .................................................................................................... 124
6.2. Materials and methods ................................................................................... 126
6.2.1. Ti6Al4V ................................................................................................... 126
6.2.2. Bacterial strains and culture...................................................................... 127
6.2.3. Adhesion experiments .............................................................................. 128
6.2.4. Viability tests on adhered bacteria ............................................................ 128
6.2.4.1. Staining-based methods ..................................................................... 128
6.2.4.2. Serial dilution method ....................................................................... 129
6.2.4.3. Direct transfer of bacteria to solid agar .............................................. 129
6.2.5. Biofilm formation on the Ti6Al4V surface ............................................... 129
6.2.6. Evaluation of changes on the Ti6Al4V surface after irradiation ................ 130
6.2.6.1. Photocatalysis on the irradiated surface ............................................. 130
6.2.6.2. Duration time of the post-radiation effect .......................................... 131
6.2.6.3. Radiation emission from the irradiated surface .................................. 131
6.2.6.4. Electrical interaction potential at the irradiated surface ...................... 132
6.2.6.5. Surface charge excess on the irradiated surface ................................. 132
6.2.7. Statistical analysis .................................................................................... 132
6.3. Results ............................................................................................................ 133
6.3.1. Quantification of adhered bacteria to the control and irradiated surface .... 133
6.3.2. Evaluation of the viability of adhered bacteria to the control and irradiated
surface ............................................................................................................... 134
6.3.3. Assessment of bacterial biofilm growth on the control and irradiated surface
.......................................................................................................................... 137
6.3.4. Insights into the post radiation antimicrobial mechanisms ........................ 138
6.3.4.1. Photocatalysis on the irradiated surface ............................................. 138
6.3.4.2. Duration time of the post-radiation effect .......................................... 138
6.3.4.3. Radiation emission ............................................................................ 140
6.3.4.4. Electrical interaction potential at the irradiated surface ...................... 141
6.3.4.5. Surface charge excess on the irradiated surface ................................. 141
6.4. Discussion ...................................................................................................... 142
6.4.1. The antimicrobial effect ........................................................................... 142
6.4.2. Mechanisms underlying the antimicrobial effect ....................................... 143
6.5. Conclusions .................................................................................................... 145
6.6. References ...................................................................................................... 146
Capítulo 7: Explaining the bactericidal behavior of the UV-treated Ti6Al4V
substrata through electrochemical surface analysis .............................................. 152
7.1. Introduction .................................................................................................... 153
7.2. Materials and methods ................................................................................... 155
7.2.1. Ti6Al4V ................................................................................................... 155
7.2.2. Solution electrolyte .................................................................................. 155
7.2.3. Electrochemical characterization .............................................................. 156
7.2.4. Electrochemical Impedance Spectroscopy ................................................ 156
7.2.5. Potentiodynamic polarization curves ........................................................ 156
7.2.6. Mott-Schottky curves ............................................................................... 157
7.3. Results and discussion .................................................................................... 157
7.3.1. Electrochemical Impedance Spectroscopy ................................................ 158
7.3.1.1. EIS for Ti6Al4V before and after the UV irradiation treatment ......... 158
7.3.1.2. EIS for Ti6Al4V after different post-radiation times .......................... 161
7.3.2. Potentiodynamic polarization curves ........................................................ 163
7.3.3. Mott-Schottky experiments ...................................................................... 164
7.4. Conclusions .................................................................................................... 167
7.5. References ...................................................................................................... 167
Capítulo 8: Conclusiones generales ........................................................................ 173
1
CAPÍTULO 1
Introducción
2
Esta Tesis se ha realizado dentro del Grupo de Investigación Superficies e Interfases de
la Universidad de Extremadura. Ha supuesto una progresión en su línea de investigación
de adhesión bacteriana en biomateriales, ya que se ha extendido el estudio de superficies
poliméricas a superficies metálicas, ampliando la metodología utilizada y colaborando
activamente con otros centros de investigación. Concretamente, en esta memoria se
presentan los resultados obtenidos de la aleación Ti6Al4V a la que se ha modificado su
superficie para mejorar su respuesta como biomaterial, mediante la implantación de iones
de silicio o mediante irradiación con luz ultravioleta. Esta última es la que ha provocado
alteraciones más importantes en las propiedades superficiales de la aleación, por lo que
ha sido más extenso el trabajo que se ha realizado sobre ella. Los resultados que se han
obtenido en esta investigación, junto con los correspondientes estudios mecánicos y de
citocompatibilidad llevados a cabo por los grupos de investigación del Centro Nacional
de Investigaciones Metalúrgicas (CENIM, CSIC, Madrid) y del Instituto de
Investigación Biomédica del Hospital La Paz (Madrid) dirigidos por el Dr. González
Carrasco y la Dra. Vilaboa Díaz, se han proyectado en cinco artículos internacionales,
además de en doce comunicaciones en congresos, que se corresponden con los
Capítulos 2 a 7 de esta Tesis. Finalmente, hay que señalar que el Capítulo 1 se ha
dedicado a proporcionar una visión general de las bases sobre las que se sustenta esta
Tesis.
3
1.1. LOS IMPLANTES Y EL PROBLEMA ASOCIADO DE LAS INFECCIONES
Los implantes están diseñados para ser incorporados al sistema vivo reemplazando
alguna función de sus tejidos o de sus órganos o para ser soporte para la regeneración de
estos tejidos. Sus usos son múltiples, por ejemplo, para sustituciones permanentes o
para fijaciones temporales en el tejido duro, óseo, (hombros, dedos, rodillas, caderas,
etc) o en el tejido blando, muscular (hernias inguinales) o como elementos permanentes
en el sistema cardiovascular (válvulas cardíacas, marcapasos, arterias y venas
artificiales), en el sistema respiratorio (laringe, tráquea y bronquios, diafragma,
pulmones), en el sistema digestivo (esófago, conductos biliares e hígado), en el sistema
genitourinario (riñones, uréter, uretra, vejiga) y en los órganos sensitivos (córneas,
oídos…) [1].
El origen de los materiales que se emplean para estos implantes puede ser biológico o
artificial. Los primeros son soportes habituales para las cada vez mayores aplicaciones
de la ingeniería de tejidos, pero el concepto de biomaterial está restringido a aquellos
“materiales no viables empleados para la fabricación de dispositivos médicos diseñados
para interaccionar con los sistemas biológicos” [2].
Para elegir correctamente el biomaterial con el que se debe construir el implante, es
esencial entender las relaciones existentes entre su estructura, sus propiedades y la
función que va a desarrollar. En este sentido, se acepta ampliamente que los requisitos
que debe cumplir un biomaterial sean [1]:
Ser biocompatible, es decir, debe ser aceptado por el organismo, no provocar
que éste desarrolle mecanismos de rechazo ante su presencia.
Ser químicamente estable, es decir, no presentar degradación en el tiempo y ser
inerte.
Tener una resistencia mecánica adecuada.
Tener un tiempo de fatiga compatible con las necesidades de su uso.
Tener densidad y peso apropiados a su aplicación.
Tener un diseño de ingeniería correcto; esto es, el tamaño y la forma del
implante deben ser acordes a su finalidad.
4
Los cuatro tipos generales de biomateriales son los metálicos, los cerámicos, los
poliméricos y los compuestos. Concretamente el interés de esta Memoria se centra en
uno de los biomateriales metálicos más habituales, la aleación de titanio Ti6Al4V.
Aunque los biomateriales metálicos tienen distintos usos, su empleo en implantes
estructurales es básico, reemplazando determinados componentes del cuerpo humano,
fundamentalmente cuando se requiere soportar cargas (implantes dentales, huesos y
articulaciones), o también como elementos de fijación temporales. Por tanto, además de
ser biocompatibles, es necesario que tengan buenas propiedades mecánicas y una gran
resistencia a la corrosión.
Una de las complicaciones que puede aparecer relacionada con el uso de implantes,
prótesis u otros elementos artificiales es el desencadenamiento de procesos infecciosos
originados por su colonización por microorganismos. La incidencia de este tipo de
procesos infecciosos es bastante habitual en dispositivos como catéteres vasculares o
sondas vesicales alcanzando, tras 1000 días de uso, el 5-15 % de los casos, dependiendo
del área de hospitalización que se analice. En el caso de implantes metálicos en
ortopedia, si bien la tasa de infección oscila entre un 2-4 % cuando se trata de implantes
fijos, se eleva hasta un 45 % en el caso de los clavos empleados como fijadores
externos. El aumento progresivo del número de implantes hace que los episodios de
infección se estén incrementando de forma absoluta con un coste humano, además del
económico, muy elevado, ya que el proceso asociado a una sustitución de un implante
originado por una infección supone un gasto de unos 50000 € por caso [3].
La causa directa del desarrollo de un proceso infeccioso en un implante es la formación
de una biocapa sobre la superficie del material. Una biocapa se puede definir como un
conjunto de células eucariotas y procariotas distribuidas sobre la superficie de un
determinado material, bien sea inerte o vivo, y embebidas en una matriz orgánica de
origen biológico [4]. Los estudios han revelado que las biocapas están presentes en
todos los ecosistemas. De hecho, en la mayoría de los ambientes naturales la asociación
de los microorganismos con una superficie formando biocapas es el “estilo de vida”
prevalente. Actualmente se admite que en ambientes naturales entre el 95-99% de los
microorganismos se encuentran en biocapas [5] y que la forma de vida planctónica no es
más que un medio de traslado de una superficie a otra [6]. La capacidad de formación
5
de biocapa no parece restringida a ningún grupo específico de microorganismo y se
considera que bajo condiciones ambientales adecuadas se podrían encontrar cepas
formadoras de biocapas de cualquiera de ellos. [7].
Es interesante mencionar que, incluso restringiéndonos al ámbito de la salud, la
presencia de una biocapa no es necesariamente una circunstancia perjudicial. Así, por
ejemplo, las biocapas de lactobacilos presentes en la vagina previenen la colonización
por microorganismos patógenos como Gardnerella vaginalis y otros microorganismos
anaerobios [8]. También la biocapa formada sobre la superficie de los dientes protege
frente a la colonización por otros patógenos exógenos, siempre que las especies
bacterianas que la constituyan se mantengan en equilibrio [8]. Sin embargo, en el
ámbito de este trabajo, la formación de biocapas en la superficie de implantes
esqueléticos es un proceso perjudicial, que se trata de evitar en la mayor extensión
posible, y en el que están implicadas mayoritariamente cepas de Staphylococcus
epidermidis y Staphylococcus aureus.
La formación de la biocapa es un proceso dinámico complejo que, aunque algunos
autores describen asociado a condiciones de flujo turbulento [9], es muy similar
independientemente del ambiente en el que habiten los microorganismos, pero en
cualquier caso ha de comenzar con la adhesión inicial de algunos microorganismos a la
superficie del material.
Se ha propuesto una particular metáfora patogénica para explicar la situación que ocurre
después del proceso de inserción de un implante: “La carrera por la superficie” [10]. De
acuerdo con este concepto, tanto la adhesión microbiana como la integración de los
biomateriales con el tejido del hospedador son procesos que se llevan a cabo de forma
similar, aunque las consecuencias finales en cada caso sean muy diferentes, de modo
que si el implante está adecuadamente integrado con el tejido consigue tener una mejor
defensa frente a la contaminación bacteriana. Aunque esta “carrera por la superficie” se
ha descrito en términos de microorganismos y células eucariotas del hospedador,
conviene señalar que después de la implantación tiene lugar una secuenciación de etapas
con participación activa de otros agentes biológicos que culminan con la formación de
la biocapa madura [11-13]:
6
Es importante señalar que inmediatamente después de implantar el material y contactar
con el medio fisiológico tiene lugar una cascada de procesos en su superficie que
originan la interfase entre el material y el medio. Nada más ponerse en contacto el
implante con los fluidos fisiológicos, las moléculas de agua, al ser las mayoritarias en
estos medios, son las primeras en alcanzar y acomodarse a la superficie del material,
formando sobre este una monocapa o bicapa, cuya estructura es diferente a la del agua
líquida (Fig. 1.1a). En líneas generales el agua interacciona de manera diferente con las
superficies de acuerdo a las propiedades de mojabilidad de éstas; en una superficie
hidrofílica las moléculas de agua pueden disociarse formando una superficie terminada
en grupos –OH o bien se adsorben fuertemente en forma molecular. En cambio, en una
superficie hidrofóbica, las moléculas de H2O, sin disociarse, se adsorben débilmente a la
superficie [13].
De forma casi simultánea y debido a la ionización de la superficie al ponerse en
contacto con el líquido, la interfase se enriquece con iones del medio biológico, como
Na+ y Cl
-, formando lo que se denomina doble capa eléctrica (se describirá con mayor
detalle más adelante) cuya extensión depende fundamentalmente de las propiedades
electrostáticas de la superficie del implante y de la fuerza iónica del medio (Fig. 1.1b).
A continuación, proteínas, como la albúmina, la fibronectina, el fibrinógeno, la
vitronectina o la laminina, y otras macromoléculas se acercan a la interfase donde se
adsorben en base a su concentración relativa en el medio, su tamaño, las propiedades de
la superficie y el grado de hidratación tanto de la superficie como de la proteína. (Fig.
1.1c). Es interesante destacar que este proceso de formación de la interfase biomaterial-
fluido fisiológico es un proceso muy dinámico y en el que la competición por la
superficie entre proteínas provoca que la composición y estructura de la interfase se
vaya modificando en el transcurso del tiempo, desplazando unas proteínas a otras de la
capa adsorbida y forzando su reorientación, lo que se conoce como efecto Vroman [14].
7
Fig. 1.1. Secuencia de eventos que ocurren al colocar un biomaterial dentro del cuerpo humano
[13].
Si nos centramos en los procesos de adhesión bacteriana, el siguiente evento tras la
formación de esta película acondicionadora de proteínas en la superficie del material es
la aproximación de los posibles microorganismos planctónicos. Los mecanismos a
través de los cuales son transportados hacia la superficie del hospedador incluyen el
movimiento Browniano, la sedimentación, la difusión forzada y natural y el transporte
activo mediado por actividad flagelar que puede incluir o no quimiotaxis [15]. Una vez
que los microorganismos planctónicos están próximos a la superficie del implante,
comienza el proceso de formación de la biocapa propiamente dicho, que se puede
resumir en cuatro etapas: adherencia primaria o inespecífica, adherencia secundaria o
específica, acumulación y separación o dispersión (Fig. 1.2).
En la fase de adherencia primaria, también conocida como fase reversible o
inespecífica, los microorganismos se retienen sobre el sustrato. Existen bacterias Gram
negativas como Pseudomonas aeruginosa, Vibrio cholerae, Escherichia coli,
Salmonella enterica en las que los flagelos suponen un elemento decisivo para alcanzar
la superficie ya que la motilidad ayuda a la bacteria a aproximarse al sustrato
contrarrestando posible fuerzas repulsivas. Sin embargo, aunque la motilidad ayuda al
proceso no parece ser un requisito esencial, pues muchas bacterias Gram positivas
inmóviles como Estafilococos, Estreptococos y Micobacterias son igualmente capaces
de formar biocapas. La retención de los microorganismos tiene lugar a través de fuerzas
físico-químicas como son las fuerzas de van der Waals, hidrofóbicas o relacionadas con
(a) (b) (c)
8
la polaridad y eléctricas. Una vez que los microorganismos se han aproximado al
sustrato, en una segunda fase conocida como fase irreversible o específica, los
microorganismos retenidos comienzan a producir adhesinas (MSCRAMMs), que son
proteínas de superficie implicadas en la interacción permanente con la película
precursora de proteínas adsorbidas [16, 17].
A continuación, comienzan los procesos de adherencia intercelular que consolidan la
biocapa, es lo que se conoce como fase de acumulación. Después de adherirse al cuerpo
extraño, las bacterias se multiplican y se acumulan como conglomerado de células en
múltiples capas. Las células adheridas generan una gran cantidad de componentes
extracelulares que interaccionan con las moléculas orgánicas e inorgánicas del medio
ambiente más inmediato para formar una matriz extracelular, denominada glicocálix o
slime. Esta matriz extracelular en la que se encuentran embebidas las bacterias,
constituye aproximadamente el 85 % de la totalidad de la biocapa y está compuesta por
una mezcla de materiales tales como polisacáridos, proteínas, ácidos nucleícos, etc y se
considera como el “cemento” que mantiene las células unidas, ayudando a atrapar y
retener nutrientes para el crecimiento de la biocapa y protegiendo a las células de la
deshidratación y de la acción de los antimicrobianos.
Fig. 1.2. Etapas de un proceso de formación de una biocapa.1 Adherencia primaria o
inespecífica; 2 Adherencia secundaria o específica; 3 y 4 fase de acumulación y 5 fase de
separación [18].
Por último, algunas células se liberan al medio líquido posibilitando la dispersión a
otras superficies. Un mecanismo propuesto, particularmente cuando se trata de biocapas
situadas en zonas expuestas al flujo del fluido, es por disgregación mecánica, por
9
fuerzas de cizalla. Sin embargo, estudios más recientes sugieren que la separación,
llamada frecuentemente “dispersión” o “disolución”, es un proceso activo que está
regulado por las células adheridas, las cuales son capaces de comunicarse entre ellas
dentro de la biocapa con mecanismos conocidos como quorum sensing [19, 20] que
estimulan la salida de bacterias desde la biocapa hacia el medio, lo que les permite
colonizar otras zonas alejadas de la biocapa primaria.
1.2. LA ALEACIÓN TI6AL4V
El Ti es un metal de transición muy atractivo debido al buen balance entre sus
propiedades mecánicas, su alta resistencia a la corrosión y su buena biocompatibilidad,
con módulo de Young de 110 GPa, más cercano que otros metales al del hueso cortical,
tensión de ruptura de 1 GPa y densidad de 4,4 g/cm3. A pesar de ello, se ha dopado el
titanio con ciertos elementos químicos con el fin de mejorar aún más sus propiedades.
En este sentido, la aleación Ti6Al4V es hoy la usada más frecuentemente entre las
aleaciones de titanio para aplicaciones biomédicas. Hay que señalar que la aleación
Ti6Al4V posee ciertas ventajas frente al Ti comercialmente puro (Ti cp). Esta aleación
es más resistente que el Ti cp, es decir tiene un mayor límite elástico, mayor resistencia
máxima a la tracción, mayor resistencia a la fatiga y a la corrosión y además posee una
excelente biocompatibilidad [21, 22].
El titanio es el único metal ligero que presenta dimorfismo, ya que su estructura
compacta hexagonal (fase α) presenta una transformación alotrópica a 882 ºC, pasando
a una microestructura cúbica centrada en el cuerpo (fase β) [23]. El dimorfismo del
titanio ofrece la posibilidad de obtener aleaciones con microestructuras de tipo α, β ó
α/β, dependiendo de los elementos aleantes, que estabilizan una u otra fase a
temperaturas diferentes de las de equilibrio para el Ti puro.
Según la capacidad de los aleantes de estabilizar la fase α o β, se definen tres tipos de
aleaciones de titanio. Las aleaciones tipo α son aquellas que se preparan con elementos,
como el aluminio, el oxígeno, el carbono y el nitrógeno, que incrementan la temperatura
a la cual la fase α es estable. Las aleaciones tipo β son aquellas en que la fase β es
estable a temperaturas inferiores a la temperatura de transición (882ºC) y se obtienen
por la adición de aleantes como el vanadio, el molibdeno y el tántalo, y también el
hierro, el manganeso, el cromo, el cobalto, el níquel, el cobre y el silicio a través de la
10
formación de sistemas eutectoides con el titanio. Las aleaciones tipo α/β, entre las que
se encuentra la Ti6Al4V, tienen una estructura tal que a temperatura ambiente las fases
α y β se encuentran mezcladas y estabilizadas. La manipulación de estas variaciones
cristalográficas mediante adición de aleantes y procesos termomecánicos da lugar a un
amplio rango de aleaciones y propiedades [23].
1.2.1. Pasivado superficial del Ti6Al4V
El titanio es un metal muy reactivo que cuando se expone a un ambiente oxidante, como
la atmósfera, reacciona prácticamente de forma inmediata a sus estados oxidados. Este
proceso que ocurre en su superficie da lugar a una película superficial de óxido o capa
de pasivado. Con el tiempo, la capa de óxido continúa creciendo espontáneamente y
después de 2 h de exposición al aire y a temperatura ambiente su espesor llega a ser de
aproximadamente 1,7 nm. Después de 70 días puede alcanzar los 5 nm y después de 4
años los 25 nm, aproximadamente. A través de tratamientos como anodización,
aplicación de plasma de oxígeno o calentamiento es posible obtener capas más gruesas
en menos tiempo [24]. Esta capa es muy estable, con una adherencia muy buena y
protege al Ti y a sus aleaciones frente a los agentes oxidantes. El óxido mayoritario en
la capa de pasivado es TiO2, pero otros óxidos tales como Ti2O3 y TiO también están
presentes [24], encontrándose en general en estado amorfo, aunque en ocasiones,
dependiendo del proceso de pasivación seguido, puede hallarse en estado
microcristalino.
El dióxido de titanio cristaliza principalmente en tres estructuras: rutilo, anatasa y
brookita. Sin embargo, el rutilo y la anatasa son las formas más estables del TiO2, las
que juegan un papel más importante en sus aplicaciones y las que comúnmente son
observadas en películas delgadas; en la Fig 1.3 se representa la estructura unidad de
ambos cristales.
Una de las propiedades más interesantes del TiO2 es su carácter semiconductor. Su
banda de valencia está formada por los orbitales 2p del oxígeno y su banda de
conducción por los orbitales 3d del titanio. Esta distribución de bandas da lugar a una
banda prohibida de aproximadamente 3.2 eV capaz de activarse con luz de longitudes
de onda ≤413nm para el rutilo y ≤388nm para la anatasa. En semiconductores
ordinarios, tras la activación, los pares electrón-hueco se recombinan rápidamente, sin
11
embargo, en el óxido de titanio este proceso es relativamente lento, por lo que después
de la fotoexcitación su superficie sigue siendo estable.
Fig. 1.3. Estructura cristalina de los óxidos de titanio: (a) TiO2-anatasa y (b) TiO2-rutilo.
1.3. LA SUPERFICIE Y SU RELEVANCIA EN LA ADHESIÓN MICROBIANA
La imposibilidad de alterar in situ la superficie de los microorganismos para hacerlos
menos adhesivos, hace que la estrategia más extendida contra la adhesión bacteriana sea
actuar sobre las superficies a las que se van a adherir, modificando sus propiedades
superficiales. Algunas de las propuestas de modificación superficial también llevan
asociadas el objetivo básico de desarrollar biomateriales con propiedades de resistencia
a la corrosión, fatiga y al desgaste mejoradas.
Kasemo y Gold [25] establecen que las modificaciones superficiales pueden dividirse
básicamente en tres categorías: (a) las modificaciones topográficas, tales como tamaño y
distribución de poros, rugosidad, etc.., (b) modificaciones de las propiedades
bioquímicas de la superficie; liberación de especies químicas, adsorción de
biomoléculas, etc.., y (c) modificaciones de las propiedades micro-mecánicas o
viscoelásticas de la superficie.
De este modo se busca controlar la topografía, ya que diferentes estudios han
demostrado que dicha propiedad, tanto a escala micrométrica como nanométrica, tiene
efectos relevantes en el comportamiento microbiano y celular en general [26-31].
Aunque es conveniente señalar que, en relación a la adhesión bacteriana no existe una
norma clara en cuanto al papel de la rugosidad, se ha descrito en diferentes ocasiones
(a) (b)
TiO2-anatasa TiO2-rutilo
12
que el incremento de la rugosidad en un sólido produce un aumento de la adhesión
bacteriana, debido no tanto a cambios drásticos en sus propiedades físico-químicas sino
al aumento del área superficial disponible para la adhesión [32], sin embargo otros
autores encuentran que ciertas topografías a escalas nanométricas dificultan la adhesión
bacteriana.
Además el objetivo es lograr una superficie activa que interactúe fuertemente con el
medio circundante y que mejore la vida útil del biomaterial. Ejemplos característicos
son, el desarrollo de biomateriales con recubrimientos diseñados para la liberación
controlada de fármacos [33-35], los cuales son inyectados directamente al torrente
sanguíneo actuando de forma eficaz contra los microorganismos presentes, o superficies
modificadas mediante implantación iónica, es decir, mediante la introducción de átomos
de un elemento seleccionado, dentro de las capas subsuperficiales del biomaterial para
conseguir, entre otros efectos, mayor resistencia al desgaste y mejoras en la corrosión y
fatiga.
La Tabla 1.1 presenta un resumen de las técnicas más habituales de modificación
superficial, muchas de ellas son también aplicables a otras áreas con gran éxito, por
ejemplo en la industria mecánica donde la modificación superficial de metales ha
duplicado la vida útil de componentes o piezas de maquinaria [36-39].
Tabla 1.1. Métodos de modificación superficial clasificados de acuerdo al proceso: mecánico,
litográficos, químico o físico. Así como según el efecto de dicha modificación sobre las
propiedades superficiales.
Método Capa Modificada Objetivo Técnica
Mecánicos Modificación de la topografía Diferentes rugosidades Pulido, blasting, grinding
a escalas micrométricas
Litográficos Modificación de la topografía Diferentes rugosidades Fotolitografía, Láser, haz
a escalas micro y nanométricas y patrones de electrones
Químicos Alteración de la superficie Inmersión en ácidos,
químicamente peróxidos
Depósitos de películas delgadas Propiedades físico-químicas Sol-Gel
por Sol-Gel diferentes
Depósitos de películas delgadas Propiedades físico-químicas CVD, PECVD
por evaporización térmica diferentes
Adsorción y autoensamblado Métodos bioquímicos
13
de biomoléculas
Físicos Depósito de películas delgadas Películas porosas Spray Térmico, flama
en forma de spray
Depósito de películas delgadas Películas densas, duras y Sputtering, Arco
por evaporación física resist. al desgaste y corros. Catódico, haz de iones
Modificación de las capas sup. Implantación iónica Nitruración, iones Si, P
Modificación sup. Titanio Grupos hidroxilos Exposición a luz UV-C
Funcionalización Grupos amino, carboxilos Descargas gaseosas
1.3.1. Propiedades superficiales
En principio, cualquier característica de una superficie puede ser un factor que afecte al
proceso de adhesión bacteriana. La topografía del material, es decir, su mayor o menor
rugosidad, el rango de esta o su distribución, afecta al comportamiento adhesivo
bacteria-sustrato. Incluso se ha reseñado la posibilidad de que la dureza superficial
pueda ejercer cierta influencia en el proceso. En cualquier caso, las propiedades físico-
químicas de las superficies, la hidrofobicidad, la energía de Gibbs superficial y el
potential zeta, son decisivas sobre la adhesión. En este trabajo nos centraremos sobre
estas últimas, puesto que las modificaciones que posteriormente se van a imponer a la
aleación de Ti6Al4V van a afectar principalmente a estas propiedades. Además de
dichas propiedades, en este trabajo se hará un análisis de las características corrosivas
superficiales de la aleación como medida indirecta de la posible presencia de corrientes
superficiales después de someter la aleación a uno de los tratamientos superficiales.
1.3.1.1. Hidrofobicidad
Con el término hidrofobidad se suele hacer referencia a la tendencia que muestran
ciertas moléculas o superficies a evitar el contacto con el agua. Si macroscópicamente la
interacción desfavorable entre el agua y una superficie hidrófoba está caracterizada por
un aumento de la energía libre de Gibbs interfacial, desde el punto de vista
microscópico se puede comprender que este aumento proviene de una contribución
negativa de la entropía en el proceso. El origen está en el propio comportamiento del
agua debido a la fuerte afinidad que presentan entre sí sus moléculas como
consecuencia de la intensidad y coordinación que introducen los puentes de hidrógeno
entre ellas. En el caso de moléculas hidrófobas la interacción entre éstas y las moléculas
de agua es menos favorable que entre las moléculas de agua, debido al reordenamiento
que sufren las moléculas de agua para mantener sus enlaces de puente de hidrógeno,
14
provocando una disminución de la entropía. Las moléculas o superficies hidrófilas, es
decir, aquellas en las que su interacción con las moléculas de agua es al menos similar a
la de las moléculas de agua entre sí, permiten aumentar la entropía del conjunto, por lo
que estas moléculas pueden disolverse en agua o, en el caso de las superficies, el agua
puede esparcirse sobre ellas [40]. Ahora bien, puesto que en las interacciones
moleculares participan también otra fuerzas, como las Lifshtz-van der Waals o las
ácido-base, que en su definición más amplia incluyen también las fuerzas por puente de
hidrógeno, es realmente este balance de fuerzas quien finalmente define el carácter
hidrófobo/hidrófilo de un material [41-45]. El método más directo que permite
establecer escalas de hidrofobicidad entre superficies lo constituye la medida del ángulo
que se forma en la línea de contacto entre una gota de agua y la superficie, denominado
ángulo de contacto, y que puede variar entre 0º para superficies completamente
hidrófilas hasta prácticamente 180º para las superficies completamente hidrófobas y que
se describe con más detalle en las próximas secciones.
1.3.1.2. Energía de Gibbs superficial
La energía de Gibbs superficial de un determinado material es el exceso de energía de
Gibbs de la superficie respecto del seno del material, lejos de dicha superficie. Esta
magnitud coincide, en el caso de líquidos o de sólidos isótropos con la tensión
superficial o fuerza precisa, por unidad de longitud, para aumentar la extensión de la
superficie, por lo que ambos términos, energía de Gibbs superficial y tensión
superficial, se emplean indistintamente. A pesar de que no es posible evaluar
directamente la energía de Gibbs superficial de un sólido, Young propuso que el ángulo
de contacto (θ) [46] de una gota de un líquido puro depositada sobre una superficie
sólida, rígida, lisa y homogénea es consecuencia del equilibrio de las energías de Gibbs
interfaciales de las tres fases en equilibrio mutuo:
γLV cos θ = γSV - γSL (1.1)
donde γLV (a veces también simbolizada como γL) es la tensión superficial del líquido en
equilibrio con su vapor, γSV es la tensión interfacial del sólido en equilibrio con el vapor
del líquido y γSL, la tensión interfacial sólido-líquido (Fig. 1.4).
15
Fig. 1.4. Tensiones sobre la línea triple (sólido, líquido, vapor) y ángulo de contacto.
La tensión superficial del sólido, γS, se relaciona con la tensión interfacial γSV mediante
la presión superficial, Πe, que ejerce la película del vapor del líquido que se adsorbe
sobre la superficie del sólido alrededor de la gota, mediante Πe= γS - γSV. De este
modo, la ecuación de Young se puede escribir como:
γLV cos θ = γS - γSL – Πe (1.2)
Sin embargo, se asume que para aquellos sistemas en los que el ángulo de contacto es
mayor que cero la presión superficial de la película adsorbida es prácticamente
despreciable, con lo que la ecuación de Young queda en términos de la energía de
Gibbs superficial del sólido como:
γL cos θ = γS - γSL (1.3)
Esta ecuación permitiría de forma directa conocer la energía de Gibbs interfacial sólido-
líquido si se dispusiera de los valores del ángulo de contacto de un cierto líquido sobre
la superficie del sólido, la energía de Gibbs superficial del líquido y la energía de Gibbs
del sólido. Tanto el ángulo de contacto como la energía de Gibbs del líquido son
magnitudes de acceso experimental directo, pero en cambio no lo son ni la energía de
Gibbs interfacial sólido-líquido, γSL, ni la energía de Gibbs del sólido, γS. Por este
motivo se han desarrollado varios modelos que pretenden expresar la energía de Gibbs
interfacial sólido-líquido en función de la del sólido y de la del líquido, englobados en
dos tendencias generales. De forma resumida, una de ellas supone la existencia de una
relación universal o ecuación de estado, entre la energía de Gibbs interfacial sólido-
líquido y las energías de Gibbs superficiales del sólido y del líquido. Expresando la
energía de Gibbs interfacial sólido-líquido según esta ecuación de estado en la ecuación
de Young (1.3), sería posible determinar la energía de Gibbs superficial del sólido con
sólo disponer de los datos experimentales del ángulo de contacto y la tensión superficial
de un líquido.
(
θ
γL
γSL γS
16
La segunda tendencia se basa en el análisis de las fuerzas que actúan en las interfases,
siendo el modelo o aproximación de Van Oss uno de los más utilizados, ya que predice
comportamientos experimentales con suficiente bondad [45]. Estas fuerzas incluyen a
las denominadas Lifshitz-van der Waals, que a su vez engloban a las fuerzas de Debye y
de Keeson, o interacciones dipolo-dipolo y dipolo-dipolo inducido, respectivamente, y
las dispersivas de largo alcance de London, o interacciones dipolo inducido-dipolo
inducido. También están presentes fuerzas más específicas y de corto alcance que tienen
en cuenta las contribuciones debidas a las interacciones ácido-base (según Lewis), que
incorporan las interacciones por puente de hidrógeno. La asunción que hacen los autores
que siguen esta segunda tendencia consiste en admitir que la energía de Gibbs
interfacial y superficial puede calcularse como la suma de componentes que reflejan una
contribución a esa energía proveniente de cada uno de los tipos de fuerzas que actúan en
la interfase. Esta hipótesis, proporciona predicciones correctas para muchos sistemas
además de información valiosa respecto de la extensión de las contribuciones al proceso
de cada grupo de fuerzas descritas anteriormente, que suelen concordar adecuadamente
con los resultados experimentales.
Según esto, Van Oss sugiere establecer una división de la energía de Gibbs interfacial
en dos componentes, una correspondiente a las interacciones debidas a las fuerzas
Lifshiftz-Van der Waals, γLW
, y otra a las ácido-base, γAB
, de modo que la energía de
Gibbs superficial total se puede expresar como:
γ = γLW
+ γAB
(1.4)
Además este modelo propone que la contribución ácido-base se pueda expresar como
una media geométrica de dos parámetros que cuantifican la tendencia a ceder electrones
(parámetro electrón-donador, γ-) y a aceptar electrones (parámetro electrón-aceptor, γ
+)
de modo que:
(1.5)
En base a ello, la expresión de γSL, viene dada por la siguiente relación:
(1.6)
17
Sustituyendo finalmente se obtiene que:
(1.7)
ecuación que permite obtener la componente y los parámetros
de la
energía de Gibbs superficial del sólido resolviendo el sistema de ecuaciones que se
obtiene a partir de las medidas de los ángulos de contacto sobre la superficie de al
menos tres líquidos de características conocidas, entre los que habitualmente se incluye
al agua, formamida y diiodometano, y a partir de ellos, la energía de Gibbs superficial
total del sólido γS ( Ec.(1.4)).
1.3.1.3. Potencial eléctrico de interacción. Potencial zeta
Otro factor importante que se debe considerar para poder caracterizar la interfase
microorganismo-biomaterial es la contribución de las fuerzas de tipo eléctrico que
aparecen entre ambos, microorganismos y superficie, cuando se encuentran en el medio
fisiológico. La superficie bacteriana puede describirse como un tapiz formado por
dominios, más o menos enriquecidos por unos tipos de moléculas u otros, que se
disocian cuando el microorganismo se encuentra en suspensión, de modo que cada uno
de estos dominios presenta una carga eléctrica superficial positiva o negativa. Esta
riqueza estructural permite a las bacterias acomodarse a situaciones diversas, pero para
abordar y predecir, al menos en primera aproximación, la afinidad entre bacteria y
sustrato, se suele considerar la superficie del microorganismo como un todo, con una
carga neta distribuida homogéneamente en su superficie. Del mismo modo, cuando el
biomaterial se pone en contacto con el fluido fisiológico, su superficie adquiere una
carga eléctrica neta debida, por ejemplo, a la ionización de las especies químicas
presentes en esa superficie, la liberación de iones del material o la adsorción específica
de iones presentes en la disolución. La propiedad física que mejor describe este tipo de
características eléctricas superficiales es el potencial zeta, o potencial eléctrico de
interacción, que predice el modelo de la doble capa électrica.
Como ya se ha indicado, cuando un material se sitúa en el seno de una disolución
adquiere una carga eléctrica superficial que suele ser negativa en medios fisiológicos.
Para compensar esta carga se produce una adsorción de iones positivos de la disolución
formándose, en primer lugar, una capa adsorbida en la que estos iones están unidos
18
irreversible y muy fuertemente a la superficie, de modo que, en el movimiento relativo
superficie-disolución, se desplazan solidaria y rígidamente con la superficie. Esta capa
adyacente y rígida es conocida como capa de Stern (también aproximada en muchos
casos a la capa interna de la doble capa eléctrica) donde los iones no pueden desorberse
ni desplazarse alrededor de la superficie (Fig. 1.5). Para completar la compensación
eléctrica de la superficie otros iones positivos adicionales, sensibles a la agitación
térmica, son atraídos más débilmente por la superficie negativa dando lugar a un
equilibrio dinámico que culmina con la formación de una segunda capa, también
conocida como capa difusa o capa externa de la doble capa eléctrica. La concentración
de iones en esta capa disminuye gradualmente con la distancia, hasta que se logra un
equilibrio con los contraiones en el seno de la disolución. Precisamente, la mayor o
menor extensión de esta doble capa depende de la concentración de iones, o fuerza
iónica del medio. Un esquema de la distribución iónica se representa en la Fig. 1.5.
Fig. 1.5. Esquema de la doble capa eléctrica: Capa interna y difusa. Plano de cizalladura
(localización aproximada) donde se define el potencial zeta.
La superficie negativa y su atmósfera cargada positivamente dan lugar a un potencial
eléctrico superficial relativo a la disolución. Este tiene un valor máximo en la superficie
y disminuye gradualmente con la distancia, anulándose fuera de la capa difusa. Desde el
punto de vista experimental, este potencial no es accesible en la superficie del sólido, ya
que los iones de la capa de Stern están unidos solidariamente a ella. Sin embargo,
mediante métodos electrocinéticos sí es posible determinar su valor en la superficie de
+-----------
+++++++
+
+
+
+
+
+
-- -
--
- -
-
-
-
-++
Sóli
do
Capa interna Capa difusa
Plano de cizalladura
19
separación entre las capas interna y difusa, lo que comúnmente se conoce como plano o
superficie de cizalladura. El potencial eléctrico en esa superficie es conocido como
potencial zeta (ζ) y es la propiedad física más accesible experimentalmente en base a la
cual se suelen caracterizar las interacciones eléctricas entre superficies.
1.3.1.4. Caracterización electroquímica de la superficie
Una parte del trabajo de esta tesis ha estado dedicado a conocer las consecuencias sobre
las propiedades superficiales de la aleación Ti6Al4V al ser irradiada con UV-C. El
modo en que afecta este tipo de tratamiento a la aleación está relacionado con el
carácter semiconductor del dióxido de titanio, principal componente de su capa pasiva,
cuya energía de activación está en esas longitudes de onda del UV-C. Una vez que el
proceso de irradiación finaliza, las recombinaciones electrón-hueco del semiconductor
originan la aparición de pequeñas corrientes superficiales que pueden tener un papel
relevante en las propiedades de esta aleación. Por ello se va a abordar su estudio
empleando métodos de caracterización electroquímica que son habituales en los
estudios de corrosión.
La conductividad eléctrica superficial es una propiedad del material que puede afectar a
su comportamiento interfacial y en particular a los microorganismos que puedan entrar
en contacto con el material. Diversos estudios [47, 48] han demostrado la existencia de
un efecto bactericida en las superficies sometidas a pequeñas corrientes eléctricas,
debido a una reducción de la función celular de la coenzima CoA, que conduce a la
inhibición de la respiración celular y a la muerte bacteriana.
Los biomateriales metálicos en contacto con una solución fisiológica, están compuestos
por electrodos en cortocircuito a través del cuerpo del propio metal. Las impurezas del
mismo, constituyen los electrodos que darán lugar al trasiego electrónico que provocan
las reacciones químicas anódicas y catódicas que generan un proceso electroquímico
corrosivo. El número de electrones que intervienen en dicho proceso, generará una
mayor o menor corriente de corrosión. La Fig. 1.6 representa a modo de ejemplo las
reacciones químicas de un proceso de corrosión de un biomaterial de titanio implantado
en condiciones fisiológicas.
20
Fig. 1.6. Reacciones durante la corrosión de un biomaterial metálico en condiciones
fisiológicas [49].
Una forma de evaluar la corriente de corrosión (Icorr) que circula por la película pasiva
generada en un biomaterial de aleación de titanio como el estudiado en este trabajo, es
mediante la determinación de curvas de polarización potenciodinámicas, que
básicamente consisten en el registro de la intensidad que circula a través del sistema
electroquímico de trabajo cuando se impone sobre él un barrido de potenciales, que
comienza en un potencial inferior al de corrosión. Es un ensayo destructivo que nos
permite sacar al sistema de su estado de equilibrio, polarizándolo y comparando las
diferentes respuestas en corriente conseguidas al aplicar potenciales iguales. También
resulta interesante la determinación de propiedades de la interfase película pasiva y
solución fisiológica, como la resistencia de transferencia de carga o resistencia de
polarización (RP) y la capacidad de la película pasiva (C) generada. Propiedades que nos
permiten evaluar características dieléctricas, espesores, defectos o picaduras, etc, de la
película pasiva formada. Para ello se emplea la técnica no destructiva de espectroscopia
de impedancia electroquímica, que básicamente consiste en la aplicación al sistema de
una onda sinusoidal de potencial (a distintas frecuencias) y del registro de la respuesta
de intensidad de dicho sistema, permitiendo obtener la impedancia del sistema a través
de su cociente.
Estas técnicas, aunque están enfocadas a estudios de fenómenos de corrosión, permiten
llevar a cabo un análisis en profundidad de las propiedades electroquímicas de la
película pasiva y semiconductora generada de forma espontánea en la superficie de la
Transporte
productos
Iones metálicos
Ti2+
Transporte
reactivos
Agente oxidante
O2 + H2O
BIO
MA
TE
RIA
L
TIT
AN
IO
Reacción
catódica
Reacción
anódica
Doble capa
Capa difusa Capa rígida
Transferencia
carga
Transferencia
carga
Oxidación
biomaterial
Reducción
biomaterial
e-
Transporte
productos
Reducción productos
OH-
21
aleación de titanio y constituyen además una poderosa herramienta para el estudio de
los procesos de transferencia de carga, habiendo sido aplicadas anteriormente con éxito
en el estudio de procesos de recombinación eléctrica en la interfase de la película de
óxido generada y el electrolito [50, 51].
El dispositivo experimental empleado para el estudio de los parámetros de corrosión
consiste en una celda electroquímica con configuración de tres electrodos (electrodo de
trabajo, la muestra objeto de estudio, un contraelectrodo de platino (Pt) y un electrodo
de referencia de Ag/AgCl saturado en una solución de Cloruro de Potasio (KCl)) y un
potenciostato equipado con módulo analizador de frecuencia.
La finalidad de esta tesis doctoral no es el estudio de las propiedades anticorrosivas de
la aleación Ti6Al4V, ya que existen multitud de trabajos anteriores que dan buena
cuenta de ello, sino el uso de técnicas electroquímicas propias del estudio de la
corrosión para la obtención de datos experimentales que nos permitan el entendimiento
de las modificaciones superficiales que están teniendo lugar en la superficie de la
aleación, después de ser expuesta a luz UV-C.
1.3.1.4.1. Polarización Lineal
La polarización lineal es una técnica experimental que se emplea para determinar
parámetros tan importantes de corrosión como las corrientes y velocidades de corrosión
que tienen lugar en un metal, lo que permitirá caracterizar de una manera general el
comportamiento electroquímico del material.
Tal y como hemos dicho anteriormente, las curvas de polarización consisten en el
registro de la intensidad que circula a través del sistema electroquímico de trabajo (entre
el electrodo de trabajo y un electrodo auxiliar) cuando se impone sobre él (entre el
electrodo de trabajo y un electrodo de referencia) un barrido de potenciales que empieza
en un potencial inferior al de corrosión y avanza en sentido anódico, aplicando un
"sobrepotencial" positivo para la reacción anódica y un "sobrepotencial" negativo para
la reacción catódica. La velocidad del barrido de potencial ha de ser lo suficientemente
lenta como para permitir el intercambio de cargas que se produce en la interfase del
biomaterial.
22
Al representar la corriente (I) frente al potencial (E) se obtiene el diagrama de la Fig.
1.7. La pendiente del tramo lineal en las zonas anódicas y catódicas es conocida como
pendiente de Tafel anódica, ba y catódica, bc, respectivamente.
Fig. 1.7. Curva de polarización para la estimación de parámetros de corrosión de un metal.
De acuerdo con la terminología expuesta en la norma ASTM G15 (Standard
Terminology to Corrosion and Corrosion Testing) [52], se indican las siguientes
definiciones de interés en relación a este apartado:
Potencial de Corrosión (Ecorr) y Corriente de Corrosión (Icorr): El potencial de
corrosión es el potencial de equilibrio de un metal o aleación en un electrolito
respecto de un electrodo de referencia. La corriente de corrosión es la corriente
que circula al potencial de corrosión, entre un metal o aleación y el electrodo
auxiliar.
Densidad de corriente de Corrosión (icorr): La densidad de corriente
(intensidad/superficie) que circula a través de una pila electroquímica al
potencial de corrosión es la densidad de corriente de corrosión.
Corriente de pasivación (ip): Valor de la intensidad de corriente que permanece
estable para un intervalo de potenciales (conocido como zona de pasivación), y
que se alcanza debido a la formación de una capa pasiva.
Los parámetros Ecorr e Icorr han sido obtenidos a partir de las curvas potenciodinámicas
aplicando el “Método de las Pendientes de Tafel” que equivale a interpolar la
(ba)
(bc)
Potencial (E)
Inte
nsi
dad
de
corr
ien
te (
I)
Ecorr
ip Icorr
23
intersección de las pendientes de tafel en el eje del potencial (E) e intesidad de corriente
(I).
Una vez obtenida Icorr, mediante la ecuación desarrollada por Stern y Geary [53] es
posible establecer una relación entre la densidad de corriente de corrosión (icorr) y la
resistencia a la polarización (Rp).
(1.8)
donde B se obtiene combinando las pendientes anódicas y catódicas de Tafel (ba, bc) a
través de la siguiente expresión:
(1.9)
1.3.1.4.2. Espectroscopía de Impedancia Electroquímica (EIS)
La EIS es una de las técnicas más recomendables para obtener información acerca de la
evolución del proceso de corrosión en la interfase del material y el medio acuoso. Para
ello, con la ayuda de un potenciostato, se aplica al sistema una perturbación sinusoidal a
diferentes frecuencias ( y se obtiene la respuesta en corriente (
θ , permitiendo obtener la impedancia (Z) del sistema a través de su
cociente. Donde E0 e I0 son, respectivamente, el potencial y la corriente; es la
frecuencia angular de la señal ( =2πf, siendo f la frecuencia) y θ es el ángulo de desfase
entre el potencial de perturbación y la respuesta en corriente. [54].
En muchos materiales y sistemas electroquímicos la impedancia varía con la frecuencia
del potencial aplicado debido a sus propiedades intrínsecas (su estructura física), a los
procesos electroquímicos que tengan lugar, o a una combinación de ambos. Por
consiguiente, si se hace una medida de impedancias en un rango de frecuencias
adecuado y se representan los datos obtenidos, es posible relacionar los resultados
obtenidos con las propiedades físicas y químicas de dichos materiales y sistemas.
24
Fig. 1.8. Representación de las señales de perturbación (E) y respuesta (I) en función del
tiempo, en un estudio de impedancia. Las medidas del desfase y de la amplitud de la respuesta,
proporcionan la impedancia asociada a la transferencia electroquímica del material estudiado.
Como número complejo, la impedancia (Z = Zreal + Zimg) se puede representar tanto en
coordenadas cartesianas como polares (Fig. 1.9).
Fig. 1.9. Representación de la impedancia como un número complejo
Donde,
(1.10)
(1.11)
De la parte real Zreal se puede calcular la conductancia G y de la parte imaginaria Zimg la
capacitancia C en base a la siguiente expresión,
(1.12)
Para obtener una información más precisa de la evolución electroquímica de la interfase
metal/líquido, se utilizan diferentes representaciones gráficas del espectro de
impedancia. En el análisis realizado en este trabajo se han utilizado los diagramas de
Zreal
Zimg
|Z|
θ
25
Nyquist y de Bode. Dichas representaciones permiten a su vez interpretar el sistema
objeto de estudio a través de un circuito eléctrico equivalente como veremos a
continuación.
Diagrama de Nyquist
En un diagrama de Nyquist se representan los valores de las partes real e imaginaria de
la impedancia para cada valor de frecuencia, un ejemplo típico se muestra en la Fig.
1.10.
Fig. 1.10. Diagrama de Nyquist, gráfica de las magnitudes de impedancia imaginaria (Zimg) e
impedancia real (Zreal), a diferentes frecuencias.
El valor negativo de la parte imaginaria de la impedancia se asocia a la presencia de
procesos capacitivos que ocurren en la interfase metal/líquido, mientras que la parte real
está relacionada con fenómenos resistivos en dicha interfase.
Diagrama de Bode
Existen dos tipos de diagramas de Bode, el primero relaciona el valor del módulo |Z|
frente a la frecuencia (f); mientras que el segundo relaciona el valor del ángulo de fase
(θ) frente a la frecuencia. Ambos valores, |Z| y θ, se obtienen a partir de la información
del diagrama de Nyquist.
Ejemplos típicos de ambos diagramas se encuentran recogidos en la Fig. 1.11.
1000 2000 3000 4000 5000 6000 7000
-6000
-5000
-4000
-3000
-2000
-1000
0
Z'
Z''
par_rc.zFitResult
1·103 2·10
3 3·10
3 4·10
3 5·10
3 6·10
3
7·103
-6·103
-5·103
-4·103
-3·103
-2·103
-1·103
0
Zreal (Ω)
Zim
g (
Ω)
26
Fig. 1.11. Diagrama de Bode para el módulo de la impedancia, |Z|, frente a la frecuencia, f y
ángulo de desfase, θ, frente a la frecuencia, f.
Dependiendo del comportamiento del ángulo de fase y del módulo de la impedancia, se
tendrá un comportamiento resistivo o capacitivo. En el proceso resistivo, la señal del
potencial (E) y la respuesta de corriente (I) están en fase, es decir el ángulo de desfase
es igual a cero y el valor de Zreal es igual a la magnitud del resistor; en el proceso
capacitivo puro, se tiene un ángulo de desfase de 90° a diferentes valores de frecuencia.
Los procesos resistivos, están asociados a procesos de conducción y ejemplo de ello es
la conductividad en el electrolito y/o a una transferencia de electrones a través de la
interfase metal-electrolito. En el proceso capacitivo, el desfase que se produce entre el
potencial aplicado y la corriente medida está relacionado con fenómenos de generación
de campos eléctricos en el sistema, asociados a la presencia de procesos electroquímicos
que conllevan polarización o separación de carga.
1.3.1.4.2.1. Interpretación del sistema en base a un circuito eléctrico equivalente
Los procesos capacitivos y/o resistivos que ocurren en el sistema electroquímico
comúnmente se representan mediante un circuito eléctrico equivalente que permite
modelar los fenómenos que tienen lugar en la interfase metal-electrolito a través de la
10-2 10-1 100 101 102 103 104 105103
104
Frequency (Hz)
|Z|
par_rc.zFitResult
10-2 10-1 100 101 102 103 104 105
-40
-30
-20
-10
0
Frequency (Hz)
the
ta10-2 10-1 100 101 102 103 104 105
103
104
Frequency (Hz)|Z
|
par_rc.zFitResult
10-2 10-1 100 101 102 103 104 105
-40
-30
-20
-10
0
Frequency (Hz)
the
ta
(º)
f (Hz)
|Z| (Ω
)
10-2
10-1
100 10
1 10
2 10
3 10
4 10
5
f (Hz)
103
104
-40
-30
-20
-10
0 10
-2 10
-1 10
0 10
1 10
2 10
3 10
4 10
5
27
respuesta en impedancia observada y expresada a través de los diagramas de Nyquist y
Bode.
Puede haber varios circuitos que representen el mismo espectro de impedancia, por lo
que no basta únicamente con que el circuito equivalente se ajuste a la curva, sino que,
además, debe cumplir con la característica fundamental de tener un significado físico-
químico aceptable [55,56].
El circuito equivalente representado en la Fig. 1.12 es uno de los más sencillos al que es
posible ajustar los datos experimentales. En este caso la impedancia se representa
mediante una combinación en paralelo de una resistencia Rp y una capacitancia Cp,
ambas en serie con otra resistencia Rs.
(1.13)
RS representa la resistencia del electrolito, cuyo valor se puede calcular realizando un
barrido a altas frecuencias, RP es el término de la resistencia de polarización o
resistencia de transferencia de carga y CP representa la capacidad de la doble capa
electroquímica o capa de Helmholtz, relacionada con las interacciones que tienen lugar
en la interfase electrodo/electrolito.
Fig. 1.12. Circuito equivalente eléctrico sencillo.
Partiendo de una representación de Nyquist en la que se muestra el valor absoluto de la
parte imaginaria de la impedancia frente a la real, se pueden obtener los parámetros del
circuito equivalente (Fig. 1.13).
RS
RP
CP
28
Fig. 1.13. Diagrama de Nyquist de un circuito eléctrico sencillo.
A partir de los cortes con el eje real se puede obtener el valor de la resistencia de
transferencia de carga o resistencia de polarización (Rp) y la resistencia eléctrica del
electrolito de trabajo (Rs). Asimismo, partiendo del valor de la frecuencia en el punto
máximo se puede calcular el valor de la capacitancia de la doble capa electroquímica
(Cp).
De forma similar, el diagrama de Bode puede ser utilizado para obtener los mismos
parámetros, como se observa en la Fig. 1.14.
Fig. 1.14. Diagrama de Bode de un circuito eléctrico sencillo.
1000 2000 3000 4000 5000 6000 7000
-6000
-5000
-4000
-3000
-2000
-1000
0
Z'
Z''
par_rc.zFitResult
10-2 10-1 100 101 102 103 104 105103
104
Frequency (Hz)
|Z|
par_rc.zFitResult
10-2 10-1 100 101 102 103 104 105
-40
-30
-20
-10
0
Frequency (Hz)
the
ta
10-2 10-1 100 101 102 103 104 105103
104
Frequency (Hz)
|Z|
par_rc.zFitResult
10-2 10-1 100 101 102 103 104 105
-40
-30
-20
-10
0
Frequency (Hz)
the
ta
RS + RP RS
CP = (ω RP)-1
(RS + RP)/2
f
Zimg(max)
Zimg(max)
1·103 2·10
3 3·10
3 4·10
3 5·10
3 6·10
3
7·103
-6·103
-5·103
-4·103
-3·103
-2·103
-1·103
0
Zreal (Ω)
Zim
g (
Ω)
CP=(2πf1 (RS+RP))-1
f2 f1
RS + RP
RS
CP=(2πf2RS)-1
Pendiente -1
|Z| (Ω
)
10-2
10-1
100 10
1 10
2 10
3 10
4 10
5
f (Hz)
103
104
(
º)
f (Hz)
-40
-30
-20
-10
0 10
-2 10
-1 10
0 10
1 10
2 10
3 10
4 10
5
fθmax
29
En la curva del módulo de la impedancia frente a la frecuencia es posible detectar dos
regiones, una dominada por elementos resistivos tales como Rs (resistencia de la
disolución) y Rp (resistencia de polarización), con una pendiente cero, y otra dominada
por los elementos capacitivos, caracterizada por una pendiente de valor -1.
1.3.1.4.3. Propiedades electrónicas de las vacantes anódicas y catódicas. Ensayos
Mott-Schottky
Desde el punto de vista del intercambio de electrones, las vacantes catódicas pueden ser
consideradas como aceptores de electrones y las vacantes anódicas como donadores
[57], de forma análoga a la interpretación del dopaje en los semiconductores. Para
explicar esta relación, se considera que la ausencia de un átomo de metal, en la red del
óxido, provoca una vacante catódica en la región de la imperfección. Los oxígenos
(muy electronegativos) alrededor de la vacante metálica, pueden servir de trampas de
electrones (deslocalizados) de la red del óxido; de esta forma una vacante catódica actúa
como un sitio aceptor. Por otro lado, una vacante de oxígeno, tiene a su alrededor,
cationes metálicos; sin embargo, estos cationes (poco electronegativos), no conservan
los electrones y pueden perderlos más fácilmente, por lo que las vacantes de oxígeno
actúan como donadores [58]. Con estas consideraciones sobre las vacantes metálicas y
de oxígeno, las películas de TiO2 originadas en la superficie de la aleación Ti6Al4V,
pueden ser caracterizadas mediante técnicas convencionales utilizadas en los
semiconductores, tales como las curvas Mott-Schottky, con el fin de determinar el tipo
de comportamiento semiconductor de la película pasiva, ya sea tipo n ó tipo p y las
características de sus portadores de carga [59].
Los ensayos Mott-Schottky se llevan a cabo para evaluar el comportamiento de la
capacidad interfacial de película semiconductoras (TiO2) en función del potencial en
corriente continua (potencial DC de polarización) [58].
El modelo Mott-Schottky, predice que si la película de óxido que se forma en la
superficie de un metal se comporta como un semiconductor, existirá una relación entre
la capacitancia de la región de carga espacial del film y el potencial aplicado, de
acuerdo con las ecuaciones (1.14) y (1.15) establecidas para semiconductores tipo n y
tipo p, respectivamente:
30
(1.14)
(1.15)
donde CSC es la capacitancia de la región de carga espacial, q la carga eléctrica de los
portadores, ND y NA el número de portadores de carga donadores y aceptores
respectivamente, permitividad relativa del film, 0 permitividad del vacío, V el
potencial sobreimpuesto, VFB el potencial de banda plana del semiconductor, k la
constante de Boltzman y T la temperatura [60].
De acuerdo con la aproximación Mott- Schottky, cuando se representa 1/C2
SC frente a
V, la pendiente del tramo lineal de la gráfica será 2/ 0qND. Si el signo de la pendiente
de la recta es positiva, indica el decrecimiento de la capacidad cuando se incrementa el
potencial anódico por encima del potencial de banda plana, informando de que el film
generado en la superficie del semiconductor es de tipo n, en caso contrario será tipo p.
Además, cuando kT/q es insignificante, el potencial de banda plana puede ser obtenido
por extrapolación de la parte lineal del gráfico hasta 1/C2
SC = 0. Así pues, calcular con
precisión el valor ND dependerá en gran medida de la determinación de la correcta
relación entre CSC y V [60].
1.4. MÉTODOS DE EVALUACIÓN DE LA ADHESIÓN MICROBIANA
1.4.1. Modelos teóricos de adhesión microbiana
Como ya se ha indicado con anterioridad, el proceso infeccioso que puede tener lugar en
la superficie de un biomaterial comienza con la adhesión de microorganismos a la
superficie de éste. Además, está ampliamente reconocido que este proceso adhesivo se
inicia con interacciones de tipo no-específico entre las superficies del biomaterial y del
microorganismo donde las propiedades físico-químicas de ambos son decisivas para la
aproximación y posterior anclaje.
Existen tres teorías, que se basan en los cambios de la energía de Gibbs interfacial (ΔG),
para describir las interacciones iniciales microorganismo-substrato [61]. La primera de
ellas, en orden cronológico, es la aproximación termodinámica [62,63]. En ella se
considera que si dos superficies se ponen en contacto físico en condiciones de
31
equilibrio, la intensidad de su interacción depende de los valores de la energía
superficial de Gibbs de las fases interactuantes.
La segunda de ellas se conoce como la teoría clásica de la estabilidad coloidal DLVO
(Derjaguin, Landau, Verwey y Overbeek) [64-67]. Esta teoría considera que la adhesión
de partículas cargadas a una superficie es el efecto de la unión de las fuerzas de largo
alcance de tipo Lifshitz-Van der Waals (LW) y las electrostáticas (EL) las cuales varían
en función de la distancia de separación partícula-superficie. Las fuerzas LW son
generalmente atractivas y tienen su origen en las interacciones polares entre las
moléculas que componen la superficie de las partículas coloidales y del substrato en el
medio de suspensión. Las fuerzas EL son el resultado del solapamiento entre las dobles
capas eléctricas que se forman en torno a las células y al substrato, como consecuencia
de la distribución, alrededor de su superficie, de los iones presentes en la disolución en
la que se encuentran inmersas. Según esta interpretación, la energía de interacción de
Gibbs (ΔGTOTAL
) entre dos superficies es la suma de la energía de interacción tipo
Lifshitz-Van der Waals (ΔGLW
) y la energía de interacción eléctrica (ΔGEL
).
(1.16)
Ambas aproximaciones han demostrado que son válidas para describir la adhesión
microbiana en un cierto número de casos, sin embargo, no han conseguido una
descripción generalizada válida para todos y cada uno de los sistemas microbianos [69].
El tercer modelo considerado fue propuesto por Van Oss y cols. [68,69] y se denomina
teoría DLVO extendida (XDLVO). Este modelo considera que la energía de interacción
de Gibbs (ΔGTOTAL
) es la suma de cuatro términos independientes: uno debido a
interacciones de tipo Lifshitz-Van der Waals (ΔGLW
), otro debido a fuerzas de tipo
eléctrico (ΔGEL
), otro debido al movimiento de agitación térmica de las partículas o
superficies interactuantes (movimientos browniano) (ΔGBR
) y otro debido a fuerzas de
corto alcance de tipo ácido-base (ΔGAB
).
(1.17)
Las fuerzas de tipo ácido-base tienen su origen en las interacciones entre las zonas de
carácter electrón-aceptor y electrón-donador de las moléculas polares que forman parte
de las superficies [70,71].
32
A pesar de que las tres teorías son consecutivas en el tiempo, ninguna ha sustituido, en
el estricto sentido de la palabra, a las anteriores. Existen estudios en los que se
comprueba cómo cada una de ellas es capaz de describir casos concretos de bioadhesión
[72-75].
También hay que señalar que en otras ocasiones no se encuentra una relación directa
entre el proceso de adhesión in vitro y las predicciones teóricas de cualquiera de estas
tres aproximaciones de origen físico-químico. Dichas limitaciones se basan en la
diversidad de estructuras macromoleculares heterogéneas existentes en la superficie de
las células, las cuales hacen que una teoría, desarrollada para partículas coloidales,
supuestas esféricas y con distribución homogénea de carga, no refleje ciertos
mecanismos de adhesión más complejos que pueden surgir entre la superficie de un
microorganismo y la de un determinado substrato. Algunos de estos aspectos
específicos de la adhesión microbiana son:
El concepto de distancia de separación requiere una distinción exacta entre la
superficie de la partícula y el medio; sin embargo, en el caso de los
microorganismos elegir un punto de referencia para calcular la distancia de
separación, no resulta tan fácil. En algunas ocasiones los polímeros superficiales
suponen una dificultad para llegar al mínimo de energía primario ya que la
aproximación de las células a la superficie requiere su compresión [76].
Las expresiones que se utilizan para calcular las interacciones de tipo físico-
químico en función de la distancia de separación asumen que para toda distancia
de separación las interacciones células-substrato se llevan a cabo en equilibrio
termodinámico. Sin embargo, a medida que el microorganismo se aproxima a la
superficie, se requiere un cierto tiempo para que las macromoléculas de la
interfase se reorganicen y acondicionen a las configuraciones de menor energía
[77].
Las contribuciones por factores estéricos de las macromoléculas en la interfase
que pueden llegar a ser más decisivas que las fuerzas de origen físico-químico
[78].
33
Los parámetros requeridos para la determinación de las fuerzas LW, AB y EL
pueden ser difíciles de estimar y en todo caso reflejan una propiedad promedio
de la superficie celular, mientras que las macromoléculas involucradas en el
contacto inicial pueden tener propiedades o fuerzas de interacción diferentes a
ese promedio [79].
Por estas razones no es sorprendente que el estudio físico-químico de la adhesión
proporcione resultados variados. En ningún caso se puede hablar de predicciones
cuantitativas de la adhesión microbiana, sino que nos debemos referir exclusivamente a
predicciones cualitativas de la misma [80,81].
1.4.1.1. Determinación de la energía de Gibbs de interacción.
A partir de los valores de la energía de Gibbs superficial de los microorganismos y
substratos, la determinación de las energías de interacción Lifshitz-Van der Waals,
ácido-base y eléctrica se basa en la aplicación de las ecuaciones descritas en la física de
coloides y que forman parte de la teoría de la estabilidad DLVO extendida. Por
consiguiente, los valores de ΔGLW, ΔG
AB y ΔG
EL se calculan a partir de las siguientes
expresiones:
(1.18)
Siendo A la constante de Hamaker,
a el radio del microorganismo,
d la distancia de separación microorganismo-sustrato y la energía de interacción a
la mímina distancia de separación, d0, calculada a través de las energías de Gibbs
superficiales,
(1.19)
Indicando los subíndices B, S, L, bacteria, sustrato y líquido de suspensión,
respectivamente.
Para la componente ácido-base:
(1.20)
34
Siendo la energía de interacción AB a la distancia mínima de separación,
calculada a través de las energías de Gibbs superficiales,
2L B S L L B S L
AB
do
B B B B
G
(1.21)
Y para la componente eléctrica,
2 2
0 2 2
2 1 exp( )( ) ( ) ln ln(1 exp( 2 ))
1 exp( )
EL B Sr B S
B S
kdG d a kd
kd
(1.22)
siendo 0 y r las constantes dieléctricas del vacío y la relativa del medio, ζ el potential
zeta y k la constante de Boltzman.
1.4.2. Metodología empírica: Adhesión estática y dinámica
El estudio de la adhesión de microorganismos a diferentes substratos se realiza a través
de varias técnicas experimentales. La elección se basa en las ventajas que pueda aportar
una frente a otra cuando se trata de estudiar un aspecto concreto del proceso de
adhesión. Atendiendo a las características que presente su metodología, se pueden hacer
dos grandes grupos, los sistemas estáticos de adhesión y los de flujo o dinámicos. Los
primeros generalmente son sistemas cerrados en los cuales la suspensión que está en
contacto con el substrato permanece estacionaria o en agitación suave, permitiendo la
adhesión y, en ocasiones, el crecimiento de los microorganismos con pocas
fluctuaciones mecánicas y térmicas [82,83]. Los segundos [84,85] ofrecen la posibilidad
de controlar las condiciones hidrodinámicas en función de parámetros que determinan
los mecanismos de transporte de masa. En este segundo grupo, se contempla la
monitorización del proceso de adhesión además de la posibilidad de estudiar el
crecimiento de biocapas usando un flujo rico en nutrientes [86]. Esta técnica, también
ofrece la posibilidad de poder evaluar la fuerza de adhesión bacteriana al sustrato en el
que se adhiere, relacionándolo con el paso de interfases líquido-aire, una vez finalizado
el experimento de adhesión [87]. Son varios los sistemas de flujo que se emplean para el
estudio de la adhesión [88-90], desde el uso de cámaras de flujo (Fig 1.15) que albergan
35
Fig 1.15. Cámara de flujo laminar con posibilidad de un único sustrato.
un único sustrato con el fin de poder visualizar mediante microscopía la cinética de
adhesión de las bacterias, hasta el uso de dispositivos de Robbins (Fig 1.16), capaces de
estudiar simultáneamente la afinidad bacteriana a distintos biomateriales que hayan sido
sometidos a diferentes tratamientos superficiales.
Fig 1.16. Dispositivo de Robbin con posibilidad de hasta 9 sustratos.
En los sustratos que presentan unas propiedades adecuadas para poder observar con
microscopia óptica las bacterias adheridas, la cuantificación de la adhesión bacteriana se
realiza por conteo sobre las imágenes obtenidas. Sin embargo este método está limitado
por las características del material. En aquellos casos en que no es posible el uso de esta
técnica directa, se emplean métodos bioquímicos o de recuento en placa de cultivo
bacteriano que tienen el inconveniente de que precisan una manipulación importante
36
sobre la muestra, provocando que se infraevalúe la adhesión debido a que en el proceso
las bacterias más débilmente adheridas se despeguen del sustrato.
Los métodos de tinción o colorimétricos más estándares incluyen la tinción de Gram,
empleando cristal violeta o safranina si la cepa es Gram positiva o Gram negativa,
respectivamente. Este método no es aplicable si la adhesión está producida por cepas
productoras de biocapa, dado que el colorante no es capaz de penetrar en el interior de
la biocapa quedando sin teñir las bacterias embebidas. En estos casos se emplean
métodos de bioluminiscencia, que permite estimar el número de bacterias presentes en
función del ATP producido. Las bacterias que producen CO2 como producto de su
metabolismo pueden ser cuantificadas por radiometría, que es una técnica relativamente
rápida.
Entre los métodos de tinción, es interesante destacar aquellos que además de informar
sobre el número de bacterias adheridas, permiten conocer la viabilidad de las células
tras el experimento. Estos incluyen el uso de dos moléculas con fluoróforos que tienen
diferente respuesta frente a las bacterias, por ejemplo, una de las moléculas penetra las
células independientemente de cómo se encuentra su pared, marcándolas con una
tonalidad verdosa, sin embargo el otro es capaz de penetrar solo en células cuya pared
esté suficientemente dañada marcándolas con una tonalidad anaranjada-rojiza,
reduciendo el otro fluoróforo que previamente ha sido penetrado.
También es posible cuantificar las bacterias adheridas a un sustrato extrayéndolas de la
superficie sonicando la muestra adecuadamente. Para realizar el recuento de las
bacterias viables extraídas, se utiliza el método de recuento en placas de cultivo por
diluciones seriadas, que consiste en transferir a un tubo de ensayo, al que previamente
se ha añadido un volumen concreto de solución fisiológica, un cierto volumen de la
suspensión sonicada, consiguiendo la primera dilución 10-1
, a continuación se repite el
proceso extrayendo de este tubo de ensayo un cierto volumen a otro tubo de ensayo al
que previamente se ha añadido un volumen concreto de solución fisiológica,
consiguiendo así la segunda dilución 10-2
, repitiendo el proceso tantas veces hasta
conseguir el orden de dilución deseado. A continuación, se transfiere de cada uno de los
tubos de ensayo una pequeña cantidad a placas de agar y se esparce con una espátula de
Driglasky estéril. Finalmente, después del periodo de incubación es posible observar las
37
colonias bacterianas sobre la superficie del agar en forma de unidades formadoras de
colonias (UFC). Usualmente una UFC se corresponde con una célula viable [91].
Hay que destacar, que aunque el investigador opte por una técnica u otra en base a las
características de su experimento, en algunas ocasiones una combinación de
metodologías permite entender mejor el sistema de estudio.
1.5. OBJETIVO
Esta tesis se presenta con dos objetivos fundamentales. El primero de ellos es la
caracterización de la superficie del biomaterial Ti6Al4V a través de su hidrofobicidad,
energía libre de Gibbs, potential zeta y propiedades electroquímicas tanto en su estado
original al recibirse del suministrador, como tras ser modificada mediante dos técnicas,
implantación iónica e irradiación UV, evaluándose los cambios que aportan tales
alteraciones.
El segundo objetivo analizará los beneficios que dichas modificaciones introducen en
diferentes procesos de adhesión bacteriana, con cepas de las familias Staphylococcus
epidermidis y Staphylococcus aureus, responsables de un alto porcentaje de infecciones
nosocomiales en implantes metálicos. Para ello se emplearán distintos tests de adhesión,
tanto estáticos como dinámicos, y se realizará un enfoque que englobe la interacción
bacteria-sustrato y la capacidad bactericida de las superficies modificadas.
1.6. BIBLIOGRAFÍA
[1] Costerton JW, Stoodley P, Shirtliff ME, Pasmore M, Cook G, in Ratner BD,
Hoffman AS, Schoen FJ, Lemons JE (Eds.), Biomaterials Science, Elsevier Academic
Press, San Diego, USA, 2004.
[2] Williams, DF, Definitions in Biomaterials. Proceedings of a Consensus Conference
of the European Society for Biomaterials, Chester, England, March 3–5, Vol. 4,
Elsevier, New York,1986.
[3] Vila J, Soriano A, Mensa J. Bases moleculares de la adherencia microbiana sobre los
materiales protésicos. Papel de las biocapas en las infecciones asociadas a los materiales
protésicos. Enfer. Infecc. Microbiol. Clin. 2008;26(1):48-55.
38
[4] Savage DC, Fletcher M, Bacterial adhesion. Mechanisms and physiological
significance, Plenum Press, New York, NY, USA, 1985.
[5] Nikolaev YA, Plakunov VK. Biofilm—“City of Microbes” or an Analogue of
Multicellular Organisms? Microbiology 2007;76:125–138
[6] Watnick P, Kolter R. Biofilm, city of microbes. J. Bacteriol. 2000;182:2675–2679
[7] Parsek MR, Fuqua C. Biofilms 2003: emerging themes and challenges in studies of
surface-associated microbial life. J. Bacteriol. 2004;186:4427-4440.
[8] Lasa I, Del Pozo JL, Penadés JR, Leiva J. Bacterial biofilms and Infection. An. Sist.
Sanit. Navar. 2005;28:163-175
[9] Jiang X, Pace JL, Microbial biofilms, in Pace JL, Rupp ME, Finch RG (Eds.),
Biofilms, Infection and Antimicrobial Therapy, Taylor and Francis, Boca Raton FL,
USA, 2006.
[10] Gristina AG, Naylor PT, Webb LX. Molecular mechanisms in musculoskeletal
sepsis: the race for the surface. Instr. Course Lect. 1990;39:471-482.
[11] Kasemo B, Gold J. Implant surfaces and interface processes. Adv. Dent. Res.
1999;13:8-20.
[12] Kasemo B, Lausmaa J. Biomaterial and implant surfaces: A surface science
approach. Int. J. Oral Maxillofac. Implants 1988;3:247-259.
[13] Rodil SE. Modificación superficial de biomateriales metálicos. Revista
Latinoamericana de metalurgia y materiales 2009;29(2):67-83
[14] Leonard EF, Vroman LJ. Is the Vroman effect of importance in the interaction of
blood with artificial materials? Biomater. Sci. Polym. Ed. 1991;3(1): 95-107.
[15] Van Loosdrecht MC, Lyklema J, Norde W, Zehnder AJ. Influence of interfaces on
microbial activity. Microbiol. Mol. Biol. R. 1990;54(1):75- 87.
[16] Arciola CR, Bustanji Y, Conti M, Campoccia D, Baldassarri L, Samorì B,
Montanaro L. Staphylococcus epidermidis-fibronectin binding and its inhibition by
heparin. Biomaterials 2003;24:3013-3019.
39
[17] Kanzaki H, Arata J. Role of fibronectin in the adherence of Staphylococcus aureus
to dermal tissues. J. Dermatol. Sci. 1992;4:87-94.
[18] Monroe D, Looking for Chinks in the Armor of Bacterial Biofilms. PLoS Biol,
2007:5,307-311.
[19] Miller MB, Bassler BL. Quorum sensing in bacteria. Annu. Rev. Microbiol.
2001;55:165-199.
[20] Voung C, Gerke C, Somerville GA, Fischer ER, Otto M. Quorum-sensing control
of biofilm factors in Staphylococcus epidermidis. J. Infect. Dis. 2003;188(5):706-718.
[21] Black J, Hastings G, Handbook of Biomaterial properties, in Black J, Hastings G
(Eds.), Titanium and titanium alloys, Chapman & Hall, London, UK, 1998, p. 179-200.
[22] Oldani CR, Domínguez AA. Simulación del comportamiento mecánico de un
implante de cadera. XV Congreso argentino de Bioingeniería, Universidad Nacional de
Córdoba, Argentina, 2005.
[23] Welsh G, Boyer R, Collins EW, Material properties Handbook: Titanium alloys,
ASM Ohio, USA, 1994.
[24] Rossetti F, Can atomically flat titanium oxide surfaces be prepared by the template
stripping method? [PhD Thesis], Laboratory of Surface Science and Technology, ETH
Zürich, Switzerland, 2001.
[25] Kasemo B, Gold J. Implant surfaces and interface processes. Adv. Dent. Res.
1999;13:8-20.
[26] Jäger M, Zilkens C, Zanger K, Krauspe R. Significance of nano- and
microtopography for cell-surface interactions in orthopaedic implants. J. Biomed.
Biotechnol. 2007;2007:69036-69055.
[27] Andersson AS, Backhed F, von Euler A, Richter-Dahlfors A, Sutherland D,
Kasemo B. Nanoscale features influence epithelial cell morphology and cytokine
production. Biomaterials 2003;24:3427-3436.
40
[28] Bigerelle M, Anselme K, Noel B, Ruderman I, Hardouin P, Iost A. Improvement
in the morphology of Ti-based surfaces: a new process to increase in vitro human
osteoblast response. Biomaterials 2002;23:1563-1577.
[29] Zhao G, Raines AL, Wieland M, Schwartz Z, Boyan BD. Requirement for Both
Micron and Submicron Scale Structure for Synergistic Responses of Osteoblasts to
Substrate Surface Energy and Topography. Biomaterials 2007;28:2821-2829.
[30] Lim JY, Dreiss AD, Zhou Z, Hansen JC, Siedlecki CA, Hengstebeck RW, Cheng J,
Winograd N, Donahue HJ. The regulation of integrin-mediated osteoblast focal
adhesion and focal adhesion kinase expression by nanoscale topography. Biomaterials
2007;28:1787-1797.
[31] Katsikogianni M, Missirlis YF, Katsikogianni M, Missirlis YF. Concise review of
mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating
bacteria-material interactions. European Cells and Materials 2004;8:37-57.
[32] Terada A, Yuasa A, Tsuneda S, Hirata A, Katakai A, Tamada M. Elucidation of
dominant effect on initial bacterial adhesion onto polymer surfaces prepared by
radiation-induced graft polymerization. Colloids Surf.B: Biointerfaces 2005;43:99–107.
[33] Lao LL, Venkatraman SS. Adjustable paclitaxel release kinetics and its efficacy to
inhibit smooth muscle cells proliferation. J. Control. Release 2008;130:9-14.
[34] Kathuria YP. Some aspects of drug eluting stents. Int. J. Cardiol. 2007;119:380-
383.
[35] Wykrzykowska JJ, Onuma Y, Serruys PW. Advances in stent drug delivery: the
future is in bioabsorbable stents. Expert. Opin. Drug Deliv. 2009;6:113-126.
[36] Diaz C, Lutz J, Mandl S, Garcia JA, Martinez R, Rodriguez RJ. Rodríguez,
Improved tribocorrosion of biomedical alloys by Ion implantation techniques. Nucl.
Instrum. Meth. B 2009;267:1630-1633.
[37] Variola F, Vetrone F, Richert L, Jedrzejowski P, Yi JH, Zalzal S, Clair S,
Sarkissian A, Perepichka DF, Wuest JD, Rosei F, Nanci A. Improving biocompatibility
of implantable metals by nanoscale modification of surfaces: an overview of strategies,
fabrication methods, and challenges. Small 2009;5:996-1006.
41
[38] Engel E, Michiardi A, Navarro M, Lacroix D, Planell JA. Nanotechnology in
regenerative medicine: The material side. Trends Biotechnol. 2008;26:39-47.
[39] Denkbas EB, Vaseashta A. Nanotechnology in Medicine and Health Sciences.
Nano 2008;3:263-269.
[40] Israelachvili J, Intermolecular and surface forces, Academic Press Limited,
London, UK, 1992.
[41] Van Oss CJ, Gillman CF, Newmann AW. The hydrophobic effect: essentially a
Van der Waals interaction. Colloid. Polym. Sci. 1980;258:424-427.
[42] Duncan-Hewitt WC, Nature of the hydrophobic effect, in Doyle RJ, Rosenberg M
(Eds.), Microbial cell surface hydrophobicity. ASM Publications, Washington DC,
USA, 1990, p 39-73.
[43] Ducker WA, Xu Z, Israelachvili JN. Measurements of hydrophobic and DLVO
forces in bubble-surface interactions in aqueous solutions. Langmuir 1994;10: 3279-
3289.
[44] Wood J, Sharma R. How long is the long-range hydrophobic attraction? Langmuir
1994;11: 4797-3289.
[45] Van Oss CJ, Interfacial forces in aqueous media, Marcel Dekker, New York, NY,
USA, 1994.
[46] Donoso MG. Energía libre superficial de biomateriales metálicos: Diferentes
aproximaciones a partir de la goniometría de ángulo de contacto [Master Degree].
Departamento de Física. Universidad de Extremadura, Badajoz, Spain, 2004.
[47] Wai-Kin Liu. Mechanisms of the bactericidal activity of low amperaje electric
current (DC). Journal of Antimicrobial Chemotherapy 1997;39:687-695.
[48] Liu, W-K. The effects of electric current on bacteria colonising intravenous
catheters. Journal of Infection 1993;27:261–269.
[49] Landolt, D. Corrosion and surface chemistry of metals, EPFL Press, Lausanne,
Switzerland, 2007.
42
[50] Pedraza Avella JA, Acevedo Peña P, Pedraza Rosas JE. Photocatalytic oxidation of
cyanide on TiO2: An electrochemical approach. Catal. Today 2008;133-135:611-618;
[51] Leng WH, Zhang Z, Zhang JQ, Cao CN. Investigation of the kinetics of a TiO2
photoelectrocatalytic reaction involving charge transfer and recombination through
surface states by electrochemical impedance spectroscopy. J Phys. Chem. B
2005;109:15008-15023.
[52] ASTM G-15, Standard Terminology Relating to Corrosion and Corrosion Testing,
2004.
[53] Stern M, Geary AL. Electrochemical Polarization: I. A theoretical analysis of the
shape of polarization curves. J. Electrochem. Soc. 1957;104(1):56-53.
[54] Macdonald JR, Johnson WB, Fundamentals of impedance spectroscopy.
Barsoukov E, Macdonald JR (Eds.), Impedance Spectroscopy. Theory, experiment and
applications (2nd
Edition), John Wiley & Sons Inc., New Jersey, USA, 2005, pp. 1-26
[55] Boukamp BA. A package for impedance/admittance analysis. Solid State Ionics
1986;18:136-140.
[56] Macdonald JR, Potter LD. A flexible procedure for analyzing impedance
spectroscopy results: Description and illustrations. Solid State Ionics 1987;24:61-79.
[57] Sikora E, Sikora J, Macdonald DD. A new method for estimating the diffusivities
of vacancies in passive films. Electrochim. Acta, 1996:41;783-789.
[58] Morrison SR, Electrochemistry at semiconductor and oxidized metal electrodes,
Plenum Press, New York, USA, 1980.
[59] Vázquez Huerta G, Caracterización de películas de óxidos crecidos
potenciostáticamente sobre superficies de Nb, Ta y W en medio ácido y alcalino [PhD
Thesis], Departamento de Química, Área de Electroquímica, México DF, México, 2008.
[60] Harrington SP, Devine TM. Analysis of electrodes displaying frequency dispersion
in Mott-Schottky tests. J. Electrochem. Soc. 2008;155(8):381-386.
43
[61] Bos R, Van der Mei HC, Busscher HJ. Physico-chemistryof inicial microbial
adhesive interactions – its mechanisms and mhetods fot study. FEMS Microbiol. Rev.
1990;23:179-230.
[62] Absolom DR, Lamberti FV, Policova Z, Sing W, Van Oss CJ, Neumann AW.
Surface thermodynamics of bacterial adhesion. Appl. Environ. Microb. 1983;46:90-97.
[63] Busscher HJ, Weerkamp AH, Van der Mei HC, Van Pelt AWJ, de Jong HP,
Arends J. Measurement of the surface free energy of bacterial cell surfaces and its
relevance for adhesion. Appl. Environ. Microb. 1984:48:980-983.
[64] Derjaguin BV, Landau LD. Theory of the stability of strongly charged lyophobic
sols and the adhesion of strongly charged particle in solutions of electrolytes. Acta
Phys. Chim. USRR 1941;14:633-662.
[65] Verwey EJW, Overbeek GTG, Theory of stability of lyophobic colloids, Elsevier,
Amsterdam, Holland, 1948.
[66] Rutter PR, Vincent B, The adhesion of micro-organisms to surfaces: physico-
chemical aspects, in Berkeley RCW, Lynch JM, Melling J, Rutter PR, Vincent B (Eds.),
Microbial adhesion to surfaces, Ellis Horwood Limited, London, UK, 1980.
[67] Tadros ThF. Particle-surface adhesion, in Berkeley RCW, Lynch JM, Melling J,
Rutter PR, Vincent B (Eds.), Microbial adhesion to surfaces, Ellis Horwood Limited,
London, UK, 1980.
[68] Van Oss CJ, Good RJ, Chaudhury MK. Additive and nonadditive surface tensions
components and the interpretation of contact angles. Langmuir 1988;4:884-891.
[69] Van Oss CJ, Good RJ, Chaudhury MK. The role of Van der Waals forces an
hydrogen bonds in hydrophobic interactions between biopolymers and low energy
surfaces. J Colloid Interf. Sci. 1986;111:378-390.
[70] Wood J, Sharma R. How long is the long-range hydrophobic attraction? Langmuir
1995;11:4797-4802.
[71] Elimelech M. Indirect evidence for hydratation forces in the deposition of
polystyrene latex colloids on glass surfaces. J. Chem. Soc. Faraday T. 1990;86:1623-
1624.
44
[72] Briandet R, Herry HM, Bellon-Fontaine MN. Determination of the Van der Waals,
electron donor and electron acceptor surface tension components of static gram-positive
microbial biofilms. Colloid Surface B 2001;21:299-310.
[73] Millsap KW, Bos R, Busscher HJ, Van der Mei HC. Surface aggregation of
Candida albicans on glass in the absence and presence of adhering Streptococcus
gordonii in a parallel-plate flow chamber: a surface thermodynamical analysis based on
acid-base interactions. J. Colloid Interf. Sci 1999;212:495-502.
[74] Poortinga AT, Bos R, Busscher HJ. Electrostatic interactions in the adhesion of an
ionpenetrable and an ion-impenetrable bacterial strains to glass. Colloid Surface B
2001;20:105-117.
[75] Hayashi H, Tsuneda S, Hirata A, Saaki H. Soft particle analysis of bacterial cells
and its interpretation of cell adhesión behaviours in terms of DLVO theory. Colloid
Surface B 2001;22:149-157.
[76] Jucker BA, Zehnder AJB, Harms H. Quantification of polymer interactions in
bacterial adhesion. Environ. Sci. Technol. 1998;32:2909-2915.
[77] Klein J, Luckham P. Forces between two adsorbed polyethylene oxide layers
immersed in a good aqueous solvent. Nature 1982;300:429-421.
[78] de Gennes PG. Model polymers at interfaces, in Bongrad P. (Ed.), Physical basis of
cell-cell adhesion, CRC Press, Boca Raton, FL, USA, 1988.
[79] Dickinson RB, Nagel JA, McDevitt D, Foster TJ, Proctor RA, Cooper SL.
Qualitative comparision of clumping factor and coagulase-mediated Staphylococcus
aureus adhesion to surface-bound fibrinogen under flow. Infect. Immun. 1995;63:3143-
3150.
[80] Gallardo-Moreno AM, Méndez-Vilas A, González-Martín ML, Nuevo MJ, Bruque
JM, Garduño E, Pérez-Giraldo C. Comparative study of the hydrophobicity of Candida
parapsiolis 294 through macroscopic and microscopic analysis. Langmuir
2002;18:3639-3644.
45
[81] Van der Mei HC, Wwerkamp AH, Busscher HJ. A comparison of various methods
to determine hydrophobic properties of streptococcal cell curfaces. J. Microbiol. Meth.
1987;6:277-287.
[82] Lyklema J, Norde W, Van Loosdrecht MCM, Zehnder AJB. Adhesion of bacteria
to polystyrene surfaces. Colloid Surface 1989;39:175-187.
[83] Reid G, Beg HS, Preston CAK, Hawthorn LA. Effect of bacterial, urine and
substratum surface tension properties on bacterial adhesion to biomaterials. Biofouling
1991;4:171-176.
[84] Rijnaarts HHM, Norde W, Bouwer EJ, Lyklema J, Zehnder AJB. Bacterial
adhesion under static and dynamic conditions. Appl. Environ. Microb. 1993;59: 3255-
3265.
[85] Yang J, Bos R, Belder GF, Engel J, Busscher HJ. Deposition of oral bacteria and
polystyrene particles to quartz and dental enamel in a parallel plate and stagnation point
flow chamber. J. Colloid Interf. Sci. 1999;220:410-418.
[86] Chavant P, Gaillard-Martinie B, Talon R, Hébraud M, Bernardi T. A new device
for rapid evaluation of biofilm formation potential by bacteria. J. Microbiol Meth.
2007;68:605-612.
[87] Gallardo Moreno AM, Pacha-Olivenza MA, Saldaña L, Pérez-Giraldo C, Bruque
JM, Vilaboa N, González-Martín ML. In vitro biocompatibility and bacterial adhesion
of physico-chemically modified Ti6Al4V surface by means of UV irradiation. Acta
biomaterialia 2009;5:181-192.
[88] Bos R, de Jonge HH, Van de Belt-Gritter B, de Vries J, Busscher HJ. Interaction of
two oral streptococcal strains with physicochemical characterized fluorosilane diffusion
gradient surfaces. Langmuir 2000;16:2845- 2850.
[89] Duddridge JA, Kent CA, Laws JF. Effect of surface shear stress on the attachment
of Pseudomonas fluorescens to stainless steel under defined flow conditions.
Biotechnol. Bioeng. 1982;24:153-164.
46
[90] Pedersen K, Holmström C, Olsson AK, Pedersen A. Statistic evaluation of the
influence of species variation, culture conditions, surface wettability and fluid shear on
attachment and biofilm development of marine bacteria. Arch. Microbiol. 1986;145:1-8.
[91] NCCLS, Methods for dilution antimicrobial susceptibility test for bacteria that
grow aerobically - Sixth edition: approved atandard M7-A6. NCCLS, Waune, PA,
USA, 2003.
47
MODIFICACIÓN SUPERFICIAL BASADA EN
IMPLANTACIÓN CON SILICIO
48
CAPÍTULO 2
Si+ ion implantation reduces the bacterial
accumulation on the Ti6Al4V surface
Este capítulo es una reproducción de la publicación: "A.M. Gallardo-Moreno, M.A.
Pacha Olivenza, J. Perera-Núñez, J.L. González-Carrasco, M.L. González-Martín.
Journal of Physics: Conference Series 2010;252:012017" realizada en colaboración con
el grupo del Dr. J.L. González-Carrasco del Centro Nacional de Investigaciones
Metalúrgicas, CENIM-CSIC (Madrid).
49
ABSTRACT
Ti6Al4V is one of the most commonly used biomaterials in orthopedic applications due
to its interesting mechanical properties and reasonable biocompatibility. Nevertheless,
after the implantation, microbial adhesion to its surface can provoke severe health
problems associated to the development of biofilms and subsequent infectious
processes. This work shows a modification of the Ti6Al4V surface by Si+ ion
implantation which reduces the bacterial accumulation under shear forces. Results have
shown that the number of bacteria remaining on the surface at the end of the adhesion
experiments decreased for silicon-treated surface. In general, the new surface also
behaved as less adhesive under in vitro flow conditions. Since no changes are observed
in the electrical characteristics between the control and implanted samples, differences
are likely related to small changes observed in hydrophobicity.
2.1. INTRODUCTION
Bacteria prefer a community-based surface-bound, sedentary lifestyle to a nomadic
existence. There may be an obvious explanation for bacterial adhesion, because
nutrients in an aqueous environment tend to concentrate near a solid surface [1].
As an oversimplified rule of thumb, primary adhesion between bacteria and abiotic
surfaces is generally mediated by nonspecific interactions, whereas adhesion to living or
devitalized tissue is accomplished through specific molecular (lectin, ligand or
adhesion) docking mechanisms. Primary adhesion is reversible and defines the further
adhesion between the bacterial cell surface and the conditioned surface of interest. Once
the microorganisms reach critical proximity to the surface, the final determination of
adhesion depends on the net sum of attractive or repulsive forces generated between
both surfaces, including electrostatic and hydrophobic interactions [2].
The ability to control microbial adhesion is of enormous importance in healthcare,
particularly in modern surgery where postoperative implant-associated infections are
still an unresolved and serious complication. This challenge is compounded by the ever-
increasing problem of antibiotic resistant hospital and community acquired infections
[3, 4]. For this reason new strategies are focused on minimizing microbial surface
colonization by modifying the biomaterial surface and antibacterial coatings of Ti6Al4V
have been recently proposed by different research groups [5-7].
50
In this line, this work shows a new modification of the Ti6Al4V surface by Si+ ion
implantation which reduces the bacterial accumulation under in vitro flow conditions.
Surface characterization is carried out by means of hydrophobicity and isoelectric point.
2.2. MATERIALS AND METHODS
2.2.1. Ti6Al4V surface modification
Ion implantation was carried out by using a F4Si precursor on the surface of 20 mm
diameter disks of Ti6Al4V kindly supplied by Surgival SA, Spain. Ti6Al4V without
implantation will be denoted as “control” and Ti6Al4V implanted will be denoted as
“implanted”.
2.2.2. Surface characterization
Hydrophobicity: It was quantified through the water contact angle (θW) on the sample
surface.
Surface Gibbs Energy: It was evaluated from the contact angles of water, formamide
(F) and diiodomethane (D) by applying the Young Equation [8] and Van Oss approach
[9]. The surface Gibbs energy (Total
) was the sum of the Lifshitz-van der Waals
component (LW
) and acid-base component (AB
) which, in turn, is the geometric mean
of the electron-donor (-) and electron-acceptor (
+) parameters.
Isoelectric point: zeta potential () was measured as pH function and isoelectric point
(IEP) was obtained as the pH at which was zero [10].
2.2.3. Bacterial strains
Three gram-positive strains with different EPS-production were used: Staphylococcus
aureus ATCC29213 (S. aureus), Staphylococcus epidermidis ATCC35984 (S.
epidermidis4), Staphylococcus epidermidis HAM892 (S. epidermidis2).
2.2.4. Adhesion experiments
Bacterial adhesion experiments were carried out at 37 ºC in a parallel plate flow
chamber and the results were analysed in terms of flow or static conditions by selecting
a constant flow rate or stopping the bacterial flow. Different protocols were followed:
Dynamic adhesion: the flow chamber was placed so that the alloy surface was on the
top side of the flow channel and the bacteria were allowed to attach while flowing at a
51
constant rate for 20 min. The number of bacteria at the end the experiments dynamic
was denoted by ND-20min. It was also analysed the initial adhesion rate to the surface (jD).
Static adhesion: the sample was placed on the bottom side of the flow channel, the flow
was stopped and the bacteria were left to deposit on the surface for different time
interval periods. Static adhesion rates (jS) were obtained and compared to that of
dynamic.
Shear forces: At the end of the experiments two consecutive air-liquid interfaces were
passed through the flow channel and the number of bacteria remaining on the surface
were analysed in order to check the strength of the bacterial retention.
2.3. RESULTS AND DISCUSSION
Table 2.1 shows the surface characterization of both control and implanted samples.
There are small changes in contact angles but the implanted surface is slightly more
hydrophilic than the control. This also implies a higher polarity of the treated surface
since the polar liquids (W, F) present lower contact angles and AB
is higher. There is no
difference between samples in the IEP within experimental error.
Fig. 2.1 presents interesting information from the bacterial adhesion tests. It is observed
that initial adhesion rates to control and implanted are not statistically different (Fig.
2.1a). Only in the case of S. epidermidis2 a decrease in jD is observed in the implanted
sample respect to control. This strain also presents the highest decrease in the final
number of adhered bacteria after dynamic experiments (Fig. 2.1b). ND-20min also
diminishes for S. aureus in the implanted. Information provided by static experiments
are different to that of dynamic, as showed when comparing Figs 2.1a and 2.1c. jS is
always higher than jD, indicating that static adhesion is highly influenced by
sedimentation processes. Relationships between control and implanted in both
experiments are also different. In the case of static adhesion Si+ ion implantation never
reduces adhesion (Fig. 2.1c) on the contrary; it is increased for both strains of S.
epidermidis. An interesting observation is that after the passage of two air-liquid
interfaces the bacterial detachment is more effective in silicon-treated surface than in
control (Fig. 2.1d), which could enhance the applicability of this technique in those
orthopaedic applications in which shear forces are present.
52
Table 2.1. Contact angles of water (θW), formamide (θF) and diiodomethane (θD), Lifshitz-Van
der Waals (LW) and acid-base (AB
) components as well as electron-acceptor (+) and electron-
donor (-) parameters and the total surface Gibbs energy (Total
) for control and implanted
samples. Isoelectric points (IEP) are also represented.
Contact Angle Surface Gibbs Energy IEP
W F D LW
AB
+
-
Total
Control 58.6
±1.8
48.7
±1.6
40.6
±1.4
32.4
±0.7
8.1
±1.7
0.7
±0.2
24
±3
40.5
±2.4
5.5
±0.9
Implanted 53.5
±0.7
42.5
±0.7
38.3
±0.7
33.1
±0.6
10.9
±0.8
1.14
±0.11
26.1
±1.1
44.0
±1.4
4.8
±0.8
Fig. 2.1. Bacterial adhesion experiments. jD: initial dynamic adhesion rate locating the sample
at the top of the flow channel (a). ND-20min: number of adhered bacteria at the end of the dynamic
adhesion experiment (b). jS: static adhesion rate locating the sample at the bottom of the flow
channel (c). Percentage of remaining bacteria after passing two air-liquid interfaces (d).
Relationships between physico-chemical surface parameters and bacterial adhesion have
been extensively studied [2, 7, 11]. However it is difficult to establish the exact
contribution of each magnitude to any particular adhesion process involving
microorganisms. Electrostatic interactions tend to favor repulsion, because most
bacteria and inert surfaces are negatively charged. Stenotrophomonas maltophilia is one
exception to this rule, and the overall positive surface charge of this organism, at
physiological pH can promote primary adhesion to negatively charged materials such as
0
2
4
6
8
10
12
1 2 3
ND
-20m
in (
ba
cte
ria
/cm
2)·
10
-4
control
implanted
S. aureus S. epidermidis2 S. epidermidis4
(b)
ND
.20
min
(b
acte
ria
/cm
2)x
10
-4
0
100
200
300
400
500
600
700
800
900
1000
1 2 3
j S (
ba
cte
ria
/cm
2 s
)
control
implanted
(c)
j S (
ba
cte
ria
/cm
2s)
S. aureus S. epidermidis2 S. epidermidis4
0
50
100
150
200
250
1 2 3
j D (
ba
cte
ria
/cm
2 s
)
control
implanted
j D (
bacte
ria
/cm
2s)
(a)
S. aureus S. epidermidis2 S. epidermidis4
0
20
40
60
80
100
120
1 2 3
Rem
ain
ing
bacte
ria a
fter
air
-liq
uid
sh
ear
forc
es (
%) control
implanted
(d)
Rem
ain
ing
bacte
ria
aft
er
Air
-liq
uid
sh
ear
force
s (%
)
S. aureus S. epidermidis2 S. epidermidis4
53
Teflon [12]. In our case, the IEP obtained for both surfaces indicates that they are
negatively charged at physiological pH (about pH=7). As indicated by different research
groups, hydrophobic-hydrophilic interactions probably have greater influence on the
outcome of primary adhesion [7, 13, 14]. In the present research the small changes in
the hydrophobicity of the surface after implantation seems to be related to the lower
retention of the three strains (Fig. 2.1d). Silicon-treated surface become more
hydrophilic and this means a higher affinity for water under shear conditions, reducing
the final bacterial accumulation on the surface.
2.4. REFERENCES
[1] Dunne WMJr. Bacterial adhesion: seen any good biofilms lately?. Clin.
Microbiol. Rev. 2002;15:155-166.
[2] An Y H, Friedman R J (ed) Handbook of bacterial adhesion: principles, methods
and applications. Humana Press, Totowa, NJ, 2000.
[3] Gould IM. Costs of hospital-acquired methicillin-resistant Staphylococcus aureus
(MRSA) and its control. Int. J. Antimicrob. Ag. 2006;28:379-384.
[4] Moellering RCJr. The growing menace of community-acquired methicillin-
resistant Staphylococcus aureus. Ann. Intern. Med.2006;144:368-370.
[5] Heidenau F, Mittelmeier W, Detsch R, Haenle M, Stenzel F, Ziegler G,
Gollwitzer H. A novel antibacterial titania coating: metal ion toxicity and in vitro
surface colonization. J. Mater. Sci. 2005;16:883-888.
[6] Sarró MI, Moreno DA, Ranninger C, King E, Ruiz J. Influence of gas nitriding of
Ti6Al4V alloy at high temperature on the adhesion of Staphylococcus aureus.
Surf. Coat. Technol. 2006;201:2807-2812.
[7] Gallardo-Moreno AM, Pacha-Olivenza MA, Saldaña L, Pérez-Giraldo C, Bruque
JM, Vilaboa N, González-Martín ML. In vitro biocompatibility and bacterial
adhesion of physico-chemically modified Ti6Al4V surface by means of UV
irradiation. Acta Biomater. 2009;5:181-192.
[8] Young T. An essay on the cohesion of fluids. Philos. Trans. R. Soc. London
1805;95:65-87.
54
[9] Van Oss CJ. Interfacial forces in aqueous media. Marcel Dekker, New York,1994.
[10] Shaw DJ. Introduction to colloid and surface chemistry. Butterworths,
London,1989.
[11] Bos R, Van der Mei HC, Busscher HJ. Physico-chemistry of initial microbial
adhesive interactions – its mechanisms and methods for study. FEMS Microbiol.
Rev. 1999;23:179-230.
[12] Jucker BA, Harms H, Zehnder JB. Adhesion of the positively charged bacterium
Stenotrophomonas (Xanthomonas) maltophilia 70401 to glass and Teflon. J.
Bacteriol. 1996;178:5472-5479.
[13] De Morais LC, Bernardes-Filho R, Assais OBG. Wettability and bacteria
attachment evaluation of multilayer proteases biofilm for sensor application.
World J. Microb. Biot. 2009;25:123–129.
[14] Mazumder S, Falkinham JO, Dietrich AM, Puri IK. Role of hydrophobicity in
bacterial adherence to carbon nanostructures and biofilm formation. Biofouling
2010;26:333-339.
55
MODIFICACIÓN SUPERFICIAL BASADA EN
IRRADIACIÓN CON LUZ UV-C
56
CAPÍTULO 3
Effect of UV irradiation on the surface Gibbs
energy of Ti6Al4V and thermally oxidized
Ti6Al4V
Este capítulo es una reproducción de la publicación: "M.A. Pacha Olivenza, A.M.
Gallardo-Moreno, A. Méndez-Vilas, J.M. Bruque, J.L. González-Carrasco, M.L.
González-Martín. Journal of Colloid and Interface Science 2008;320:117-124",
realizada en colaboración con el grupo del Dr. J.L. González-Carrasco del Centro
Nacional de Investigaciones Metalúrgicas, CENIM-CSIC (Madrid).
57
ABSTRACT
Thermal oxidation of Ti6Al4V increases thickness, modifies the structure, and changes
the amount of alloying elements of the surface titanium dioxide layer in respect to the
spontaneous passive layer of Ti6Al4V. The effect on the surface properties of Ti6Al4V
and thermally oxidized Ti6Al4V after different periods of UV irradiation have been
studied by the measurement of water, formamide and diiodomethane contact angles.
The rate of modification of the water contact angle with the irradiation time is
dependent on the surface treatment, but the water adhesion work, after an initial
energetic step, follows a similar trend for both. Application of Young equation together
with the van Oss approach allowed the evaluation of the surface Gibbs energy of the
alloys. Similarly to the water adhesion work, the surface Gibbs energy dependence with
the irradiation time follows a similar trend for both samples and it is due to the change
of the electron-donor parameter of the acid-base component. Also, a linear relationship
common for both samples have been obtained between the cosines of the water contact
angles and formamide or diiodomethane contact angle. These facts indicate that the
surface modification continuously produced by the UV irradiation is similar all along
the process and similar for both samples after an energetic threshold for the thermally
oxidized sample. It has been also tested that the hydrophilic-hydrophobic conversion is
reversible for Ti6Al4V and Ti6Al4V thermally treated.
3.1. INTRODUCTION
Ti6Al4V is the most frequently used Ti alloy for medical purposes due to its interesting
mechanical properties and biocompatibility. It is an α/β titanium alloy and, as cp Ti,
once exposed to the atmosphere, a passive layer is formed on its surface, whose
thickness can reach up to 6 nm after one year of exposure to normal ambient
temperatures [1]. Weakness of this alloy is that adherence of the passive film is rather
poor, and it may be disrupted at very low shear stresses, even rubbing against soft
tissues [2]. This feature may account for the accumulation of released ions in the tissues
around the implant [3]. Different treatments on this alloy have been proposed to
overcome this shortage. Among them, it has been probed that heating the Ti6Al4V alloy
above 450 ºC changes the thickness of the passivated layer up to several tens of
nanometres [4]. Also, corrosion resistance is improved whereas the good
citobiocompatibility is not affected [5,6] and a decrease in the ion release has been
observed in vitro [7].
58
TiO2, the main component of the passive layer, [8] is a photoactive n-type
semiconductor oxide [9,10] which modifies temporarily its hydrophilicity after being
exposed to UV radiation. Depending on the procedure followed to prepare the passive
layer, titanium dioxide can be found as amorphous or as microcrystalline rutile or
anatase. Each crystalline form of TiO2 has different properties; in particular, the band
gap is 3.2 eV for anatase and 3.0 eV for rutile [9]. Also, the rate of surface photo-
dependent modifications and the evolution after UV irradiation are dependent on the
crystalline TiO2 structure and on the crystal face exposed [11].
Nevertheless, the passive layer of Ti6Al4V is composed not only by titanium oxides,
but the alloying elements are also present in a proportion which is dependent on the
previous treatment of the alloy. Different authors have found about a two- to three-fold
increase of the Al concentration in the oxide film in comparison to the corresponding
value in the bulk metal, with a tendency to be more strongly enriched at the outermost
surface [10]. The presence of Al as been found as Al3+
, suggesting that it exists either in
the form Al2O3 in the naturally passivated layer. In the natural oxide film V is in a
concentration below to that in the bulk material (aprox. 0.3 to 1 wt%). On the other
hand, thermal treatments, particularly at temperatures above 450 ºC, change the
proportion of alloying elements, as well as the microstructural properties of the oxide
film that becomes formed mainly of microcrystalline rutile [10].
Surface properties of a biomaterial dominate its interaction with the surrounding fluids,
cells, microorganisms, etc. in this sense wettability and, more generally, surface Gibbs
energy are the basic information needed to predict the adhesion and adsorptive
behaviour of a material. Its knowledge is also interesting because some authors have
suggested that it modulates bone cell maturation and differentiation [12]. It has been
reported that UV radiation affects wettability of titanium dioxide [11], however, there is
a lack in detailed studies either on the effect of progressive UV radiation on the surface
Gibbs energy of Ti6Al4V or after thermal treatment. On this basis, the purpose of this
research has been to characterize the effect of different exposure times to UV radiation
on the wettability and surface Gibbs energy of the Ti6Al4V alloy, and the Ti6Al4V
alloy thermally oxidized at 475 ºC, as well as the evolution of these properties once the
irradiation is ceased.
59
3.2. MATERIALS AND METHODS
3.2.1. Sample characterization
Discs of Ti6Al4V were cut from a single bar of 50 mm in diameter kindly supplied by
SURGIVAL S.A. (Valencia, Spain). It was a TIMETAL 6-4 ELI alloy processed as a
hot rolled annealed bar at 700ºC for 2h, then air cooled. Table 3.1 shows the chemical
composition supplied with the product certificate. Discs were abraded on successively
finer silicon carbide papers, mechanically polished with diamond paste, and finishing
with silica colloidal (denoted as TiAlV). After this preparation, some discs were
oxidized by heating at 475ºC for 1 hour in air (denoted as TiAlVOx).
Table 3.1. Chemical analysis (mass %) of Ti6Al4V alloy. Data supplied by the manufacturer
Ti V Al Fe C O
Bal. 4.10 - 4.15 6.07 - 6.09 0.17 0.005 - 0.01 0.12 - 0.13
For microstructural characterization some samples were etched with a solution
containing 2 ml HF, 3 ml HCl and 5 ml HNO3 in 90 ml H2O to reveal the -phase. The
analysis was performed by using a scanning electron microscope (SEM) equipped with
a field emission gun (FEG) emitter coupled with an energy dispersive X-ray (EDX)
system for chemical analysis. To minimize the uncertainties of the EDX measurements
several zones were analysed with measuring times of about 100 s.
Surface composition and oxide-layer thickness of samples have been analyzed by XPS,
with a K-Alpha (Thermo, UK), using an Al K monochromatic X-ray source, with spot
size of 400 m. Profiling was done using a 3kV Ar+ ion beam which generates a beam
current of 1.5 A.mm-2
at the surface. The atomic percentages of elements were
calculated using software and atomic sensitivity factors included with the instrument
data system.
Surface roughness was evaluated with an Autoprobe CP atomic force microscope (Park
Scientific Instrument, Geneva, Switzerland, now a part of Veeco), equipped with a
scanner of maximum ranges of 100 μm in the x and y directions and 7 μm in the z
direction. The images were acquired by using silicon nitride cantilevers with a nominal
probe curvature radius of 10 nm, as supplied by the manufacturer. Images were acquired
at a resolution of 512 x 512 pixels, and were subjected to first-order flattening.
60
3.2.2. Experimental procedures
Before each experimental run, samples were carefully cleaned with distilled water at
60ºC, vigorously rubbed with a smooth cotton cloth, then rinsed repeatedly, first with
distilled water and finally with distilled and deionized water (MiliQ system); immersed
in a beaker with distilled and deioniezed water and sonicated for 10 min, rinsed again
with distilled and deionized water, dried in an oven at 40ºC for 1h and stored in a
disiccator for no more than 24 h. This cleaning procedure was also followed to study the
recovery of the samples after UV irradiation. By this reason it has not been used any
chemical compound during the procedure to avoid any possible interference of them on
the surface energy modifications studied.
TUV TL-D 15W SLV lamps, kindly provided by Philips (Philips Ibérica, SAU, Madrid,
Spain), emitting predominantly at a wavelength of 257,7 nm were used for UV
irradiation of samples. Glass of lamp has filtering to avoid the production of ozone,
which is produced by wavelengths lower than 200 nm. Discs were positioned exactly
centred 10 cm below the lamp, receiving an intensity of 2,6 mW cm-2
, during fixed time
intervals. The whole irradiation installation was inside an opaque wood chamber to
avoid interferences from room or day light and any damage to users.
Table 3.2. Surface tension, , Lifshitz-van der Waals, LW, and acid-base, AB
, components,
electron-acceptor, +, and electron-donor, -
, parameters of the test liquids used for contact
angle measurements, in mJ.m-2
.
LW AB + - Ref.
Water 72.8 21.8 51.0 25.5 25.5 [13]
Formamide 58 39 19 2.28 39.6 [13]
Diiodomethane 50.8 50.8 0 0.72 0 [14]
Water (distilled and deionized from a Milli-Q Plus system), formamide (puriss > 99.0%,
Fluka, Switzerland) and diiodomethane (purum > 98%, Fluka, Switzerland) contact
angles on discs were measured using the sessile drop technique with the aid of a G20
goniometer (Krüss, Germany). Drops were placed on the central part of the surface of
discs. Surface tension and its components and parameters [13,14] are listed in Table 3.2
The results presented are averaged values of at least three independent runs.
61
3.3. RESULTS AND DISCUSSION
Fig. 3.1 shows a representative secondary electron image (SEI) of the sample, where it
can be seen the expected biphasic microstructure of the alloy, consisting of phase
(dark zones), Al richer, and phase (bright zones), V richer, arranged in a cellular way.
The relative composition of Ti, Al and V elements in the passive and thermally oxidized
layers obtained from XPS analysis are summarized in Table 3.3, as well as the relative
composition of the bulk material obtained from the analysis certificate supplied by the
manufacturer. Vanadium has a lower presence in the passive layer (sample TiAlV) than
in the bulk. However, the presence of both alloying elements is higher in relation to Ti
in the oxide layer of TiAlVOx sample than in TiAlV and than in the bulk material, but
V is depleted in relation to Al in both passive layers. These results are in the same line
than those obtained by MacDonald et al [4], despite these authors obtained higher
concentrations of Al and V in relation to Ti on Ti6Al4V heated at 600º C.
Fig. 3.1. SEI image of the alloy obtained on polished and etched specimens. Dark zones
correspond to alpha phase and bright zones to beta phase.
The higher oxidation temperature with regards to that of the present study (475 ºC)
would likely yield higher diffusion of the alloying elements to the passive layer.
Analysis of the XPS spectra obtained (not shown) indicates that in both samples Al and
V are present in the passive layers in the form of oxides, although, for sample TiAlV
some metallic V and Ti were detected, probably due to the thin passive layer of this
sample (less than 4 nm, against the value of ca. 90 nm obtained for sample TiAlVOx),
in accordance with other authors results [10, 15-17].
62
Table 3.3. Relative atomic composition of the samples surface, and bulk material
(Reproducibility better than ±10%).
Al/Ti V/Ti V/Al
TiAlV 0.12 0.014 0.12
TiAlVOx 0.70 0.16 0.23
Bulk 0.12 0.044 0.36
Fig. 3.2 shows the AFM topographical images of both samples, TiAlV and TiAlVOx.
TiAlV sample has a topographically homogeneous surface, in contrast with the terraced
surface shown after the oxidation treatment (sample TiAlVOx). Two roughness
parameters have been used to characterize the topography of the samples at different
scales, from 1 to 50 m: the root mean square (Rrms) surface roughness and the average
roughness (Ra), described elsewhere [18]. Table 3.4, that lists the results obtained for
both samples, indicates that sample TiAlVOx has a roughness slightly higher than
TiAlV for scanning lengths of 1, 5 and 10 microns.
Fig. 3.2. AFM topographical images of TiAlV and TiAlVOx samples.
Wettability and surface Gibbs energy evaluations used to be done through the
measurement of the equilibrium contact angle of liquid drops on the solid. This
equilibrium contact angle needs to be determined on perfectly smooth surfaces, but
most of the real surfaces do not fit this requirement. Wenzel´s equation [19], cos obs = r
cos s, relates the observed contact angle on a real surface, obs, with the contact angle
that would be measured on a perfectly smooth surface of the same material, s, r being
the Wenzel’s roughness factor, which gives the relation between the real surface area to
the projected area below the drop, and whose value is higher than 1. In order to evaluate
63
the possible influence of the samples roughness on the measured contact angles, r has
been directly derived from AFM measurements, and listed in Table 3.4. The Wenzel’s
factor indicates that the thermally oxidized surface always displays a roughness-
developed area (with respect to the projected area) larger than that of TiAlV, at all
scales, following the same general tendency than Rrms and Ra. The values obtained are
in accordance with other studies using TiAlV [20], and they are much lower than values
reported for other materials as, for example polymer surfaces [21]. The extremely low
absolute values of the computed Wenzel´s factor for both surfaces at all scales indicate
that surface roughness has not any relevant effect on the contact angles. In consequence,
differences between obs and s calculated by Wenzel´s equation are well below the
experimental uncertainty in contact angles measurement, so no correction for roughness
will be made.
Table 3.4. Root mean square roughness, Rrms, average roughness, Ra, and Wenzel roughness
factor, r, of samples determined from AFM measurements, at different dimensional scales.
Dimensional
scale
(m)
Rrms
(nm)
Ra
(nm)
r
TiAlV TiAlVOx TiAlV TiAlVOx TiAlV TiAlVOx
50 6±1 7±2 4.2±0.7 5±1 1.0002±2 10-4
1.0008±1 10-4
25 4±2 6.0±0.9 3±1 4.3±0.5 1.0002±2 10-4
1.0014±7 10-4
10 2±2 5.4±0.4 1.3±0.8 4.0±0.3 1.0002±1 10-4
1.0028±5 10-4
5 1±1 5.3±0.4 0.8±0.6 4.0±0.4 1.0004±1 10-4
1.0038±5 10-4
1 0.5±0.4 3±1 0.3±0.2 2.0±0.9 1.004±2 10-3
1.012±4 10-3
Fig. 3.3 depicts the water contact angle on TiAlV and TiAlVOx samples after different
irradiation dosages. Before irradiation (t=0) both samples have a very markedly
hydrophobic character as their water contact angle indicates. It must be highlighted that
values of water contact angle found in bibliography on different metallic oxides,
including titanium, range from nearly 90º down to 0º. However, these large differences
are related to procedures followed in the preparation (liquids and protocols of cleaning,
passivation, sterilization process, etc) and the roughness (Rrms and Ra) of the sample
[4, 17, 22, 23]. Nevertheless, the high contact angle values obtained on samples are also
related to the presence of hydrocarbon contamination from atmosphere [10]. It should
be expected that a perfectly clean surface must be composed by atoms of titanium,
oxygen and eventually alloying elements, with a very high surface energy. However,
64
such kind of “clean” surface is not likely to be found in a medical implant in surgery or
in laboratory because it takes a few seconds to 1 min in atmosphere before this high
energy surface becomes contaminated by a layer of hydrocarbons and inorganic
impurities, modifying its chemical surface composition and hydrophilicity [12].
Fig. 3.3. Water contact angle on TiAlV () and TiAlVOx () against the exposure time to UV
irradiation.
As UV irradiation time increases, the surfaces of both samples change from
hydrophobic to hydrophilic, but at different rates. The decrease of the water contact
angle on TiAlV is very much sharper than on TiAlVOx until the irradiation time was 50
min. Nevertheless a further decrease down to 11±5º was measured for an exposure time
of 900 min for both samples. This behaviour is readily related to the presence of TiO2 in
the passive layer of the alloy. UV radiation on TiO2 provokes the creation of pairs hole-
electron. Electrons tend to reduce Ti4+
surface sites to Ti3+
while the generate holes
reacts with the oxygen resulting in oxygen vacancies [11]. Water adsorption on different
faces of rutile have been extensively studied, despite it continues to be controversial. On
rutile (100) it is expected that water adsorbs both dissociatively and molecularly,
independently of steps, point defects and the Ti3+
sites presents, followed by molecular
adsorption at higher coverage. Experimentally rutile (110) does not seem to dissociate
water, except at defect sites, despite of theoretical studies that predict dissociative
adsorption. On (110) and (100) surfaces there are two kinds of oxygen, threefold –as in
the bulk– and twofold coordinated, this last protruding from the crystal surface is called
bridging site oxygen and it is more reactive than the threefold coordinated. However,
bridging sites do not exist on (001) surfaces. On the other hand, for the less studied
anatase, it is suggested that water adsorbs molecularly on anatase (101), but theoretical
0
15
30
45
60
75
90
0 60 120 180 240
t (min)
W
(º)
900
//
65
calculations predict dissociative adsorption on the anatase (001). Extensive revisions on
the present knowledge on water adsorption on TiO2 can be found in the works of
Diebold [9] and Henderson [24] and references herein.
The influence of alloying elements, impurity constituents or the interaction with the
environment lead to a probably more defective passive layers on both TiAlV and
TiAlVOx samples [25], that also increased their oxygen defective vacancies after UV
radiation, being healed by water molecules to give hydroxyl groups adsorbed.
The passive layer on TiAlV is essentially amorphous, but it is likely a certain degree of
short-range order in the nanometer scale, which allows assuming, in first order
approximation, this passive layer as a rutile (110) surface [10]. On the other hand the
oxide layer of the TiAlVOx sample has shown to be composed by microcrystalline
rutile without specific orientation, so then exposing different crystallographic rutile
surfaces [10].
Studies on the effect of UV irradiation on pure TiO2 rutile have proved that the duration
of UV illumination required to get the (001) plane fully hydrophilized was three times
longer than for the (110) plane [11]. In consequence the observed differences in the rate
of the wettability change with the irradiation time in our samples can be related to the
differences in the crystalline state of the passive layer, probably due to the presence of
rutile microcrystallites with the (001) plane exposed in the thermally oxidized alloy, or
to the different ratio of alloying elements respect to Ti shown in the passive layer of
both samples.
Despite water contact angle gives a straightforward measure of the water wettability of
a given surface, when changes in this property comes from water adsorption on the
surface, adhesion work of water provides a more clear information of the modification
of the surface layer. Adhesion work measures the Gibbs energy change needed to create
a unit of interfacial area between the solid and the liquid [26]. It is given by the
expression
adh L LW (1 cos ) (3.1)
γL and θL being the surface tension of the liquid and its contact angle on the solid,
respectively. Fig. 3.4 presents the water adhesion work as a function of the UV
66
irradiation time for both samples (left axis reads for TiAlV sample and the right one for
TiAlVOx sample).
Fig. 3.4. Water adhesion work on TiAlV () and TiAlVOx () against the exposure time to UV
irradiation.
As it can be expected from the measured dependence of water contact angles, the
general trend of the adhesion work for both samples differ each other, moreover as θW
approaches zero, Wadh tends to 145.6 mJ m-2
, the water cohesion work, for both.
Nevertheless, after the 20 first minutes of UV irradiation, the rate of variation of Wadh
presents a similar dependence for both TiAlV and TiAlVOx, up to values close to 140
mJ m-2
. This dependence suggests that the amorphous layer on TiAlV sample can be
treated as composed by at least two different kinds of patches. One kind of patches
should expose easily accessible oxygen positions, so that the amount of energy given
during the first twenty minutes of irradiation is enough to activate them and then being
hydroxyl groups or water adsorbed. This confers to TiAlV a Wadh value of 116 mJ m-2
after 20 min irradiation, larger than the value of 83 mJ m-2
for TiAlVOx after the same
time. A second kind of patches on the amorphous layer on TiAlV should behave as
microcrystalline rutile does, as the trend of Wadh for irradiation times longer than 20 min
is similar to the TiAlVOx sample, that is, it is needed a similar amount of energy for
both samples to get a given increase in their oxygen activation as the amount of
adsorbed water shows. Of course, once Wadh approaches closely to the cohesion work of
water the behaviour of TiAlV differs from that of TiAlVOx at the same irradiation time,
probably because of the completion of a layer of physisorbed water on it.
105
140
175
0 60 120 180 240
t (min)
Wad
h(m
J m
-2)
70
Wad
h (mJ m
-2)140
//
105
900
67
Taking into account the acute effect that different UV irradiation times induce on the
water wettability of these samples, it is important to elucidate how their surface Gibbs
energy is modified along this process, because it is expected that these changes alters
the interaction that these materials can have with proteins, bacteria or cells.
Young equation together with the van Oss approach allows evaluating the surface Gibbs
energy, and provides information about the contribution of van der Waals and acid-base
interactions to the total surface Gibbs energy of the solid, as it is expressed in Eq (2.2).
1 2 1 2 1 2
LW LW
L L S L S L S Lcos 1 2 2 2 (3.2)
where γLW
is the Lifshitz-van der Waals component, γ+ is the electron-acceptor and γ
- is
the electron-donor part of the acid-base component, 1 2
AB 2 , of the total surface
Gibbs energy LW AB . To solve Eq. (3.2) for the solid components it is needed the
measured contact angle of drops of three liquids deposited on the solid. Water,
formamide and diiodomethane are commonly utilized to this purpose, and the results
obtained with them on the alloys after different irradiation times are shown in Table 3.5.
Table 3.5. Contact angles of water (θW), formamide (θF) and diiodomethane (θD) on TiAlV and
TiAlVOx alloys, as a function of the exposure time to the UV radiation.
Samples t
(min)
W
(º)
F
(º)
D
(º)
TiAlV
0 83±2 68±2 54±2
20 54±3 38±3 41±2
40 24±3 22±3 37±2
60 18±4 11±4 36±2
TiAlVOx
0 84±2 73±2 54±2
20 82±2 67±3 52±2
40 64±2 58±3 47±2
60 52±2 39±4 43±2
The decrease that exposure to UV radiation induces on the water contact angle is also
reflected on the formamide and diiodomethane contact angles, despite on a lesser
extension for the apolar than for the polar liquids, but in a similar way for both samples.
This particular behaviour suggests checking if there is any relationship between the
68
changes of the contact angles of the liquids and the time that samples were under UV
exposure. Fig. 3.5 shows the plot of cos θF and cos θD against cos θW for both samples at
the different irradiation times. This plot indicates a good correlation, within the
experimental error, between the changes in cos θW with those of cos θF and cos θD, no
matter the sample selected. Linear minimum square fits of data were:
cos θF = 0,26 + 0,76 cos θW (r2 = 0,97)
cos θD = 0,57 + 0,26 cos θW (r2 = 0,97) (3.3)
These straight lines have been also plotted in Fig. 3.5. The existence of such
relationships indicates again that the kind of modification that UV radiation induces is
the same independently on how long the exposure to the UV irradiation last and the
kind of sample; however, as it has been stated, the extension of this modification
depends on the exposure time in a different way for TiAlV and for TiAlVOx.
Fig. 3.5. Relationship between water-formamide contact angles (continue line, filled symbols)
and water-diiodomethane (doted line, open symbols), for TiAlV (round symbols) and TiAlVOx
(square symbols).
As a further consequence, the linear relationships found allow evaluating the surface
Gibbs energy using the van Oss model from the single information of the water contact
angle. Fig. 3.6 shows the dependence of T, LW
, AB
, and the parameters - and
+ with
the water contact angle. Points in this figure were obtained solving the three equation
system that corresponds to the application of Eq. (3.2) to the water, formamide and
diiodomethane contact angles measured after each irradiation time, and lines (except the
0,00
0,20
0,40
0,60
0,80
1,00
0,00 0,20 0,40 0,60 0,80 1,00
cos W
cos F
, co
s D
69
straight line on T) correspond to the values obtained using water contact angle and the
relationships given in Eq. (3.3). As it can be expected from the existence of these
relations, lines in this figure approximate accurately the calculated points.
It is clear that the total surface Gibbs energy of any of both samples increases when UV
irradiation increases its duration (indicated by the change in the water contact angle),
due to the change in the electron-donor parameter of the acid-base component of the
surface Gibbs energy. Changes in + and
LW are not so important in the studied range.
This behaviour is in accordance with a progressive surface coverage with hydroxyl
groups or chemisorbed water in a preferential orientation [9,24]. Despite the change of
the total surface Gibbs energy with water contact angle seems to follow a linear
dependence in the whole range of water contact angles (straight line in Fig. 3.6), the
results obtained show some deviations at the extreme values of the water contact angle.
Fig. 3.6. Total surface Gibbs energy (T, ), Lifshitz-van der Waals (LW, ) and acid-base (AB
,
) components and electron-donor (-, ) and electron-aceptor (+
, ◊) parameters dependence
on the water contact angle. Lines were calculated using Eq. 3 for the contact angles of
formamide and diiodomethane. Extrapolation of the linear part of the total surface Gibbs
energy has been plotted as (- - -).
However, it can not be discarded, at the highest contact angles, the influence of some
unavoidable hydrocarbons contamination coming from the ambient that are adsorbed on
the surface. Deviation observed at the lowest water contact angle corresponds to surface
states highly hydrated, probably being covered with a layer of physisorbed water. In
such a case, it could be expected that the surface pressure could be not negligible.
0
20
40
60
80
0 20 40 60 80 100
W (º)
(
mJ
m-2
)
T
+
LW
AB
-
70
However it is not clear any different behaviour of formamide and diiodomethane
contact angle in respect to the water contact angle in such range.
Although TiO2 has been assumed that does not suffer from photocorrosion in
comparison to other photocalysts, it is needed to know if the presence of the alloying
elements in the passive layer of our samples could modify this behaviour. To this
purpose, the samples, after 60 min of UV exposure, were recovered following the same
procedure as the cleaning procedure described in the experimental section, and
subsequently, the contact angle of the three liquids were measured. It is noted that the
procedure followed did not involve the use of any chemical compound, but only the use
of hot water (60 ºC). This process (recovering-contact angle measurement) was repeated
for four times, and the results for water contact angles have been plotted in Fig. 3.7.
Fig. 3.7. Water contact angle measured on TiAlV () and TiAlVOx () after 60 min UV
irradiation, as a function of the number of recovery cycles.
This Figure shows that samples recover the water contact angle previous to the UV
irradiation after the fourth cycle. Also, after the first recovery cycle both samples
behave similarly. Miyauchi et al. [27] studied the recovery after UV irradiation of TiO2
samples by irradiation with VIS-IR light (above 430 nm), which can not be absorbed by
TiO2, for a fixed time, without photoexcitation. That process accelerates the elimination
of surface hydroxyl groups, resulting in a quickly conversion of the hydrophilic surfaces
to hydrophobic. They concluded that the hydrophobic conversion without an inter-band
transition is caused by the elimination of surface hydroxyl groups by the thermal
process, but the structure below the outermost layer, where most of the electron-hole
pairs are generated, remains stable free from changes in chemical conditions. In our
0
15
30
45
60
75
90
0 1 2 3 4
number of recovery cycles
w
(º)
71
case, it can be expected that the first recovery cycle was able to remove the hydroxyl
groups adsorbed on surface, and also it helps to reconvert part of the structure below of
our samples.
The similar behaviour of both samples on recovery can be seen on Fig. 3.8 where cos F
and cos D have been plotted against cos W for each of the recovery cycles. Also, it has
been found that cos F and cos D change linearly with cos W accordingly to
cos θF = 0,33 + 0,72 cos θW (r2 = 0,97)
cos θD = 0,58 + 0,25 cos θW (r2 = 0,87) (3.4)
These linear relationships (also plotted in Fig. 3.8) are very similar to those found for
the contact angles measured during the UV irradiation, but with a lower slope in the
case of formamide and a bigger dispersion of the contact angles in the case of
diiodomethane. However these differences can not be related to physical changes on the
evolution of the samples surfaces, or to differences between samples, including
differences in Ra as Miyauchi et al. [27] found differences in the hydrophobic
conversion depending on the roughness Ra of the samples. In consequence, from the
results obtained it can be stated that the hydrophobic-hydrophilic modifications are
reversible for these samples, without any evidence of photocorrosion, within the
experimental errors.
Fig. 3.8. Relationship between water-formamide contact angles (continue line, filled symbols)
and water-diiodomethane (doted line, open symbols), for TiAlV (round symbols) and TiAlVOx
(square symbols) in the recovery cycles.
0
0,2
0,4
0,6
0,8
1
0 0,2 0,4 0,6 0,8 1
cos W
co
s
F, co
s
D
72
3.4. CONCLUSIONS
Passive layers formed spontaneously and after thermal treatments, suffer different
modifications of their hydrophilicity and surface Gibbs energy for a given UV
irradiation time. This behaviour could be related to the different structure of the titanium
dioxide and/or to the different amount of alloying elements present in the passive layer.
Nevertheless the modification of the surface Gibbs energy for a given change of the
hydrophilicity of the surface (estimated through the water contact angle) is the same for
both samples, which suggests that the mechanisms by which surfaces became
hydrophilic are reversible and the same for both kinds of samples. This result implies a
less probable influence of the different amount of alloying elements on the UV effect on
these samples.
Interestingly, common linear relationships between the cosines of formamide and
diiodomethane, and water contact angles have been found for both samples. It indicates
that the mechanisms for increasing the hydrophilic character of the surface is the same
in the range studied. Modifications of the surface Gibbs energy comes from the -
parameter of the acid-base component, which is related to the progressive coverage of
the surface with hydroxyl groups and oriented water on the surface.
3.5. REFERENCES
[1] Sitting C, Textor M, Spencer ND, Wieland M, Vallotton PH. Surface
characterization of implant materials c.p. Ti, Ti–6Al–7Nb and Ti–6Al–4V with different
pretreatments. J. Mater. Sci. Mater. M. 1999;10:35-46.
[2] Lilley PA, Walker PS, Blunn GW. Wear of titanium by soft tissue. Transaction of
the 4th Word Biomaterials Congress, Berlin, 1992, p. 227.
[3] Mu Y, Kobayashi T, Sumita M, Yamamoto A, Hanawa T. Metal ion release from
titanium with active oxygen species generated by rat macrophages in vitro. J. Biomed.
Mater. Res. 2000;49:238-243.
[4] MacDonald DE, Rapuano BE, Deo N, Stranick M, Somasundaran P, Boskey AL.
Thermal and chemical modification of titanium-aluminum-vanadium implant materials:
effects on surface properties, glycoprotein adsorption, and MG63 cell attachment.
Biomaterials 2004;25:3135-3146.
73
[5] Saldaña L, Vilaboa N, Vallés G, González-Cabrero G, Munuera L. Osteoblast
response to thermally oxidized Ti6Al4V alloy. J. Biomed. Mater. Res A 2005;73:97-
107.
[6] García-Alonso MC, Saldaña L, Vallés G, González-Carrasco JL, González-Cabrero
J, Martinez ME, Gil-Garay E, Munuera L. In vitro corrosion behaviour and osteoblast
response of thermally oxidised Ti6Al4V alloy. Biomaterials 2003;24:19-26.
[7] Saldaña L, Barranco V, Garcia-Alonso MC, Valles G, Escudero ML, Munuera L,
Vilaboa N. Concentration-dependent effects of titanium and aluminium ions released
from thermally oxidized Ti6Al4V alloy on human osteoblasts. J. Biomed. Mater. Res. A
2006;77:220-229.
[8] Vörös J, Wieland N, Ruiz-Taylor L, Textor M, Brunette DM, in Brunette DM,
Tengvall P, Textor M, Thomsen P (Eds.), Springer-Verlag, Berlin, 2001, p. 87.
[9] U. Diebold. The surface science of titanium dioxide. Surf. Sci. Rep. 2003;48:53-229.
[10] Textor M, Sittig C, Frauchiger V, Tosatti S, Brunette DM, in Brunette DM,
Tengvall P, Textor M, Thomsen P (Eds.), Springer-Verlag, Berlin, 2001, p. 172-224.
[11] Watanabe T, Nakajima A, Wang R, Minabe M, Koizumi S, Fusjishima A,
Hasimoto K. Photocatalytic activity and photoinduced hydrophilicity of titanium
dioxide coated glass. Thin Solid Films 1999;351:260-263.
[12] Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J, Cochran DL, Boyan
BD. High surface energy enhances cell response to titanium substrate microstructure. J.
Biomed. Mater. Res. A 2005;74:49-58.
[13] van Oss CJ, Good RJ, Busscher HJ. Estimation of the polar surface tension
parameters of glycerol and formamide, for use in contact angle measurements on polar
solids. J. Disper. Sci. Technol. 1990;11:75-81.
[14] Jańczuk B., Kerkeb M.L., Białopiotrowicz T., González-Caballero F. Surface free
energy of cholesterol and bile salts from contact angles. J. Colloid Interf. Sci.
1992;151:333-342.
[15] Ask M, Lausmaa J, Kasemo B. A surface spectroscopic study of nitrogen-
implanted Ti and Ti6Al4V wear against UHMWPE. Appl. Surf. Sci. 1989;35:283-336.
74
[16] Hernández de Gatica NL, Jones GL, Gardella Jr. JA. Surface characterization of
titanium alloys sterilized for biomedical applications. Appl. Surf. Sci. 1993;68:107-120.
[17] Roessler S, Zimmermann R, Scharnweber D, Werner C, Worch H. Characterization
of oxide layers on Ti6Al4V and titanium by streaming potential and streaming current
measurements. Colloid Surface B 2002;26:387-395.
[18] Méndez-Vilas A, Bruque JM, González-Martín ML. Sensitivity of surface
roughness parameters to changes in the density scanning points in multi-scale afm
studies. Aplication to a biomaterial surface. Ultramicroscopy 2007;107:617-625.
[19] Wenzel RN. Resistance of solid surfaces to wetting by water. Ind. Eng. Chem.
Fund. 1936;28:988-994.
[20] Bren L, English L, Fogarty J, Policor R, Zsidi A, Vance J, Drelich J, Istephanous
N, Rohly K. Hydrophilic∕electron-acceptor surface properties of metallic biomaterials
and their effect on osteoblast cell activity. J. Adhes. Sci. Technol. 2004;18:1711-1722.
[21] Kamusewitz H, Possart W. Wetting and scanning force microscopy on rough
polymer surfaces: Wenzel`s roughness factor and the thermodynamic contact angle.
Appl. Phys. A 2003;76:899-902.
[22] Kern T, Yang Y, Glover R, Ong JL. Effect of heat-treated titanium surfaces on
protein adsorption and osteoblast precursor cell initial attachment. Implant Dent.
2005;14:70-76.
[23] Jańczuk B, Bruque JM, González-Martín ML, Moreno del Pozo J. Determination
of Components of Cassiterite Surface Free Energy from Contact Angle Measurements.
J. Colloid Interf. Sci. 1993;161:209-222.
[24] Henderson MA. The interaction of water with solid surfaces: fundamental aspects
revisited. Surf. Sci. Rep. 2002;46:1-308.
[25] Xiao S-J, Kenausis G, Textor M, in Brunette DM, Tengvall T, Textor M, Thomsen
P (Eds.), Springer-Verlag, Berlin, 2001, p. 417.
[26] Adamson AW, Gastz AP, Physical Chemistry of Surfaces, Wiley-Interscience,
New York, 1997.
75
[27] Miyauchi M, Kieda N, Hishita S, Mitsuhashi T, Nakajima A, Watanabe T,
Hashimoto K. Reversible wettability control of TiO2 surface by light irradiation. Surf.
Sci. 2002;511:401-407.
76
CAPÍTULO 4
Influence of slight microstructural gradients
on the surface properties of Ti6Al4V
irradiated by UV
Este capítulo es una reproducción de la publicación: "A.M. Gallardo-Moreno, M.
Multigner, M.A. Pacha Olivenza, M. Lieblich, J.A. Jiménez, J.L. González-Carrasco,
M.L. González-Martín. Journal of Colloid and Interface Science 2008;320:117-124",
realizada en colaboración con el grupo del Dr. J.L. González-Carrasco del Centro
Nacional de Investigaciones Metalúrgicas, CENIM-CSIC (Madrid).
77
ABSTRACT
Ti6Al4V alloy is one of the most widely used materials for biomedical implants.
Among its properties, it is remarkable the photoactivity displayed by its passive layer,
which is mainly composed by titanium dioxide. However, variations in the processing
conditions may yield to differences in the microstructure which can be reflected on the
surface properties of the machined product. From contact angle measurements on
different zones of discs cut from a commercial bar of Ti6Al4V, it has been shown that
the modifications of the surface Gibbs energy suffered by the alloy under UV irradiation
have a radial dependence. This behaviour is related to slight microstructural changes of
the alloy, particularly with an increase in the volume fraction of the β-phase when
moving to the interior of the specimen, altering the composition and/or microstructure
of the passive layer along the radius of the discs. This study shows that gradients in the
microstructure and physical properties are sample size dependent and are likely related
to thermal gradients during processing.
4.1. INTRODUCTION
Ti6Al4V ELI (Extra-Low Interstitial) is commonly used as biomaterial, particularly for
implantable components, due to the combination of good mechanical properties with a
reasonable biocompatibility. This alloy is a high purity version of the Ti6Al4V
originally developed for aerospace applications. The lower specified limits for iron and
the interstitial elements C and O confers to the alloy superior damage tolerance (fracture
toughness and fatigue crack growth rate) and better mechanical properties at cryogenic
temperatures. An additional advantage is that it is easy to process and machine, thus a
wide range of products (bars, plates, tubes, sheets,..) are available with a reasonable
cost. Processing route and dimensions of Ti6Al4V ELI wrought products are obviously
selected considering factors like shape and size of the components. Variations in the
processing conditions (temperature, annealing time, cooling rate) may yield differences
in the microstructure that are also manifested in the mechanical properties of the bulk
[1]. Therefore, properties can vary from one product to other and often mechanical
properties are only valid for a certain range of products and dimensions.
In absence of compositional variations, changes in the microstructure should not
influence the biological performance since it is controlled by the surface properties.
However, recent studies performed with ultrafine grained Commercially Pure (CP) Ti
78
[2] and Ti6Al4V [3] have shown an enhanced osteoblast adhesion, compared to a
regular grained counterpart. The advantages of an ultrafine grained microstructure in Ti
alloys have been also pointed out in terms of corrosion resistance [4]. More recently, it
has been reported that the presence of a predominant crystallographic orientation of the
substrate, i.e. texture, may affect the pre-osteoblast response of pure Ti [5] and the
Ti6Al4V alloy [6].
Surface of Ti6Al4V alloy, as other Ti-base materials, is covered by a passive layer of
amorphous TiO2 that it is responsible for most of the alloy surface properties. Especially
relevant is that TiO2 is a semiconductor whose band gap can be overcome under UV
radiation. In a previous study we showed that the extension of wettability and surface
Gibbs energy modification on the Ti6Al4V surface is depending on the exposure time to
UV irradiation, until a complete wettability is reached [7]. For a given irradiation time,
the wettability was different depending on the thermal oxidation process applied to the
surface, which affects the microcrystalline structure of the titanium dioxide layer. This
behaviour was related to the fact that the electronic gap of titanium dioxide is slightly
different depending on the crystalline form of the oxide, that is, rutile, anatase or
brookite. Relevance of this finding for the biomaterials field is that an increased surface
wettability or hydrophilicity would enhance protein adsorption [8] and cell response [9-
11]. In a recent work of the group it has been demonstrated that UV irradiation of
Ti6Al4V decreases the bacterial adhesion strength without compromise the
biocompatibility in vitro, which enhances the potential of this technique as a part of the
production process prior for instance to the implantation event [12].
The effect of UV on the wettability was confirmed in small discs (20 mm in diameter)
and large discs (50 mm in diameter). However, only in the latter case, for a given
irradiation time, wettability variations were observed from one place to other of the
same sample. This feature triggered off a systematic study of the physical surface
properties in the radial direction of large discs. The aim of this study is to correlate
changes in wettability and surface Gibbs energy with the microstructure and
microhardness of the underlying substrate. A deeper knowledge of this issue should be
obviously relevant when considering performance of relative large components.
79
4.2. MATERIALS AND METHODS
4.2.1. Material
Hot rolled and annealed bar of Ti6Al4V ELI used for medical applications was kindly
delivered by SURGIVAL S.A. (Valencia, Spain). According to the product certificate,
the billet of 50 mm in diameter and 3800 mm long was annealed at 700 ºC for 2 h, and
then air cooled. The chemical composition supplied with the product certificate is
shown in Table 4.1. The scatter of the values observed in these tables was related to
differences found between samples taken from the top or bottom of the bar.
Table 4.1. Heat chemical analysis (mass %). Data supplied by the alloy manufacturer.
Ti V Al Fe C O
Bal. 4.10 - 4.15 6.07 - 6.09 0.17 0.005 - 0.01 0.12 - 0.13
4.2.2. Microstructural characterization
Slices of Ti6Al4V bar were cut, grinded and polished down to a mirror like finished by
using a final step with silice colloidal. Some samples were also etched with a solution
containing 2 ml FH, 3 ml ClH and 5 ml NO3H in 90 ml H2O to reveal the -phase.
Microstructural characterization was performed by using a scanning electron
microscope (SEM) equipped with a field emission gun (FEG) emitter coupled with an
energy dispersive X-ray (EDX) system for chemical analysis. To minimize the
uncertainties of the EDX measurements several zones were analysed with acquisition
times of about 100 s. Depending on the analysis, secondary (SEI) or backscattered
electron images (BEI) were selected.
Volume fraction of the -phase was determined by using the image analysis software
image-Pro Plus. BEI images were obtained at a magnification of X3000 on longitudinal
sections of specimens in the as-polished condition. Etched samples were not suitable for
this evaluation because they lead to overestimation of -phase, as they protrude above
the α-matrix. Five random pictures from both positions, the centre and close to the
border of the bar, were digitally treated to remove noise and to get a black and white
contrast.
Samples for X-Ray Diffraction (XRD) studies were cut perpendicular to the
longitudinal bar direction. These samples were grinded and fine mechanically polished
with 1 micron diamond paste and colloidal silica (400 nm) to remove the deformed
80
layer. In order to evaluate changes in texture XRD measurements were performed at the
centre and near the edge of the bar. The X-ray diffractometer was equipped with a
single Goebel mirror and an incident beam collimator of 1 mm diameter. The sample
alignment and area selection for the measurements was performed with the help of a
video microscope and a focussed laser. For evaluation of the phases present, the
measurements were performed under a conventional /2 scanning using a point
scintillation detector. In this study, we have used the version 4.0 of Rietveld analysis
program TOPAS (Bruker AXS) for the XRD data refinement. Refinement analyses
were carried out using space groups and crystallographic information from the ICDD
database of the phases previously identified. The refinement protocol included also the
background, the scale factors, and the global-instrumental, lattice, profile and texture
parameters.
Texture measurements were performed in the back-reflected mode using a HI-STAR
two dimensional multiwire proportional counter (General Area Detector Diffraction
System, Bruker AXS). A Cu-tube operating at 40 kV and 30 mA was used. The detector
was centred at 2 = 37.60º and 61.80º, with the centre of the detector position
coupled with the beam’s incident angle . At each 2 position enough planar frames
were collected for covering the entire pole sphere. From the normalized and corrected
X-ray data, the (100), (002), (101), (102), (110) and (103) pole figures were
reconstructed. The sample reference system was fibre, corresponding to the symmetry
imposed by the extrusion process. The incomplete pole figures were smoothed, and it
was recalculated the whole pole figures using the harmonic series expansion method (l
max = 22) and ghost correction.
Mechanical properties were evaluated by Vicker hardness tests performed on polished
surfaces. Indentations were made with 1-kg load and 15-seconds dwell time.
4.2.3. Surface wettability characterization
Wettability and surface Gibbs energy of the samples were evaluated from contact angle
measurements of sessile drops of water (W), distilled and deionized from a Milli-Q Plus
system, formamide (F), puriss > 99.0%, Fluka, Switzerland and diiodomethane (D),
purum > 98%, Fluka, Switzerland, with the aid of a G20 goniometer (Krüss, Germany).
Surface Gibbs energy and its components and parameters of these liquids are listed in
Table 4.2. [13,14] To study the possible dependence of the liquid contact angles on the
81
microestructural characteristics of the sample, drops of each one of the three liquids
were placed on three different concentric zones of the disc. Zone A, a circle of radius
0.8 cm, refers to the most inner area; zone B, an annulus of radius 0.8 and 1.6 cm,
corresponds to the intermediate area of the disc and zone C, an annulus of radius 1.6 cm
and 2.3 cm, matches the external area of the disc. To avoid edge effects on the contact
angle measurements, the outermost annulus of radius 2.3 cm and 2.5 cm was not tested.
Results of contact angle are averaged values of at least 30 symmetrical drops measured
on at least three different discs.
Table 4.2. Surface tension, , Lifshitz-van der Waals, LW, and acid-base, AB
, components,
electron-acceptor, +, and electron-donor, -
, parameters of the test liquids used for contact
angle measurements, in mJ.m-2
.
LW
AB
+
- Ref.
Water 72.8 21.8 51.0 25.5 25.5 [13]
Formamide 58 39 19 2.28 39.6 [13]
Diiodomethane 50.8 50.8 0 0.72 0 [14]
Before each experimental run, samples were carefully cleaned with distilled water at
60ºC, vigorously rubbed with a smooth cotton cloth, then rinsed repeatedly, first with
distilled water and finally with distilled and deionized water; immersed in a beaker with
distilled and deionized water and sonicated for 10 min, rinsed again with distilled and
deionized water, dried in an oven at 40ºC for 1h and stored in a desiccator for no more
than 24 h. [7]
Ultraviolet irradiation was carried out with UV lamps kindly provided by Philips (TUV
TL-D 15W SLV), emitting predominantly at a wavelength of 257.7 nm. Glass of the
lamp has a filter that avoids the production of ozone, which is produced by wavelengths
lower than 200 nm. For irradiation the discs were centred 10 cm below the lamp,
receiving an intensity of 2.6 mW cm-2
. The whole irradiation installation was inside an
opaque wood chamber to avoid interferences from room or day light and any damage to
users. Depending on the experiment, Ti6Al4V discs were irradiated for 20 min, 40 min
or 60 min.
82
4.3. RESULTS AND DISCUSSION
Wettability was measured on the three zones of the disc previously described. Before
UV irradiation contact angles of the three liquids did not show any dependence on the
zone of the disc tested (Fig. 4.1); for water they were comprised between 82±2º and
84±2º, corresponding to a hydrophobic surface, for formamide between 68±2º and
69±2º, and for diiodomethane between 53±2º and 54±2º. After exposition to UV
radiation (Fig. 4.1), water contact angle decreases in a way depending on the length of
the exposure time and on the zone of the disc tested, being this diminution greater on
the inner than the outer zones of the discs. A similar behaviour was observed for
formamide and diiodomethane contact angles, although the radial dependence was weak
for diiodomethane. The lowest differences between the inner and the outer zones were
observed after being the samples under UV irradiation during the longest period studied.
Fig. 4.1. Contact angle of water (θW), formamide (θF), and diiodomethane (θD), measured on
the three areas of the disc before UV radiation (♦) and after 20 min (), 40 min () and 60 min
() exposure to UV radiation.
Despite wettability modifications are relevant, surface Gibbs energy allows a deeper
knowledge of the capability of the surface to interact with its surrounding. These
interactions are mediated by van der Waals and short range forces, as those coming
from acid-base interactions. The van Oss approach [13, 15-19] considers total surface
Gibbs energy, γ, as the addition of two independent contributions coming from
noncovalent long-range Lifshitz-van der Waals interactions, γLW
, and from Lewis acid-
base interactions, γAB
,
0
30
60
90
-0,5 0,5 1,5 2,5
W
(º)
A B C
zone zone zone
0
30
60
90
-0,5 0,5 1,5 2,5
F
(º)
A B C
zone zone zone
30
40
50
60
-0,5 0,5 1,5 2,5
D
(º)
A B C
zone zone zone
83
LW AB (4.1)
and it assumes that the γAB
component can be expressed as a function of the geometric
mean of an electron-acceptor, γ+, and an electron-donor, γ
-, parameters
1 2
AB + -γ = 2 γ γ (4.2)
Then, on the frame of the van Oss approach, the interfacial solid-liquid Gibbs energy is
given by
1 2 1 2 1 2
LW LW + - - +
SL S L S L S L S Lγ = γ + γ -2 γ γ - 2 γ γ - 2 γ γ (4.3)
From Eq (4.3) and Young’s equation, it is possible to evaluate the surface Gibbs energy,
and get insight on the contribution of each of these interactions to the total surface
Gibbs energy of the solid, as it is expressed in Eq (4.4).
1 2 1 2 1 2
LW LW + - - +
L L L S L S L S Lγ cosθ +1 +Πe = 2 γ γ +2 γ γ +2 γ γ (4.4)
If the surface pressure, ΠeL, value is known, it is possible to determine from Eq. (4.4)
the Gibbs surface energy components of a solid from the measurement of the contact
angles θL of three different liquids of known surface tension components and
parameters, by solving a three equations system in the unknowns γLW
, γ+ and γ
-.
Commonly it is assumed that if θL>0 the ΠeL value can be neglected [13, 15-19].
Applying Eq. (4.4) to water, formamide and diiodomethane, the following equations
system is obtained
1/2 1/2 1/2
LW LW
W W S W S W S Wcos 1 2 2 2
1/2 1/2 1/2
LW LW
F F S F S F S Fcos 1 2 2 2
1/2 1/2
LW LW
D D S D S Dcos 1 2 2 (4.5)
From Eqs. (4.1), (4.2) and (4.5) the total solid surface Gibbs energy and its Lifshitz-van
der Waals and acid-base components, and its electron-donor and electron-aceptor
parameters have been determined and plotted in Fig. 4.2 for each zone of the disc after
84
each period of UV irradiation. This figure clearly shows that the modification of the
surface Gibbs energy by the UV irradiation is consequence of the changes of the acid-
base component, being the long-range, Lifshtiz-van der Waals forces, nearly unaffected
by irradiation.
Fig. 4.2. Surface Gibbs energy (γ), Lifshitz-van der Waals component (γLW
, open symbols), acid-
base component (γAB
, closed symbols) and its parameters electron-acceptor (γ+, open symbols),
and electron-donor (γ-, closed symbols) of the three areas of the disc before UV radiation (♦)
and after 20 min (), 40 min () and 60 min () exposure to UV radiation.
20
40
60
-0,5 0,5 1,5 2,5
(m
J m
-2)
A B C
zone zone zone
0
10
20
30
40
-0,5 0,5 1,5 2,5
L
W,
AB(m
J m
-2)
A B C
zone zone zone
0
20
40
60
-0,5 0,5 1,5 2,5
+
,
- (m
J m
-2)
A B C
zone zone zone
85
On the other hand, the acid-base component modification is a consequence of the
change of the electron-donor parameter, since the electron-acceptor parameter is nearly
unaffected by the UV irradiation. This behaviour is in accordance with a progressive
surface coverage with hydroxyl groups or chemisorbed water in a preferential
orientation [20, 21].
These results show that before to UV irradiation, the alloy surface presents a low and
uniform energetic value, ca. 30 mJm-2
, probably due to the unavoidable organic
contamination from the surrounding. UV treatment activates the surface considerably,
turning it into a high energetic surface after 60 min of irradiation, with surface Gibbs
energy values near to 60 mJm-2
for any of the measured zones, within experimental
error. However, for shorter irradiation times, it is interesting to note that zone A gets a
more energetic state than zone B or zone C. After 20 min irradiation, the most internal
zone (A) reaches a value of 46±3 mJm-2
but the intermediate and external zones (B and
C) get both the same value of 40±3 mJm-2
. After 40 min irradiation, surface Gibbs
energy in zone A increases up to 54±3 mJm-2
, in zone B up to 50±4 mJm-2
but in the
most external zone C it is not possible to detect variations in its surface Gibbs energy in
respect to its value after 20 min of UV irradiation.
Variation of the physical surface properties with UV irradiation is obviously related to
the photocatalytic activity of titanium dioxide forming the passive layer. However, a
question still arises about the microstructural reasons behind the change in the physical
surface properties along the radial direction. First evidence of microstructural gradients
within the samples was provided by the hardness experiments. Fig. 4.3 shows the
hardness variation as a function of depth in the radial direction. Data for smaller discs of
20 mm in diameter are also included for comparative purposes. As it can be seen,
hardness of the 50 mm discs decreases when moving to the interior, being differences at
the centre out of the scatter band of results. However, the tests on the smaller discs
failed to show any significant variation in hardness, which denotes that variation in
hardness on large discs is a size sensitive effect. The observed trend in the large discs
was confirmed by employing different discs of the same diameter coming from different
positions of the same bar, which discards the influence of any artefact introduced during
the preparation of the discs for these determinations.
86
0 5 10 15 20 25220
240
260
280
300
320
R=10 mm
R=25 mm
Vic
ker
s H
ard
nes
s (H
V1
)
radious(mm)
Fig. 4.3. Hardness Vickers as a function of depth for Ti6Al4V discs removed from bars of
different diameters.
Interpretation of the decrease in hardness in the centre of the specimen is complex since
several causes for softening are possible. Ti6Al4V is a biphasic alloy, therefore, in
addition to the effect of grain size and crystallographic texture, factors related to the
discontinuous -phase (size, volume fraction, and dispersion) should be considered.
Fig. 4.4 shows representative SEM images of both cross and longitudinal sections. As it
can be seen, the alloy exhibits the expected biphasic microstructure, consisting of
(dark zones) and (bright zones) phases arranged in a cellular way. Cross sectional
views at the border and centre of the specimens reveals a quite uniform equiaxial
microstructure with a similar values for the equivalent grain size of about 10 m (Fig.
4.4a and 4.4b). However, longitudinal sections (Fig. 4.4c and 4.4d) evidence clear
differences in the -phase distribution and size between areas located at the centre of the
bar (Fig. 4.4c) and close to the border (Fig. 4.4d). In the later zone, the -phase areas
are more elongated and the i particles smaller.
Recent nanoindentation experiments performed by Rack and Qazi in an ultrafine
grained Ti-6Al-4V alloy have shown that hardness of the phase is lower than that of
the phase (up to about 25% less), irrespective of the annealing temperature [22].
Therefore, an initial possible explanation for the gradient in hardness concerns the
variation in the radial direction of the volume fraction of phase. To assess this
consideration, a quantitative metallographic analysis was performed on both cross and
87
longitudinal views, but only results corresponding to the longitudinal sections will be
presented (Fig. 4.5).
Fig. 4.4. SEI (a,b) and BEI (c,d) images corresponding to the centre (a,c) and border (b,d) of
selected cross (a,b) and longitudinal (c,d) sections. SEI images (a,b) were obtained on polished
and etched specimens.
0 2 4 6 8 10 12 14 16 18 20 220
10
20
30
40
50
60
70
80
90
100
% P
art
icle
s
Particle size (m2)
CENTRE
0 2 4 6 8 10 12 14 16 18 20 220
10
20
30
40
50
60
70
80
90
100
% P
arti
cles
Particle size (m2)
BORDER
Fig. 4.5. Distribution and size of the -particles at the centre and border of the discs.
(a)
=
(b)
=
(c)
=
(d)
=
88
As it can be seen, the distribution and size of the particles slightly differs from the
centre to the border of the bar. Determination of the volume fraction of the -phase in a
radial direction revealed moderated higher values at the centre, 9.5 ± 1.8%, than in areas
close to the border, 7.8 ± 0.8%. Both values are in the range predicted by the
equilibrium diagram, and in good agreement with the values determined by the Rietveld
refinement of the X ray diffraction patterns obtained from the centre and near the bar
edge. Fig. 4.6 shows that in both zones the material mainly consists of -Ti and a small
amount of -Ti. The intensity of the (110) -Ti peak is lower in the pattern obtained
close to the edge, and would indicate a lower volume fraction of this phase. However,
again only a slight difference of -Ti volume fraction was determined from these
patterns by the Rietveld refinement (10.5% and 9.5% at the centre and close to the
border, respectively).
Fig. 4.6. X-ray diffraction patterns of a slice of Ti6Al4V bar recorded near the border and in the
centre.
This difference in the volume fraction of phase along the radial direction could result
from segregations in the chemical composition. Consequently microanalysis was
performed on several small areas of about 600 µm2
placed close to the border (5 mm
far) and around the centre of the specimen. Additionally, point microanalysis was
performed on selected zones where size was large enough (> 1µm) to minimize
contribution of the matrix ( phase). Average values are presented in Table 4.3. As it
can be seen, chemical composition obtained by microanalysis fits with the nominal
values for the bulk alloy. With regards to the composition of the phase, vanadium
content is well above that of the nominal composition, as expected. Differences in the
89
vanadium content (stabilizer element) are slightly higher in the centre of the
specimen, which is counterbalanced by a moderated decrease in the Ti content.
However, due to the scatter band this result cannot be conclusive. The high scattering
may result from the relative small size of the phase, which makes difficult to avoid the
contribution of the matrix (phase).
Table 4.3. Chemical composition (wt%) determined by EDS
Another factor that could be responsible for variations in the Vickers hardness across
the diameter is a difference in crystallographic texture. The phase pole figures
measured at the centre and near the bar edge show that the texture component is the
same in both cases, but slightly stronger in intensity near the edge, as observed in Fig.
4.7. This figure shows a well-defined fiber texture with the plane normal direction (1 0
0) aligned with the longitudinal axis.
From the above analyses if follows that the observed variation in hardness in the radial
direction can be mostly related to the slight increase in the volume fraction of the -
phase and the bigger size of the Ti particles in the disc interior. Significant changes
in the chemical composition of the phase were not observed and, therefore, changes
in the volume fraction associated to a microchemical segregation cannot be invoked. In
consequence the different behaviour of the surface Gibbs energy of each of the three
studied zones against UV irradiation suggests that the passive layer changes its
composition and/or microstructure along the radius of the disc. It is known that the
presence of alloying elements can modify the surface properties of the passive layer in
respect to cp Ti. It can be considered the possibility that differences in α and β phases,
[23] and then the Al and V concentration differences can affect the surface properties as
these elements can be displaced to the passive layer.
Zone microanalysis - phase
Ti Al V Ti Al V
Border 90.20 ± 0.08 6.10 ± 0.11 3.71 ± 0.16 82.43 ± 0.62 3.80 ± 0.14 13.77± 0.73
Centre 89.94 ± 0.08 6.07 ± 0.08 3.9 ± 0.11 82.89 ± 1.10 3.92 ± 0.13 13.19 ±1.20
90
Fig. 4.7. Recalculated (100) pole figure of a slice of Ti6Al4V bar (a) near the border and (b) in
the centre.
Microstructural gradients can be associated to the processing history, although single
valued functions remain unclear. Optimization of the mechanical properties relies on an
appropriated control of the microstructure, particularly the grain size, the alpha/beta
volume fraction and morphology. Overall, varying the solution annealing temperature,
duration, cooling rate and final aging temperature a lamellar, equiaxed or bimodal
91
microstructure can be developed. Advantages of each microstructure in terms of
strength, ductility and fracture toughness have been recently summarized by Rack and
Qazi. [22] It is well known that cooling method may affect tensile strength after
annealing and aging [1] giving rise to complex phase transformation in Ti-based alloys.
Here it is worth to remark that all thermal treatments involve an stage around the
transus (970-980ºC) and a further stage around 700ºC for relative short exposure times.
It is very well known that thermal conductivity of Ti6Al4V is rather low. Therefore, a
different thermal cycling can be experienced at the centre of the specimens. The absence
of such radial dependence in a disc of smaller (20 mm) diameter having a similar
microstructure indicates that the observed effect is size dependent.
4.4. CONCLUSIONS
Thermal gradients during processing of wide bars of Ti6Al4V are likely responsible of
the slight gradients found in its microstructure, particularly of an increase in the volume
fraction of the -phase (V-rich) when moving to the interior of the specimen. This
feature has been found to affect the photoactivity of the titanium dioxide passive layer
on the surface of discs cut from wide bars. In consequence, the rate at which wettability
and surface Gibbs energy of discs change after being exposed to UV radiation, mainly
due to the modification suffered by the acid-base component of the surface Gibbs
energy, is dependent on the distance to the centre of the disc. However, if the exposure
time of the samples to the UV radiation is longer enough, the observed differences in
wettability and surface Gibbs energy along the radius of discs tend to be softened.
4.5. REFERENCES
[1] da Rocha SS, Adabo GL, Vaz LG, Henriques GE. Effect of thermal treatments on
tensile strength of commercially cast pure titanium and Ti-6Al-4V alloys. J. Mater. Sci.
Mater. M. 2005;16:759-766.
[2] Faghhi S, Zhilyaev AP, Szpunar JA, Azari F, Vali H, Tabrizian M. Nanostructuring
of a Titanium Material by High-Pressure Torsion Improves Pre-Osteoblast Attachment.
Adv. Mater. Res. 2007;19:1069-1073.
92
[3] Yao C, Qazi JL, Rack HJ, Slamovich EB, Webster TJ. Improved bone cell adhesion
on ultrafine grained titanium and Ti6Al4V. Ceramic Nanomaterials and Nanotecnology
III, 106th Acers Transactions, USA, 2004, p. 159.
[4] Balyanov A, Kutnyakova J, Amirkhanova NA, Stolyarov VV, Valiev RZ, Liao XZ,
Zhao YH, Jiang YB, Xu HF, Lowe TC, Zhu YT. Corrosion Resistance of Ultrafine
Grained Ti. Scripta Mater. 2004;51:225-229.
[5] Faghihi S, Azari F, Bateni MR, Szpunar JA, Vali H, Tabrizian M. A prospective
study to improve osseointegration of Cp-titanium considering its crystallographic
textura. Thirtieth annual meeting and exposition of Society of Biomaterials (SFB),
Menphis, TN, USA, 2005.
[6] Faghihi S, Azari F, Li H, Bateni MR, Szpunar JA, Vali H, Tabrizian M. The
significance of crystallographic texture of titanium alloy substrates on pre-osteoblast
responses. Biomaterials 2006;27:3532-3539.
[7] Pacha-Olivenza MA, Gallardo-Moreno AM, Mendez-Vilas A, Bruque JM,
González-Carrasco JL, González-Martín ML. Effect of UV irradiation on the surface
Gibbs energy of Ti6Al4V and thermally oxidized Ti6Al4V. J. Colloid Interf. Sci.
2008;320:117-124.
[8] Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R. Specific proteins mediate
enhanced osteoblast adhesion on nanophase ceramics. J. Biomed. Mater. Res.
2000;51:475-483.
[9] Altankov G, Grinnell F, Groth T. Studies on the biocompatibility of materials:
fibroblast reorganization of substratum-bound fibronectin on surfaces varying in
wettability. J. Biomed. Mater. Res. 1996;30:385-391.
[10] Schakenraad JM, Busscher HJ, Wildevuur CR, Arends J. The influence of
substratum surface free energy on growth and spreading of human fibroblasts in the
presence and absence of serum proteins. J. Biomed. Mater. Res. 1986;20:773784.
[11] Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J,Cochran DL, Boyan
BD. High surface energy enhances cell response to titanium substrate microstructure. J.
Biomed. Mater. Res. A 2005;74:49-58.
93
[12] Gallardo-Moreno AM, Pacha-Olivenza MA, Saldaña L, Pérez-Giraldo C, Bruque
JM, Vilaboa N, González-Martín ML. In vitro biocompatibility and bacterial adhesion
of physicochemically modified Ti6Al4V surface by means of UV irradiation. Acta
Biomater. 2009:5:181-192.
[13] van Oss CJ, Good RJ, BusscherH. Estimation on the polar surface tension
parameters of glycerol and formamide, for use in contact angle measurements on polar
solids. J. Disper. Sci. Technol. 1990;11:75-81.
[14] Jańczuk B., Kerkeb M.L., Białopiotrowicz T., González-Caballero F. Surface free
energy of cholesterol and bile salts from contact angles. J. Colloid Interf. Sci.
1992;151:333-342.
[15] van Oss CJ, Good RJ, Chaudhury ML. Mechanism of DNA (southern) and Protein
(western) Blotting on Cellulose Nitrate and Other Membranes. J. Chromatogr.
1987;391:53-65.
[16] van Oss CJ, Chaudhury MK, Good RJ. Interfacial Lifshitz-van der Waals and polar
interactions in macroscopic systems. Chem. Rev. 1988;88:927-941.
[17] van Oss CJ, Good RJ. On the mechanism of “hydrophobic” interactions. J. Disper.
Sci. Technol. 1988;9:355-362.
[18] van Oss CJ, Ju L, Chaudhury MK, Good RJ. Estimation of the Polar Parameters of
the Surface Tensions of Water by Contact Angle Measurements on Gels. J. Colloid
Interf. Sci. 1989;128:313-319.
[19] Good RJ, Chaudhury MK, van Oss CJ in: Lee LH (Ed.), Plenum, New York, 1991,
p. 153.
[20] U. Diebold. The surface science of titanium dioxide. Surf. Sci. Rep. 2003;48:53-
229.
[21] Henderson MA. The interaction of water with solid surfaces: Fundamental aspects
revisited. Surf. Sci. Rep. 2002;46:1-8.
[22] Rack HJ, Qazi JI. Titanium alloys for biomedical applications. Mater. Sci. Eng. C
2006;26:1269-1277.
94
[23] Sittig C, Hähner G, Marti A, Textor M, Spencer ND, Hauert R. The Implant
Material, Ti6Al7Nb: Surface Microstructure, Composition and Properties. J. Mater. Sci.
Mater. M. 1999;10:191-198.
95
CAPÍTULO 5
In vitro biocompatibility and bacterial
adhesion of physico-chemically modified
Ti6Al4V surface by means of UV irradiation
Este capítulo es una reproducción de la publicación: "A.M. Gallardo-Moreno, M.A.
Pacha Olivenza, L. Saldaña, C. Pérez-Giraldo, J.M. Bruque, N. Vilaboa, M.L.
González-Martín. Acta Biomaterialia 2009;5(1):181-192", realizada en colaboración
con el grupo del Dr. J.L. González-Carrasco del Centro Nacional de Investigaciones
Metalúrgicas, CENIM-CSIC (Madrid) y el grupo de la Dra. N. Vilaboa del Hospital
Universitario La Paz, FIB-HULP (Madrid).
96
ABSTRACT
UV irradiation leads to a “spontaneous” wettability increase of the Ti6Al4V surface
while preserving bulk properties of the alloy that are crucial for its performance as an
orthopaedic and dental implant. We hypothesized that UV treatment of Ti6Al4V may
impair bacterial adhesion without compromising the good response of human bone-
forming cells to this alloy. The in vitro biocompatibility of the Ti6Al4V surface, before
and after UV irradiation, was analyzed by using human cells related to the osteoblastic
phenotype. The adhesion processes of bacterial strains related to clinical orthopaedic
infections, i.e. S. aureus and S. epidermidis, were studied theoretically and in vitro,
under dynamic and static conditions as well as in the presence or absence of shear
forces. While human cell adhesion was not altered by UV irradiation of Ti6Al4V alloy,
this treatment reduced not only initial bacterial adhesion rates but also bacterial
adhesion strength to the surface. The analysis of G as a function of the interaction
distance explained the bacterial adhesion-retention processes to the surfaces. Secondary
adhesion minima were responsible for the landing of bacteria onto UV treated or
untreated surfaces. Primary adhesion minima, present in non-irradiated surfaces,
promote a firmer anchorage of bacterial cells, while energy barriers, present in
irradiated surfaces, are responsible for loose adhesion. This study proposes the use of
UV treatment prior to implantation protocols as an easy, economic and effective way of
reducing bacterial adhesion on the Ti6Al4V surface without compromising its excellent
biocompatibility.
5.1. INTRODUCTION
Microbial infection is one of the most destructive complications related to orthopaedic
implants because antimicrobial therapy usually lacks efficacy at the point when the
infection process is detected [1,2]. Controlled antibiotic release from the biomaterial
and antibiotic loading on the biomaterial surface are strategies employed to overcome
this problem, but there is concern about the increased microbial resistance to antibiotics
that these procedures may induce. An alternative approach in the fight against bacterial
adhesion focuses on the modification of physico-chemical surface properties of the
biomaterial, such as hydrophobicity, surface tension and electrical surface potential,
because they are crucial in the initial approach and further retention of bacterial cells to
various surfaces [3-5]. On the other hand, adequate adhesion of osteoblasts and their
progenitors to the implant surface ultimately influences their capacity to proliferate and
97
perform their specific functions. Surface characteristics of materials, such as chemistry
and surface energy, play an essential role in cell adhesion to biomaterials. Thus, any
modification of the surface characteristics introduced in order to diminish adhesion of
microorganisms to a biomaterial should not compromise bone-forming cell adhesion.
The Ti6Al4V alloy is widely used in orthopaedic and dental applications due to its low
density, excellent mechanical and anti-corrosive properties, and good biocompatibility
[6,7]. The spontaneous passivation of this alloy forms a thin outer layer, predominantly
composed of amorphous or poorly recrystallised TiO2. We have recently shown that the
presence of TiO2 changes the physico-chemical surface properties of Ti6Al4V after UV
irradiation [8]. Interestingly, titanium, TiO2 surfaces and anodized titanium alloy with a
thin film of anatase show anti-bacterial properties under UV treatment [9-11]. However,
to date there is no information available on the affinity of human cells and bacteria for
physico-chemically modified Ti6Al4V surfaces after UV treatment. We hypothesized
that UV treatment of Ti6Al4V may impair bacterial adhesion without compromising the
behaviour of human bone-forming cells on this alloy. The present study reports on the
in vitro biocompatibility of the Ti6Al4V surface after UV irradiation by evaluating the
behaviour of three types of human cells related to the osteoblastic phenotype, including
the osteoblastic Saos-2 cell line, mesenchymal cells from bone marrow (hMSC) and
primary osteoblasts (hOB). We address the in vitro initial adhesion behaviour and
further retention of three staphylococci strains directly involved in implant-related
infections, i.e., S. aureus ATCC29213, S. epidermidis ATCC35984 (producer of an
extracellular polysaccharide substance, EPS) and S. epidermidis HAM892 (mutant of S.
epidermidis ATCC35984, non-producer of EPS) [12-14]. The behaviours of human
cells and bacterial strains on the titanium alloy surface after irradiation are extensively
discussed in relation to UV-induced physico-chemical changes on the surface.
5.2. MATERIALS AND METHODS
5.2.1. Ti6Al4V
Discs of Ti6Al4V were cut from bars of 25 mm or 20 mm in diameter kindly supplied
by SURGIVAL S.A. (Valencia, Spain). A TIMETAL 6-4 ELI alloy was processed as a
hot rolled annealed bar at 700ºC for 2 h, then air cooled. The discs were abraded on
successively finer silicon carbide papers, mechanically polished with diamond paste,
and finished with silica colloidal.
98
Prior to their use, the Ti6Al4V discs were carefully cleaned with distilled water at 60ºC,
vigorously rubbed with a smooth cotton cloth, then rinsed repeatedly, first with distilled
water and finally with distilled and deionised water (MilliQ system); they were then
immersed in a beaker with distilled and deionized water and sonicated for 10 min,
rinsed again with distilled and deionized water, dried in an oven at 40ºC for 1 h and
stored in a desiccator for no longer than 24 h. Samples used as controls were not
subjected to further treatment. A second set of samples was exposed to an UV source
for 15 h. This period was sufficient to guarantee a complete hydrophilization of the
surface, as we have recently shown [8]. G15-T8 UV lamps were kindly provided by
Philips (Philips Iberica, Spain). The lamps emitted predominantly at a wavelength of
257.7 nm and the lamp glass avoided the production of ozone, which is produced by
wavelengths lower than 200 nm. The discs were positioned at 10 cm from the light
source and centred, receiving an intensity of 2.6 mW/cm2. The irradiation installation
was inside an opaque wood chamber to prevent interference from the room or daylight,
or damage to the users.
5.2.2. Cell culture
Human osteosarcoma Saos-2 cells (ECACC, Salisbury, Wiltshire, UK) were grown in
DMEM medium supplemented with 10% (v/v) heat-inactivated foetal bovine serum
(FBS), 500 UI/ml of penicillin and 0.1 mg/ml of streptomycin. Human mesenchymal
stem cells from bone marrow (hMSC) were purchased from Cambrex Bio Science
(Verviers, Belgium) and maintained in growth medium (Cambrex). Primary bone cells
(hOB) were obtained from human bone specimens aseptically collected during
orthopaedic knee surgery and were cultured as has been described previously [15]. Bone
samples were obtained from 4 patients aged 725 years old. Each bone sample was
processed in a separated primary culture and experiments were performed using cultures
obtained from independent patients. Patients enrolled in this research signed Informed
Consent and all procedures using human tissue designated "surgical waste" were
approved by the Human Research Committee of Hospital La Paz (Date of Approval: 02-
14-2007). hOB were cultured in DMEM containing 15% (v/v) FBS, 500 UI/ml of
penicillin and 0.1 mg/ml of streptomycin. Cells were maintained at 37°C under 5% CO2
and 95% air in a humidified incubator.
For cell culture, Ti6Al4V discs of 20 mm in diameter were used. Ti6Al4V samples
were routinely sterilised under UV light in a laminar flow hood for 15 min on each side
99
and stored until use. Immediately before use in cell culture experiments, parallel sets of
samples were left untreated or treated with UV light as described above.
5.2.3. Cell spreading assays
Cells were seeded on Ti6Al4V samples treated or untreated with UV light in 12-well
plates (1.5×105 Saos-2 cells/well, 6×10
4 hMSC/well and 6×10
4 hOB/well) as described
above and cultured for 3 or 24 h. Attached cells were fixed with 4% formaldehyde in
PBS and permeabilized with 0.1% Triton X-100 in PBS. The cells were then stained
with PBS containing 410-7
M phalloidine-TRITC (Sigma) to visualize filamentous
actin and with PBS containing 310-6
M 4,6-diamidino-2-phenylindole (DAPI, Sigma)
for nuclear DNA. Cells were examined using a fluorescence microscope (Leica
AF6000, Wetzlar, Germany). High resolution fluorescence images from representative
fields of each sample were captured and digitally deconvolved using Huygens software
(Scientific Volume Imaging, Hilversum, The Netherlands). A total of 30 cells randomly
selected from 5 representative images per sample were manually outlined, and cell areas
were measured using ImageJ v1.34 image analysis software.
5.2.4. Bacterial strains and culture
S. aureus ATCC29213 (S. aureus), S. epidermidis ATCC35984 (S. epidermidis4) and S.
epidermidis HAM892 (S. epidermidis2) were stored at –80 ºC in porous beads
(Microbank, Pro-Lab Diagnostics, Austin, Texas, USA). S. epidermidis HAM892 is a
negative extracellular polysaccharide substance producer (EPS-negative) mutant
derived by acriflavin 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 ºC to obtain cultures. From these cultures, tubes of 4 ml
of Trypticase Soy Broth (TSB) (BBL, Becton Dickinson, Cockeysville, Maryland,
USA) were inoculated for 10 h at 37 ºC and then 25 l of this pre-culture was used
again to inoculate 50 ml of TSB at 37 ºC for 14 h. This amount of time was sufficient to
guarantee that all strains were at the stationary phase of growing (previously checked
with the growing curves for the three strains).
The bacteria were then harvested by centrifugation for 5 min at 1000g (Sorvall TC6,
Dulont, Newtown, Pennsylvania, USA) and washed three times with PBS conditioned
at 37ºC. Then the bacteria were re-suspended in PBS at a concentration of 3108
100
bacteria/ml for flow experiments and contact angle assays, and of approximately 107
bacteria/ml for zeta potential tests.
5.2.5. Bacterial adhesion-retention experiments
Flow experiments were carried out at 37 ºC in a parallel plate flow chamber previously
described in detail [16] by using an inverted metallographic microscope (Olympus
GX51, Barcelona, Spain). Ti6Al4V disks of 25 mm were fixed in the centre of one of
the plates of the chamber to ensure laminar conditions for the liquid flow. The adhesion
process was analyzed by using two different experimental settings. In order to study the
dynamic adhesion of bacteria on Ti6Al4V, the flow chamber was placed so that the
alloy surface was on the top side of the flow channel and the bacteria were allowed to
attach while flowing at a constant rate of 2 ml/min for 20 min. Live adhesion was
followed as time passed by recording the images with a video camera connected to a
computer. Initial adhesion rates (j0) were given as bacteria adhered per square
centimetre and second after counting the adhered bacteria in each image. To evaluate
whether progression of adhesion was modified in static conditions, the alloy sample was
placed on the bottom side of the flow channel, the flow was stopped and the bacteria
were left to deposit on the surface for 2 min. Then the parallel plate flow chamber was
turned upside down in order to take images of the adhered bacteria in different areas of
the sample and to count them. This last procedure was repeated until 20 minutes of
adhesion was reached. At the end of each static adhesion period and after taking images
of the adhered bacteria, the flow was slowly opened in order to homogenize the
bacterial concentration inside the flow channel, and then stopped again. Previous
experiments showed that adhered bacteria were not detached during this process. We
will refer to this last experimental setting as static-time-function. Static-time adhesion
rates (j) were given as bacteria adhered per square centimetre and second.
Once the adhesion process was finished, and with a final number of bacteria nf on the
titanium alloy surface, the surface was exposed to the passing of two liquid-air
interfaces in order to check the strength of the adhesion (bacterial retention). After the
first and second liquid-air interfaces the number of remaining bacteria on the surface
was counted and denoted as nI-1 and nI-2, respectively.
101
5.2.6. Physico-chemical surface characterization
5.2.6.1. Contact angle measurements
For the surface tension evaluation, the contact angles of water (W) (Milli-Q Plus),
formamide (F) (puriss>99.0%, Fluka, Switzerland) and diiodomethane (D)
(purum>98%, Fluka, Switzerland) were determined on the Ti6Al4V surface and on
bacterial lawns using the sessile drop technique.
Bacterial lawns were prepared by filtering a bacterial suspension though 0.45 m pore
size filters (Millipore, Molsheim, France) using negative pressure [17]. Filters were left
to air dry at 37 ºC for 60 min for S. aureus, and 30 min for S. epidermidis4 and S.
epidermidis2. The time for drying was the time at which residual PBS disappeared
between the cells and the “plateau contact angles” () were obtained. This time was
checked for the three strains prior to the experiments [17]. Then the filters were
introduced into an environmental chamber G211 (Krüss, Hamburg, Germany),
connected to a thermostat to maintain the temperature at 37 ºC. Before measuring the
contact angle with a probe liquid, the chamber was allowed to become saturated with
the vapour of the liquid employed. Subsequently, the contact angles were obtained by
analysing the images captured with a computer.
5.2.6.2. Zeta potential measurements
For the electrical surface potential characterization, the zeta potentials () of bacteria
suspended in PBS were measured at 37 ºC with a Laser Doppler Velocimeter Coulter
DELSA 440 (Langley Ford Instruments, Amherst, USA), which uses scattering of
incident laser light to provide the bacterial electrophoretic mobility (e) from the
velocity of the particles inside an electric field [18]. were related to e through the
Helmholtz-Smoluchowski equation
e
4
(5.1)
where and are the medium viscosity and permittivity, respectively.
The zeta potential of Ti6Al4V was obtained from the streaming potential [18] 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 (P) between both ends of the clamping cell provoked movement of the
102
electrolyte inside the electrokinetic channel, which, in turn, was reflected in a potential
difference between both ends (V). Both magnitudes were related to provide the value
of ,
001.0
M1.0M1.0
R··P
R···V
(5.2)
with R0.001M being the resistivity of the 0.001 M KCl electrolytic solution and 0.1M and
R0,1M the electrical conductivity and resistivity of a 0.1 M solution of KCl needed for
correcting the surface conductivity of the sample.
5.2.7. X-DLVO analysis
The surface tension components, LW
(Lifshitz-van der Waals), 2AB (acid-
base, with - the electron-donor and
+ the electron-acceptor parameters) were calculated
from the contact angles by applying the Young-Dupré equation to each probe liquid (L):
LL
LW
L
LW
L 222)1(cos (5.3)
where ABL
LWLL is the surface tension of the probe liquid [19].
At 37 ºC these values were the following: for water LW
=20.3 mJ/m2,
-=25.0 mJ/m
2,
+=25.0 mJ/m
2,
AB=50.0 mJ/m
2; for formamide
LW=36.7 mJ/m
2,
-=38.3 mJ/m
2,
+=2.2
mJ/m2,
AB=18.4 mJ/m
2 for diiodomethane
LW=48.1 mJ/m
2,
-=0.0 mJ/m
2,
+=0.7
mJ/m2,
AB=0.0 mJ/m
2. [19,20]
The total interaction free energy, G, between microorganisms and substrata through
water (W) as a function of the separation distance (d) was calculated by the sum of the
Lifshitz-van der Waals (LW), the acid-base (AB) and the electrical interaction free
energies (GLW
, GAB
and GEL
, respectively) as proposed by the extended DLVO
theory (X-DLVO) and calculated as follows:
a2d
dln
a2d
a
d
a
6
A)d(GLW (5.4)
A is the Hamaker constant, LW
d
2
0 0Gd12A , a is the microorganisms radius, d0 the
distance of the closest approach between two surfaces and LW
d0G was obtained through
103
the Lifshitz-van der Waals surface free energy as follows,
2LW
PBS
LW
S
2LW
PBS
LW
B
2LW
S
LW
B
LW
d0G (5.5)
subscripts B and S being bacterium and substratum, respectively, and the surface free
energy of the suspending liquid, PBS, is considered nearly equal to that of water.
0
0AB
d0
AB
d
)dd(expGad2G
0
(5.6)
where,
SBSB
PBSSBPBSPBSSBPBSAB
d 2G0
(5.7)
and finally,
))d2exp(1ln(
)dexp(1
)dexp(1ln
2)(a)d(G
2
S
2
B
SB2
S
2
Br0
EL (5.8)
0 is the dielectric constant of the vacuum and r the relative dielectric constant of the
suspending liquid. B and S are the zeta potentials of bacteria and substrata,
respectively.
Interaction free energies predict favourable adhesion when they are negative and
absence of adhesion when they are positive.
5.2.8. Statistical analysis
The statistical analysis of the samples was undertaken using a one-tail unpaired t-test
and one-way ANOVAs. All data reported are mean S.D. of at least three independent
experiments. The confidential range selected was 95%, which gives statistical
differences when P-values<0.05
5.3. RESULTS AND DISCUSSION
The changes observed in the Ti6Al4V surface after UV irradiation have already been
discussed in a recent study by our group [8]. Table 4.1 exhibits those important changes
in W and F, the polar liquids, before and after irradiation (P<0.05). Taking into
account that W can be considered a quantitative indicator of hydrophobicity, the control
surface behaves as hydrophobic, while the UV treated surface is presented as highly
104
hydrophilic. The excitation of the semiconductor TiO2 present on the surface of
Ti6Al4V generates electron-hole pairs, which are responsible for the changes observed.
The subsequent well-described chemical reactions lead to the substitution of oxygen
atoms of the TiO2 lattice for OH- groups, making the surface hydrophilic [21-23]. Such
hydrophilicity is also detected in the values of the surface tension, mainly in the acid-
base component and the parameters - and
+. As we have shown, the increment in the
electron-donor capacity is related to the presence of hydroxyl ions on the surface [8].
Table 5.1. Physico-chemical surface characterization of the Ti6Al4V surface before (control)
and after 15 h of UV light treatment (UV) and for the three bacteria studied. Contact angles of
water, formamide and diiodomethane (W, F, D) and Lifshitz-van der Waals (LW), acid base
(AB) and total () surface free energy and electron-donor (-
) and electron-acceptor (+)
parameters for all the samples.
W
(º)
F
(º)
D
(º)
(mV)
LW
(mJ/m2)
-
(mJ/m2)
+
(mJ/m2)
AB
(mJ/m2)
(mJ/m2)
control 883 662 491 312 32.3 3.2 0.2 1.5 33.8
UV 91 3.90.2 421 302 28.2 51.6 4.9 31.7 59.9
aureus 723 582 441 -82 30.4 13.8 0.3 3.9 34.3
epidermidis4 742 602 491 -62 28.2 12.6 0.4 4.4 32.6
epidermidis2 582 652 491 102 24.8 40.0 0.01 0.7 25.5
5.3.1. Effect of UV treatment on cell behaviour
Among other surface characteristics, wettability is thought to play a pivotal role in cell
response to materials. In this sense, self-assembling monolayers (SAMs) of alkanethiols
or alkylsilanes have provided suitable model surfaces to study the influence of
wettability and surface functional groups on cell attachment. While it has been generally
assumed that increased hydrophilicity favours cell adhesion to surfaces [24], available
data on mammalian cells adhesion to SAMs are far from conclusive [25-28]. The very
diverse cell types employed to assess the influence of wettability on SAMs attachment
could explain the lack of uniformity of the data, as cells from different origins may
respond differently to materials.
In order to study whether UV-induced changes of Ti6Al4V surface affect the ability of
human bone-forming cells to adhere to the alloy, we examined the behaviour of three
105
types of cells related to the osteoblastic phenotype (Saos-2, hMSC and hOB).
Examination of microphotographs from cells incubated for 3 h on the surfaces revealed
that UV treatment had no significant effect on the adhesion ability of any tested cell
type (Fig. 5.1a). Quantification of cells attached to the surfaces by means of a standard
MTT assay confirmed these observations (data not shown). Information on the
influence of wettability on cell behaviour on titanium-based surfaces is quite limited,
mainly due to the fact that reported treatments which modify surface hydrophilicity also
lead to subtle changes in roughness. For example, heating or treatment of Ti6Al4V alloy
with peroxide results in highly hydrophilic surfaces, with increased roughness, which
does not modify the rate of attachment of human osteoblastic MG-63 cells [29].
However, rabbit osteoblasts preferentially attached to heated Ti surfaces with an
increased number of hydroxyl groups and roughness, as compared to untreated samples
[30]. Roughness of the surface was not affected after UV treatment (data not shown),
and according to our data early attachment of several culture models of human
osteoblasts is not affected by UV-induced changes in the wettability of Ti6Al4V alloy
surfaces.
Fibroblasts attachment equalled rates on hydrophobic or hydrophilic SAMs but cell
spreading was sensitive to wettability, as it was impaired on highly hydrophilic surfaces
[26]. Therefore, we considered it of interest to study whether changes in the alloy
surface after UV treatment of Ti6Al4V samples modified spreading patterns of Saos-2
cells, hMSC or hOB in the short culture period of 3 h. To this end, we quantified the
area of cells cultured on the surfaces and examined the ability of cells to form actin
stress fibers. The average cell areas of both hMSC and hOB were higher than those
measured in Saos-2 cells (Fig. 5.1a). The quantified areas of cells cultured on UV-
treated surfaces were similar to those measured on surfaces without treatment,
regardless of the cell type. Microscopic examination of actin-stained cells revealed a
higher organization of actin cyoskeleton in both hMSC and hOB than in Saos-2 cells
(Fig. 5.1a). While hMSC and hOB exhibited radially positioned bundles of actin
filaments, no defined-stress fibers appeared in Saos-2 cells. Interestingly, the pattern of
actin distribution was unaffected by UV treatment in any of the cell types. Examination
of microphotographs revealed that cells have markedly different shapes at 24 h (Fig.
5.1b).
106
Fig. 5.1. Cell behaviour on Ti6Al4V surfaces. Cells were cultured on Ti6Al4V before
irradiation (control) and after irradiation (UV) for 3h (a) or 24h (b). Actin cytoskeleton and
nuclei staining of cells cultured on Ti6Al4V surfaces. Representative images of three
independent experiments with similar results. Bars = 50 m. The graphics show the cell area
measured on Ti6Al4V surfaces and results represent the mean SD of three independent
experiments. * P<0.05 compared to hMSC and hOB.
While Saos-2 cells had a typical polygonal morphology with well-formed actin
filaments, hMSC and hOB expanded to an elongated shape in which actin filaments
organized in well-defined stress fibers mostly oriented in a parallel direction following
the main cellular axis. Actin network organization was unaffected by UV treatment on
Ti6Al4V samples. Rapid initial cell spreading detected in either hMSC or hOB was not
Sao
s-2
h
MSC
h
OB
(a) Control UV Control UV
Sao
s-2
h
MSC
h
OB
(b)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
1 2
Saos
hMSC
hOB
Control UV
Cel
l are
a (µ
m2)
* *
hOB
Saos-2 hMSC
0
500
1000
1500
2000
2500
3000
3500
4000
4500
1 2
Saos
hMSC
hOB
* *
Control UV
Cel
l are
a (µ
m2)
hOB
Saos-2 hMSC
107
significantly modified after culturing cells for 24 h (Fig. 5.1b). However, the average
area of Saos-2 cells increased greatly when the culture period was extended. At 24 h,
changes induced on Ti6Al4V samples by UV treatment did not modify the spreading of
any cell type.
Interestingly, visualization of Saos-2 cells, hMSC and hOB adhered to Ti6Al4V alloy
after 24 h indicated that cell viability was not affected by UV-induced changes of the
surface. A recently described modified sand blasted and acid etched titanium surface
(modSLA) with an extremely hydrophilic character resulted in a reduction of viability
of MG-63 cells, as compared to unmodified SLA [31]. ModSLA and SLA exhibit
similar roughness, suggesting that cell colonization of blasted surfaces could be
inversely related to surface hydrophilicity. However, under our experimental conditions
we did not detect that osteoblastic viability is impaired on polished, highly hydrophilic
UV-treated Ti6Al4V alloy. In summary, UV treatment of Ti6Al4V does not impair its
good in vitro biocompatibility.
5.3.2. Effect of UV treatment on bacterial adhesion
Fig. 5.2 displays the values of the adhesion rates for the two processes, i.e., dynamic or
j0 and static-time-function or j, respectively, before (control) and after UV irradiation
(UV). Initial adhesion rates (j0), calculated from the adhesion at the top of the surface,
are lower than the adhesion rates (j), calculated from the adhesion at the bottom,
regardless of the presence or absence of UV irradiation (P<0.05).
These data suggest that bacterial adhesion strongly depends on the relative position of
the surface and the flow conditions of the surrounding media: static adhesion, mainly
mediated by deposition of bacteria, is higher than dynamic adhesion, which avoids
deposition. Measured differences between j and j0 can be as high as fifteen-fold as
detected for S. epidermidis2 after irradiation. Some experiments were also carried out in
flow-dynamic conditions with the surface placed at the bottom plate in order to check
the contribution of the flow conditions to the observed adhesion differences.
108
Fig. 5.2. Initial adhesion rates of the three bacterial strains to the Ti6Al4V surface located at
the top of the parallel plate flow chamber, j0, (a) and adhesion rates when the sample is located
at the bottom, j, (b), before irradiation (control) or after 15 h of irradiation (UV). * P<0.05
specific comparisons inside the text.
The results (not shown) demonstrated that the final state of the substratum in flow
conditions was the same as in static-time-function conditions, which implies that only
the position of the surface, relative to the bacterial suspension, plays an important role
in adhesion tendency. This behaviour was also observed in similar experiments dealing
with glass surfaces [32], which means that deposition of bacteria plays an important role
in promoting adhesion.
Comparisons among the three strains show that the highest adhesion was detected for S.
aureus, followed by S. epidermidis4, while the lowest adhesion was for the mutant
strain S. epidermidis2. Differences were, without exception, statistically significant
0
50
100
150
200
250
300
1 2
S. aureus
S. epidermidis 4
S. epidermidis 2
(a)
UV control
j 0 (
bac
teri
a/cm
2s)
*
*
*
*
*
S. aureus
S. epidermidis 4
S. epidermidis 2
0
200
400
600
800
1000
1200
1 2
S. aureus
S. epidermidis 4
S. epidermidis 2
UV control
j (b
acte
ria/
cm2s)
(b)
* * *
*
S. aureus
S. epidermidis 4
S. epidermidis 2
109
(P<0.05). The results obtained could be in agreement with multiple data which show
that S. aureus is the dominant organism associated with infected metallic implants while
S. epidermidis is more frequently isolated from non-metallic devices [13,33,34]. The
differences observed between both S. epidermidis strains could be related to their
different EPS production. In the case of S. epidermidis2 the suppression of its EPS
production capacity makes it less adherent because EPS promotes not only adhesion to
different surfaces but also co-adhesion between bacteria [35,36]. Fig. 5.3 presents some
representative images of the final state of the Ti6Al4V surface when it is located at the
top (a) and at the bottom (b). Image analysis of each experiment suggests that bacterial
co-adhesion is the main reason for the differences between both S. epidermidis strains at
the end of the experiment. Despite sonication of all bacterial suspensions prior to flow
experiments, the number of bacteria counted in the case of S. epidermidis4 was always
increased by the large cumulus of bacterial cells in the images, while the density of
single attached bacteria seemed to be similar for S. epidermidis4 and S. epidermidis2
(Fig. 5.3b). Nevertheless, initial adhesion of S. epidermidis4 always took place with
single bacterial cells (Fig. 5.3a). The differences observed for j0 between both S
epidermidis strains were around 60% before irradiation, and 70% after irradiation (Fig
5.2a), which means that EPS production may also contribute to the higher adhesion of S.
epidermidis4 to host surfaces.
The differences between Figs 5.2a and 5.2b reveal that UV irradiation changes the
adhesion rates of the bacteria strains studied. With the exception of j for S.
epidermidis2, the adhesion of bacteria decreased on the irradiated surface compared to
the control sample regardless of the type of adhesion test carried out. However,
adhesion changes were more evident when deposition was avoided (Fig. 5.2a). Thus, j0
diminished by 16% for S. aureus, by 28% for S. epidermidis4 and by 45% for S.
epidermidis2. In an attempt to find a relationship between adhesion rates before and
after irradiation, we plotted j0 and j before UV against j0 and j after UV. As shown in
Fig. 5.4, a very good linear relationship with the slope of 1 and R2=0.9929 was found.
This means that after irradiation both j0 and j decrease by about 33 cells per square
centimetre and second (independent term of the linear fitting) as compared to their
corresponding values for the non-irradiated alloy. Although it is difficult, at present, to
account for a specific empirical relationship, it could be speculated that whatever the
properties or particular characteristics of the bacterial strain, bacteria “sense” a similar
110
Ti6Al4V surface change induced by UV irradiation.
Fig. 5.3. Representative images of the bacterial coverage state at the end of the dynamic flow
experiment at top (a) and at the end of the static-time-function adhesion experiment at bottom
(b).
(a) (b) S.aureus
S. epidermidis4
S. epidermidis2
111
Fig. 5.4. Relationship between the initial adhesion rates at top (j0, squares) and the adhesion
rates at bottom (j, rhomboids) before (control) and after 15 h of irradiation (UV). White
symbols: S. epidermidis2, grey symbols: S. epidermidis4, black symbols: S. aureus.
5.3.3. Effect of ultra-violet treatment on bacterial retention
At the end of the adhesion experiments, two consecutive liquid-air interfaces were
passed through the channel of the parallel plate flow chamber and the strength of the
adhesion, or retention, was evaluated by counting the number of bacteria that remained
on the surface. This procedure allowed us to investigate whether bacteria were firmly or
loosely attached [37,38]. The results are summarized in Fig. 5.5. With the exception of
S. epidermidis4-nI-2-UV, the number of adhered bacteria followed the same behaviour
as the adhesion rates, the highest being for S. aureus and the lowest for S. epidermidis2,
regardless of the surface characteristics of the sample (compare Figs 5.5a, 5.5b and
5.5c) (P<0.05). Although j showed small variations for control and UV-light irradiated
Ti6Al4V, nf was similar in both cases for the three strains. Interestingly, after passing
liquid-air interfaces, the coverage state of the Ti6Al4V showed important differences
between the control and the UV surfaces. The adhered bacteria were firmly attached to
the untreated alloy surface, as they could not be removed from the surface. Only S.
epidermidis4-nI-2 decreased slightly in relation to nf, probably due to the presence of the
bacterial cumulus (P<0.05). On the other hand, the UV plots showed that the irradiated
surface is not able to retain the adhered bacteria. After the passage of the second liquid-
air interface the detachment of bacteria became highly important: 76% for S. aureus,
43% for S. epidermidis4, and 93% for S. epidermidis2. EPS production may be the
reason for the highest retention observed in S. epidermidis4.
y = 1.0072x - 32.944
R2 = 0.9929
0
100
200
300
400
500
600
700
800
900
0 200 400 600 800
j0 , j (control)
(bacteria/cm2s)
j 0 ,
j (
UV
)
(ba
cte
ria
/cm
2s)
112
Fig. 5.5. Number of the final bacteria (nf) adhered to the Ti6Al4V surface at the end of the
experiment (bottom) and number of the remaining bacteria after the passing of the first liquid-
air interface (nI-1) and after the second liquid-air interface (nI-2) without irradiation (control) or
after 15 h of irradiation (UV) for S. aureus (a), S. epidermidis4 (b) and S. epidermidis2 (c). *
P<0.05 specific comparisons inside the text.
0
20
40
60
80
100
120
140
1 2
nf
nI-1
nI-2nI-2
nf
nI-1
S. aureus
n (
bac
teri
a/cm
2)
10-4
UV control
(a)
* * *
*
0
20
40
60
80
100
120
140
1 2
nf
nI-1
nI-2nI-2
nf
nI-1
n (
bac
teri
a/cm
2)
10-4
UV control
(b) S. epidermidis4
*
* *
*
0
20
40
60
80
100
120
140
1 2
nf
nI-1
nI-2nI-2
nf
nI-1
n (
bac
teri
a/cm
2)
10-4
UV control
(c)
*
*
S. epidermidis2
113
In fact, suppression of EPS production in S. epidermidis2 gave the lowest retention. Fig.
5.6 presents an illustrative image of the state of the surface at the end of the experiment
and after the passing of the two liquid-air interfaces. In the case of S. epidermidis4
cumulus of bacteria appear even after the passing of the liquid-air interfaces.
Fig. 5.6. Representative images of the bacterial coverage state after UV irradiation, at the end
of the experiment, nf, (a), after the passing of the first liquid-air interface, nI-1, (b) and after the
passing of the second liquid-air interface, nI-2, (c).
(a) (c) (b)
S. epidermidis2
S. aureus
S. epidermidis4
114
The novel results presented above suggest that despite unavoidable attachment of
bacterial cells to Ti6Al4V, irradiation of its surface with UV light prior to its use would
facilitate easy removal of most of the adhered bacteria by shear forces, which can be
present in target sites where the implants are placed.
5.3.4. Physico-chemical approach to bacterial adhesion and retention
Table 5.1 shows that W was similar for S. aureus and S. epidermidis4 (P>0.05), both of
which are hydrophobic, while W was lower for S. epidermidis2 (P<0.05). F, D and
values were very similar for all the strains. S. epidermidis2 had a very strong electron-
donor capacity compared to the other two strains. Nevertheless, due to its low + value,
AB
and were the lowest.
As a first and qualitative attempt to relate the physico-chemical parameters to in vitro
bacteria adhesion, hydrophobicity of the interacting surfaces can be taken as a predictor
of higher or lower adhesion. Although there is still some controversy about the role of
substratum hydrophobicity (or surface tension) in bacterial adhesion, [39,40] data from
the present study indicate that substratum hydrophobicity plays a crucial role in the
adhesion rates and detachment of bacteria. Regardless of the strain selected, hydrophilic
UV-irradiated Ti6Al4V presents lower adhesion rates as compared with hydrophobic
control surfaces (Fig. 5.3). In addition, the hydrophilic surface also shows a lesser
ability to retain bacterial cells, and liquid-air interfaces can remove more than three
quarters of the adhered bacteria. Working with yeast cells, we have also observed that
silicone hydrophobic surfaces have lower detachment rates compared with hydrophilic
glass surfaces [38].
Despite the shortage of X-DLVO in its application to the analysis of bacterial adhesion,
this model could give some indications on the behavior of the adhesion process. If we
consider bacteria as colloidal particles of approximately 900 nm in diameter, with
surface tension and zeta potential as evaluated from contact angle and electrophoretic
mobility measurements, interacting with a flat surface, the application of Eqs. (4.4)–
(4.8) gives the dependence of the interaction free energy with distance between particle
and surface. Fig. 5.7 presents DG, in kT units, as a function of the separation distance
for S. epidermidis4 before and after irradiation (Fig. 5.7a and 5.7b, respectively). The
other two strains had a similar pattern in the X-DLVO plots (data not shown). A
summary of the most relevant energetic information extracted from all the X-DLVO
115
figures is given in Table 5.2.
Fig. 5.7. X-DLVO plots of the total interaction free energy (G) between epidermidis4 and the
Ti6Al4V surface before (a) and after 15 h of irradiation (b). Lifshitz-van der Waals (GLW
),
acid-base (GAB
) and electrical (GEL
) components of G.
The most important feature related to the data in Fig. 5.7 is the great change in the plot
pattern after UV. For control surfaces, DG showed two favourable adhesion minima, a
secondary minimum and a primary minimum related with a potential well. For the UV-
d (A)
0 10 20 30 40 50 60
G
(kT
)
-90
-75
-60
-45
-30
-15
0
15
30
45
GEL
GAB
GLW
G
(a)
d (Å)
d (A)
0 10 20 30 40 50 60
G
(k
T)
-90
-75
-60
-45
-30
-15
0
15
30
45
GEL
GAB
GLW
G
(b)
d (Å)
116
irradiated sample, despite a secondary minimum still appearing, the primary minimum
was substituted by an energy barrier.
The assumption that bacteria are colloidal particles has the important shortcoming of
supposing that real bacteria are able to reach distances as close to the substratum surface
as 1.57 Å , which is the closest approach between two surfaces described by the
colloidal theory. For example, Li and Logan [41] have recently pointed out that
Escherichia coli bacteria approach host surfaces to a distance of within 50 Å (distance
roughly equal to the length of the core polysaccharide on Gram-negative bacteria),
while Sharma and Hanumantha assume that interaction distances should be located at
6.57 Å [42]. On this basis, the predictions of X-DLVO model at the minimal distance,
d0, seem unrealistic. Therefore, the secondary adhesion minimum has to play an
important role in the bacterial adhesion process. The favorable interaction, resulting in
adhesion to the Ti6Al4V surface, must take place when bacteria fall into the secondary
adhesion minimum at distances of 26.6 Å for S. aureus, 30.6 Å for S. epidermidis4 and
44.1 Å for S. epidermidis2, for which DG is negative in all cases, as shown in Table 5.2.
Table 5.2. Total interaction free energies (G) at the minimal separation distance (d0) and
energetic information of singular points observed in the X-DLVO graphics: presence or
absence of maxima, locations and energy of secondary minima and existence of primary
minima. Data are given for the interaction of the three bacterial strains with the Ti6Al4V
surface before irradiation (control) and after irradiation (UV).
d0 MÁX Secondary
minimum Primary
minimum G (mJ/m2)
G (kT)
d
(Å) G (kT)
d (Å)
G
(kT)
control
aureus -42.96 4504.94 16.1 -8.48 26.6 -9.92 yes
epidermidis4 -43.26 4532.99 15.1 -3.62 30.6 -7.32 yes
epidermidis2 -20.66 2084.21 10.6 28.55 44.1 -3.27 yes
UV
aureus 10.73 1065.11 no 33.1 -5.90 no
epidermidis4 9.76 961.92 no 36.1 -4.44 no
epidermidis2 28.21 2981.23 no 48.1 -2.06 no
The plot of the adhesion rates against G values of the secondary minimum in Fig. 5.8
shows a very good relationship for j0–G, whereas for j–G it is more difficult to define
such a clear tendency. This result can be related to the differences observed between
117
both adhesion rates. Bacterial adhesion to the surface located at the top of the flux can
be a consequence only of the initial adhesion tendency of bacteria, quantified by G,
while the results on the surface located at the bottom also account for deposition.
Fig. 5.8. Relationships between the initial adhesion rates at the top (j0, circles) and the
adhesion rates at the bottom (j, triangles) against the interaction free energy (G) calculated at
the minimal separation distance d0 (a) and at the secondary energy minima (b). White symbols:
S. epidermidis2, grey symbols: S. epidermidis4, black symbols: S. aureus.
After this first initial adhesion step, bacteria would strengthen their linkage to the
control surfaces. In the case of the irradiated surface, differences in the surface
properties can prevent a firm retention to the surface, making the adhered cells
vulnerable to any shear force, as proved by the results obtained after the passage of two
air–liquid interfaces (Fig. 5.5).
5.4. CONCLUSIONS
UV irradiation of the Ti6Al4V surface does not compromise the excellent in vitro
biocompatibility of the alloy, as measured by culturing human Saos-2 cells, primary
osteoblasts and mesenchymal stem cells on treated surfaces. The physico-chemical
changes occurring on the surface induce a reduction not only in adhesion rates of S.
aureus ATCC29213, S. epidermidis ATCC35984 and S. epidermidis HAM892 under
flow conditions but also in the adhesion strength between attached bacteria and the alloy
surface. A careful and detailed analysis of G as a function of the interaction distance
explains the bacterial adhesion-retention processes to the Ti6Al4V surface before and
after irradiation. Secondary adhesion minima are responsible, in any case, for the
0
50
100
150
200
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0
G at secondary minimum (kT)
j 0 (
bacte
ria
/cm
2s)
400
500
600
700
800
900
1000
j (b
acte
ria
/cm
2s)
118
landing of the bacteria onto the surface. Primary adhesion minima present in non-
irradiated surfaces promote a firmer anchorage of bacterial cells while energy barriers,
present in irradiated surfaces, are responsible for loose adhesion. From a practical point
of view, this study proposes UV treatment prior to implantation protocols as an easy,
economic and effective way of reducing the bacterial adhesion to the Ti6Al4V surface
without altering the excellent biocompatibility of the alloy.
5.5. REFERENCES
[1] Mack D, Rohde H, Harris LG, Davies AP, Horstkotte MA, Knobloch JK. Biofilm
formation in medical device-related infection. Int. J. Artif. Organs. 2006;29:343-
359.
[2] Edupuganti OP, Antoci V, King SB, Jose B, Adams CS, Parvizi J, Shapiro IM,
Zeiger AR, Hickok NJ, Wickstrom E. Covalent bonding of vancomycin to Ti6Al4V
alloy pins provides long-term inhibition of Staphylococcus aureus colonization.
Bioorg. Med. Chem. Lett. 2007;17:2692-2696.
[3] An YH, Friedman RJ. Handbook of bacterial adhesion. Principles, methods and
aplications. Totowa, NJ: Humana Press Inc; 2000.
[4] Bos R, Van der Mei HC, Busscher HJ. Physico-chemistry of initial microbial
adhesive interactions – its mechanisms and methods for study. FEMS Microbiol.
Rev. 1999;23:179-230.
[5] Méndez-Vilas A, Gallardo-Moreno AM, Calzado-Montero R, González-Martín ML.
AFM probing in aqueous environment of Staphylococcus epidermidis cells naturally
immobilised on glass: Physico-chemistry behind the successful immobilisation.
Colloid Surface B 2008;63:101-109.
[6] Brunette DM, Tengwall P, Textor M, Thomsen P. Titanium in Medicine, London:
Springer-Verlag; 2001.
[7] De Giglio E, Guascito MR, Sabbatini L, Zambonin G. Electropolymerization of
pyrrole on titanium substrates for the future development of new biocompatible
surfaces. Biomaterials 2001;22:2609-2616.
119
[8] Pacha-Olivenza MA, Gallardo-Moreno AM, Méndez-Vilas A, Bruque JM,
González-Carrasco JL, González-Martín ML. Effect of UV irradiation on the
surface Gibbs energy of Ti6Al4V and Ti6Al4V oxidized alloys. J. Colloid Interf.
Sci. 2008;320:117-124.
[9] Cho M, Chung H, Choi W, Yoon J. Different inactivation behaviours of MS-2
Phage and Escherichia coli in TiO2 photocatalytic disinfection. Appl. Environ.
Microb. 2005;71:270-275.
[10] Maness P-C, Smolinski S, Blake DM, Huang Z, Wolfrum EJ, Jacoby W.
Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of
its killing mechanism. Appl. Environ. Microb. 1999;65:4094-4098.
[11] Gopal, J, George RP, Muraleedharan P, Kalavathi S, Banerjee S, Dayal RK,
Khatak HS. Photocatalytic inhibition of microbial fouling by anodized Ti6Al4V
alloy. J. Mater. Sci. 2007;42:5152-5158.
[12] Vogely HCh. Infections in orthopaedic surgery. Clinical and experimental studies.
PhD Thesis. Universiteit Utrecht, 2000.
[13] Holgers KM, Ljungh A. Cell surface characteristics of microbiological isolates
from human percutaneous titanium implants in the head and neck. Biomaterials
1999;20:1319-1326.
[14] Oztürk O, Sudagidan M, Türkan U. Biofilm formation by Staphylococcus
epidermidis on nitrogen ion implanted CoCrMo alloy material. J. Biomed. Mater.
Res. A 2007;81:663-668.
[15] Saldaña L, Vilaboa N, Valles G, Gonzalez-Cabrero J, Munuera L. Osteoblast
response to thermally oxidized Ti6Al4V alloy. J. Biomed. Mater. Res. A
2005;73:97-107.
[16] Sjollema J, Busscher HJ, Weerkamp AH. Real time enumeration of adhering
microorganisms in a parallel plate flow cell using automated image analysis. J.
Microbiol. Meth. 1989;9:73-78.
120
[17] Busscher HJ, Weerkamp AH, Van der Mei HC, Van Pelt AWJ, de Jong HP,
Arends J. Measurements of the surface free energy of bacterial cell surfaces and
its relevance for adhesion. Appl. Environ. Microb. 1984;48:980-983.
[18] Shaw D.J. Introduction to colloid and surface chemistry. London: Butterworths;
1989.
[19] Van Oss C.J. Interfacial forces in aqueous media. New York: Marcel Dekker;
1994.
[20] González-Martín ML, Janczuk B, Labajos-Broncano L, Bruque JM. Determination
of the carbon black surface free energy components from the heat of immersion
measurements. Langmuir 1997;13:5991-5994.
[21] Diebold U. The surface of titanium dioxide. Surf. Sci. Rep. 2003;48:53-229.
[22] Wu K-R, Wang J-J, Liu W-C, Chen Z-S, Wu J-K. Deposition of graded TiO2 films
featured both hydrophobic and photo induced hydrophilic properties. Appl. Surf.
Sci. 2006;252:5829-5838.
[23] Zhang P, Tay BK, Zhang YB, Lau SP, Yung KP. The reversible wettability of Ti
containing amorphous carbon films by UV irradiation. Surf. Coat. Tech.
2005;198:184-188.
[24] Garcia AJ, Keselowsky BG. Biomimetic surfaces for control of cell adhesion to
facilitate bone formation. Crit. Rev. Eukar. Gene. 2002;12:151-62.
[25] Arima Y, Iwata H. Effect of wettability and surface functional groups on protein
adsorption and cell adhesion using well-defined mixed self-assembled
monolayers. Biomaterials 2007;28:3074-82.
[26] Faucheux N, Schweiss R, Lützow K, Werner C, Groth T. Self-assembled
monolayers with different terminating groups as model substrates for cell adhesion
studies. Biomaterials 2004;25:2721-30.
[27] Keselowsky BG, Collard DM, Garcia AJ. Surface chemistry modulates focal
adhesion composition and signaling through changes in integrin binding.
Biomaterials 2004;25:5947-54.
121
[28] Liu L, Chen S, Giachelli CM, Ratner BD, Jiang S. Controlling osteopontin
orientation on surfaces to modulate endothelial cell adhesion. J. Biomed. Mater.
Res. A 2005;74:23-31.
[29] MacDonald DE, Rapuano BE, Deo N, Stranick M, Somasundaran P, Boskey AL.
Thermal and chemical modification of titanium-aluminum-vanadium implant
materials: effects on surface properties, glycoprotein adsorption, and MG63 cell
attachment. Biomaterials 2004;25:3135-46.
[30] Feng B, Weng J, Yang BC, Qu SX, Zhang XD. Characterization of surface oxide
films on titanium and adhesion of osteoblast. Biomaterials 2003;24:4663-70.
[31] Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J, Cochran DL, Boyan
BD. High surface energy enhances cell response to titanium substrate
microstructure. J. Biomed. Mater. Res. A. 2005;74:49-58.
[32] Sjollema J, Busscher HJ, Weerkamp AH. Deposition of oral streptococci and
polystyrene lattices onto glass in a parallel flow cell. Biofouling 1988;1:101-112.
[33] Barth E, Mirivik QM, Wagner W, Gristina AG. In vitro and in vivo comparative
colonization of Staphylococcus aureus and Staphylococcus epidermidis on
orthopaedic implant materials. Biomaterials 1989;10:325-328.
[34] Fortún J, Navas E. A critical approach to the pathogenesis, diagnosis, treatment and
prevention of catheter-related bloodstream infections and nosocomial endocarditis.
Clin. Microbiol. Infec. 1999;5:2S40-2S50.
[35] Kodjikian L, Burillon C, Chanloy C, Bostvironnois V, Pellon G, Mari E, Freney J,
Roger T. In vivo study of bacterial adhesion to five types of intraocular lenses.
Invest. Ophth. Vis. Sci. 2002;43:3717-3721.
[36] Dunne WM. Bacterial adhesion: seen any good biofilms lately? Clin. Microbiol.
Rev. 2002;15:155-166.
[37] Gómez-Suárez C, Busscher HJ, Van der Mei HC. Analysis of bacterial detachment
from substratum surfaces by the passage of air-liquid interfaces. Appl. Environ.
Microb. 2001;67:2531-2537.
122
[38] Gallardo-Moreno AM, González-Martín ML, Bruque JM, Pérez-Giraldo C. The
adhesion strength of Candida parapsilosis to glass and silicone as a function of
hydrophobicity, roughness and cell morphology. Colloid Surface A 2004;249:99-
103.
[39] Gallardo-Moreno AM, Van der Mei HC, Busscher HJ, González-Martín ML,
Bruque JM, Pérez-Giraldo C. Adhesion of Enterococcus faecalis grown under
subinhibitory concentrations of ampicillin and vancomycin to a hydrophilic and a
hydrophobic substratum. FEMS Microbiol. Lett. 2001;203:75-79.
[40] Cerca N, Pier GB, Vilanova M, Oliveira R, Azeredo J. Quantitative analysis of
adhesion and biofilm formation on hydrophilic and hydrophobic surfaces of
clinical isolates of Staphylococcus epidermidis. Res. Microbiol. 2005;156:506-
514.
[41] Li B, Logan BE. Bacterial adhesion to glass and metal-oxide surfaces. Colloid
Surface B 2004;36:81-90.
[42] Sharma PK, Hanumantha Rao K. Adhesion of Paenibacillus polymyxa on
chalcopyrite and pyrite: surface thermodynamics and XDLVO theory. Colloid
Surface B 2003;29:21-38.
[43] Israelachvili J. Intermolecular and surface forces. London: Academic Press
Limited;1992.
123
CAPÍTULO 6
Bactericidal behavior of Ti6Al4V surfaces
after exposure to UV-C light
Este capítulo es una reproducción de la publicación: "A.M. Gallardo-Moreno, M.A.
Pacha Olivenza, M.C. Fernández-Calderón, C. Pérez-Giraldo, J.M. Bruque, M.L.
González-Martín. Biomaterials 2010;31:5159-5168".
124
ABSTRACT
TiO2-coated biomaterials that have been excited with UV irradiation have demonstrated
biocidal properties in environmental applications, including drinking water
decontamination. However, this procedure 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 TiO2 surface 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 S.
epidermidis and S. 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.
6.1. INTRODUCTION
Ti6Al4V is one of the most commonly used biomaterials in orthopedic applications.
The use of Al and V alloying elements stabilizes the alpha-beta microstructure, and
improves the mechanical properties in relation to the commercially pure Ti [1]. 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 [2].
This layer minimizes ion release from the implant to surrounding tissues and hence
inflammatory reactions in the body [3]. Despite these advantages, a post-operative
serious and unresolved 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 integrated antibiotics, antiseptics, metal ions [6-
125
11] most often with the integration of silver due to its antibacterial properties [12].
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
TiO2 have 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 O2
to produce superoxide ions (O2-). Both holes and OH
have a very 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 [13]. 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 [14-19]. In
these systems, selected strains used for in vitro testing include Escherichia coli [15, 20],
Lactobacillus helveticus [15], Salmonella choleraesuis, Listeria monocytogenes [19],
Pseudomonas aeruginosa [21], Bacillus anthracis [22].
Recently, and novel application is found in the textile industry. Here, apatite-coated
TiO2 particles 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 [23].
Nevertheless, although there is no doubt on the bactericidal effect of TiO2 coated
materials upon UV irradiation, there have been very few reports about its application on
bio-implant-related infections. In 1988 Cai et al [24] were one of the first researchers
who showed a new application of TiO2 photocatalyst for medical applications, by
erradicating cancer cells with ultrafine TiO2 powders [25]. 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 [28] used a similar experimental set-up to evaluate the
effect of these materials on E. coli strains.
126
Few details have been published on the TiO2 photocatalytic activity of clinical isolates,
such as Staphylococcus aureus or Staphylococcus epidermidis. Traditional TiO2
photocatalysis is effective only upon irradiation of UV-light at levels that would induce
serious damage to human cells [29]. 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. [30] found that the new developed carbon-containing TiO2 nanoparticles showed
antibacterial properties against S. aureus, Shigella flexneri and Acinetobacter baumannii
under visible-light illumination. The work of Cushnie et al. [31] gave insights on the
influence of different variables on the outcome of TiO2 photocatalysis experiments with
bacteria of clinical interest. Shiraishi et al. [32], 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 [33]. 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.
6.2. MATERIALS AND METHODS
6.2.1. Ti6Al4V
Disks of Ti6Al4V were cut from bars of 25 mm in diameter kindly supplied by
SURGIVAL S.A., Spain. A TIMETAL 6–4 ELI alloy was processed as a hot rolled
annealed bar at 700 ºC for 2 h, then air cooled. The disks were abraded on successively
127
finer silicon carbide papers, mechanically polished with diamond paste, and finished
with colloidal silica.
Prior to their use, the Ti6Al4V disks were carefully cleaned with DSF disinfectant
(DERQUIM DSF 11; Panreac Quimica S.A., Spain) at 60 ºC 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 ºC 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 [34]. 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 the users.
6.2.2. Bacterial strains and culture
Staphylococcus aureus ATCC29213 (S. aureus), S. epidermidis ATCC35984 (S.
epidermidis4), S. epidermidis HAM892 (S. epidermidis2) and Escherichia coli ATCC
25922 (E. coli) were stored at -80 ºC in porous beads (Microbank, Pro-Lab Diagnostics,
USA). S. epidermidis HAM892 is a negative extracellular polysaccharide substance
producer (EPS-negative) 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 ºC 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 ºC and then 25 l of this pre-culture was used
again to inoculate 50 ml flasks of TSB at 37 ºC 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 1000g (Sorvall TC6,
Dulont, USA) and washed three times with 0.15 M phosphate buffered saline (PBS, pH
128
7.2) pre-conditioned at 37 ºC. Then the bacteria were re-suspended in PBS at a
concentration of 3 x 108 bacteria ml
-1 for the adhesion experiments.
6.2.3. Adhesion experiments
Adhesion experiments were carried out with the help of a sterile reusable silicone
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 ºC
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.
6.2.4. 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.
6.2.4.1. 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 l of SYTO 9 and
propidium iodide and 15 l of this mixture were put in contact with the sample surface
for 15 min avoiding exposure to room light. Then, stained bacteria were observed at
randomly chosen locations under fluorescence. Live bacteria, with intact 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) (λex = 500
nm, λem = 526 nm). The stain is a DNA binding dye in which the acridin orange
fluorocrome interacts with DNA and RNA by intercalation.
129
6.2.4.2. 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.
6.2.4.3. 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.
6.2.5. Biofilm formation on the Ti6Al4V surface
Bacteria was inoculated and incubated in blood agar plates at 37 ºC 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 microorganisms
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 µl
of the bacterial suspension was cultivated on the metal surface using a sterile silicone
culture chamber in a similar way to the bacterial adhesion experiments for different
periods of time, 8 h and 24 h. After those times the cultures 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 manufacturer. 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
130
metabolically active cells and, by consequence, this method has been frequently used
for adhesion and biofilm development in vitro [35, 36]. The luminescent signal, in
relative light units, in the ATP-bioluminiscence assay is proportional 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 performed
in duplicate and repeated at least three times in order to confirm reproducibility.
Although the method was applied for the three strains studied, only S. epidermidis
ATCC35984 induced the production of biofilm in enough quantity to be analysed.
Similar to the adhesion experiments, results were analyzed with paired samples and
independent experiments, up to fourteen, were done.
6.2.6. 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.
6.2.6.1. 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,
Sigma-Aldrich, USA) and the complex (TPTZ)2Fe++
(TPTZ: 2,4,6-tripyridyl-1,3,5-
triazine, Fluka) were used by means of colorimetric methods evaluating 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. [37] was employed. Briefly, stock solutions
containing 1 g L-1
of BG dye in water were prepared, protected from light and stored at
4 ºC. For the complex (TPTZ)2Fe++
, without exhaustive details, based on the work of
131
Pilch et al. [38], 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 0,8-0,9 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, Sigma–Aldrich, USA).
This compound degrades in the presence of hydroxyl radicals through double C-C
bonds attack [39]. 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 acetonitrile–water (15/85 v/v) with
0.1% phosphoric acid (flow rate: 1ml min-1). A C18 Kromasil 15 cm long, 0.4 cm
diameter column was used. Detection was made at 264 nm. The contact between the
probe chemicals and the samples were established with the help of silicone wells and
contact time was set as 60 min.
6.2.6.2. Duration time of the post-radiation effect
Bacteria were let to adhere onto the irradiated surface of Ti6Al4V not immediately 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.
6.2.6.3. 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 l bacterial suspension droplet was deposited on their surface, allowing the
adhesion of microorganisms to the film surface for 60 min at 37 ºC. 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 analysed in terms of viable microorganisms.
132
6.2.6.4. Electrical interaction potential at the irradiated surface
Zeta potential of control and irradiated surfaces was obtained from the streaming
potential [40] 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 (P) 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 (V). Both magnitudes were
related to provide the value of ,
M001.0
M1.0M1.0
R··P
R···V
(6.1)
with R0.001M being the resistivity of the 0.001 M KCl electrolytic solution and 0.1M and
R0,1M the electrical conductivity and resistivity of a 0.1 M solution of an KCl needed for
correcting the surface conductivity of the sample.
6.2.6.5. Surface charge excess on the irradiated surface
A thin opaque conducting film (0.2 mm thickness) was placed over the irradiated
surface immediately after turning off the UV lamp (parallel experiments were
performed with control samples). Then, a 250 l bacterial suspension droplet was
deposited on their surface, allowing the adhesion of microorganisms to the film surface
for 60 min at 37 ºC. 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 analysed in
terms of viable microorganisms.
6.2.7. 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.
133
6.3. RESULTS
6.3.1. Quantification of adhered bacteria to the control and irradiated surface
The total number of adhered bacteria on the surface, no matter their viability, at the
different contact times is plotted in Fig. 6.1. 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. epidermidis2 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 of adhered bacteria was very difficult to quantify
since the aggregation of cells in big 3-D cumulus lead to the underestimation of the total
number of adhered bacteria. This fact became more relevant 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 differences).
Fig. 6.1. Total number of adhered bacteria (viable and non-viable) on the Ti6Al4V irradiated
surface as a function of contact time and on the control surface after 60 min of contact.
0
20
40
60
80
100
0 10 20 30 40 50 60 70 80
tiempo(min)
nº
ba
ct/
ca
mp
o
S. aureus
S epidermidis 2
S. epidermidis 4
Time (min)
n (
bac
teri
a cm
-2)
x 1
0-4
60
Control
S. epidermidis2
S. epidermidis4
S. aureus
134
6.3.2. Evaluation of the viability of adhered bacteria to the control and irradiated
surface
Focusing on the viability of adhered bacteria, Fig. 6.2 presents various images of typical
areas on the Ti6Al4V after 15 h of irradiation for the different contact times, 10 min, 20
min, 40 min and 60 min. Control-viability images are also shown in 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 show a progressively loss in viability. This
phenomenon seems to be a function of the contact time between bacteria and surface:
bacteria become more damaged as the time of contact between 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. 6.3.
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 ( ~ 75 %) occurs after the first
10 min of contact.
Then, after the next 10 min, a further decrease takes place of the order of 20 %, whereas
in the last two 10 min time intervals cell viability decreases by ~ 2,5 % 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 dependent 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 experiments with a gram-negative Escherichia 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 wall properties.
135
Fig. 6.2. 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 non-irradiated Ti6Al4V surface.
136
Fig. 6.3. 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.
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. [44] 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
valuable 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.
0
20
40
60
80
100
10 20 40 60 70
Tiempo (min)
po
rcen
taje
sS. aureus
S epidermidis 2
S. epidermidis 4
Time (min)
Live
bac
teri
a (
%)
S. epidermidis2
S. epidermidis4
S. aureus
60
Control
137
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 distribution 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 % (P = 0.0377) 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.
6.3.3. Assessment of bacterial biofilm growth on the control and irradiated surface
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 (r2
=
0.975, 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. 6.4. 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., very significant.
138
Fig. 6.4. Biofilm reduction (in range) associated with the number of experiments that presented
visible reductions for the strain S. epidermidis4.
After 24 h of culture, the changes in biofilm formation between the control and the
irradiated samples were lower. Though reductions 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 (P = 0.0577), which implied
not quite significant differences.
6.3.4. Insights into the post-radiation antimicrobial mechanisms
6.3.4.1. 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.
6.3.4.2. 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. 6.5 (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 a waiting time of 30 min bacteria appear
stained in red, and as this waiting time increases a higher number of green spots are
0
1
2
3
4
5
6
7
1 2 3 4
Nu
mb
er
of
exp
eri
men
ts
< 15 % 15 % - 45 %
Ranges of biofilm reduction (%)
45 % - 75 %
> 75 %
N
um
ber
of
exp
eri
me
nts
139
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 min waiting time, while in the case of S. aureus a very high proportion of green
bacteria is observed at 60 min.
Fig. 6.5. 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.
10µm 10µm
10µm 10µm
10µm 10µm
5 min
30 min
90 min
15 min
60 min
120 min
140
6.3.4.3. Radiation emission
Fig. 6.6 shows representative images for S. epidermidis2, adhered to the surface of the
different non-conducting film surfaces investigated. 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.
Fig. 6.6. Images of adhered bacteria (S. epidermidis2) to different film surfaces placed over the
Ti6Al4V surface before irradiation and immediately after irradiation.
10µm 10µm
Black plastic film
Control Irradiated
10µm 10µm
10µm 10µm
Quartz
Glass film
141
The characterization of the three films employed was carried out by obtaining the
transmission curves for different wavelengths, ranging from 254 nm to 490 nm and
results are presented in Fig. 6.7. 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.
Fig. 6.7. Emission graphs for quartz, glass film and black plastic film in the UV - visible range.
6.3.4.4. 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.
6.3.4.5. Surface charge excess on the irradiated surface
Fig. 6.8 presents the viability of bacteria adhered to the conducting 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.
0
20
40
60
80
100
200 250 300 350 400 450 500 550
Longitud de onda (nm)
Tra
ns
mit
an
cia
(%
)
glass film
quartz film
black plastic film
Wavelength (nm)
Tran
smit
tan
ce (
%)
142
Fig. 6.8. 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.
6.4. DISCUSSION
6.4.1. The antimicrobial effect
Irradiation of Ti6Al4V surfaces with UV-C light provokes a residual post-radiation
effect which directly affects the viability of adhered bacteria. Staining-based methods
have shown that bacteria attached to the irradiated surfaces present a progressively loss
in viability, reaching its maximum damage after 60 min of contact (Fig. 6.2 and Fig.
6.3). 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. non irradiated
Ti6Al4V surfaces (Fig. 6.4). The decrease in biofilm production on the irradiated
surfaces during the first hours of its development represents a valuable 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
between microorganisms, eukaryotic cells and other biological molecules yields to the
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. 6.5). Regardless of the strain selected, the antimicrobial
activation of the surface is guarantee for at least 60 min after turning off the UV lamp,
Control Irradiated
10µm 10µm
143
then it begins to attenuate. Consequently, the method here proposed for the preparation
of antimicrobial titanium surfaces could become especially useful in totally internal
implant surgery, for which the antimicrobial challenge is mainly during or shortly after
surgery [45].
6.4.2. Mechanisms underlying the antimicrobial effect
Reactive oxygen species are known as the basis of the photocatalytic disinfection
system, hydroxyl radicals being frequently assumed to be the major factor responsible
for the antimicrobial activity observed in the TiO2 photocatalysis [20]. 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 irradiated 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. 6.6) 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 TiO2 upon 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 TiO2 surface continues stable, this
aspect being decisive for the vastly use of TiO2 as photocatalytic support [47]. 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. 6.7 we have observed the nearly complete transmission
of quartz in the UV range selected and the high transmission of the glass film from
144
about 305 nm. Considering that, according to the 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 show loss in bacterial
viability. This is also consistent with the less intense colour of bacteria adhered to the
glass film in relation to the quartz surface (Fig. 6.6).
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 microorganisms 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 [51]. 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 [52-55]. A previous work of Crowley and Courcelle [56] has shown that
Escherichia 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. [57] 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 relatively slow recombination 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
[58]. Although it has not been observed changes in the zeta potential between the
control and irradiated surfaces, in a previous work of our group [34] we have shown
145
that irradiated surface displayed a huge increase in the electron-donor 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. Nevertheless, 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 after turning off the UV lamp (Fig. 6.8),
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 electron-hole recombinations must be
present at the initial steps of bacterial adhesion as shown by the enormous reduction (
95 %) in the bacterial viability during the first 20 min of adhesion experiments (Fig.
6.3). Present experiments in our group are devoted to measure such as electrical surface
currents by using more specific techniques.
6.5. CONCLUSIONS
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 electron-hole recombination process are proposed as the
mechanisms 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.
146
6.6. REFERENCES
[1] Navarro M, Michiardi A, Castaño O, Planell JA. Biomaterials in orthopaedics. J.
Roy. Soc. Interface. 2008;5:1137-1158.
[2] Sittig C, Textor M, Spencer ND, Wieland M, Vallotton PH. Surface characterization
of implant materials c.p.Ti, Ti-6Al-7Nb and Ti-6Al-4V with different pretreatments. J.
Mater. Sci. Mater. M. 1999;10:35-46.
[3] Guillemot F, Prima F, Tokarev VN, Belin C, Porté-Durrieu MC, Gloriant T, Baquey
CH, Lazare S. Ultraviolet laser surface treatment for biomedical applications of
titanium alloys: morphological and structural characterization. Appl. Phys. A
2003;77:899-904.
[4] Brunski JB, Puleo DA, Nanci A. Biomaterials and biomechanics of oral and
maxillofacial implants: current status and future developments. Int. J. Oral. Max. Impl.
2000;15:15-46.
[5] Heidenau F, Mittelmeier W, Detsch R, Haenle M, Stenzel F, Ziegler G, Gollwitzer
H. A novel antibacterial titania coating. Metal ion toxicity and in vitro surface
colonization. J. Mater. Sci. Mater. M 2005;16:883-888.
[6] Darouiche RO, Raad II, Heard SO, Thornby JI, Wenker OC, Gabrielli A, Berg J,
Khardori N, Hanna H, Hachem R, Harris RL, Mayhall G. A comparison of two
antimicrobial-impregnated central venous catheters. New Engl. J. Med. 1999;340:1-8.
[7] Gollwitzer H, Ibrahim K, Meyer H, Mittelmeier W, Busch R, Stemberger A.
antibacterial poly(D,L-lactic acid) coating of medical implants using a biodegradable
drug delivery technology. J. Antimicrob. Chemoth. 2003;51:585-591.
[8] Hench LL, Polak JM. Third-generation biomedical materials. Science 2002;295:
1014–1017.
[9] Olsson J, Van der Heijde Y, Holmberg K. Plaque formation in-vivo and bacterial
attachment in-vitro on permanently hydrophobic and hydrophilic surfaces. Caries Res.
1992;26:428-433.
[10] Raad I, Darouiche R, Dupuis J, Abi-Said D, Gabrielli A, Hachem R, Wall M,
Harris R, Jones J, Buzaid A, Robertson C, Shenaq S, Curling P, Burke T, Ericsson C.
147
Central venous catheters coated with minocycline and rifampin for the prevention of
catheter-related colonization and vloodstream infections: A randomized, double-blind
trial. Ann. Intern. Med. 1997;127:267-274.
[11] Veenstra DL, Saint S, Saha S, Lumley T, Sullivan SD. Efficacy of antiseptic-
impregnated central venous catheters in preventing catheter-related bloodstream
infection: a meta-analysis. JAMA – J. Am. Med. Assoc. 1999;281:261-267.
[12] Carbon RT, Lugauer
S, Geitner U, Regenfus A, Böswald M, Greil J, Bechert T,
Simon SI, Hümmer HP, Guggenbichler
JP. Reducing catheter-associated infections with
silver-impregnated catheters in long-term therapy of children. Infection 1999:27:S69-
S73.
[13] Matsunaga TR, Tomada R, Nakajima T, Wake H. Photochemical sterilization of
microbial cells by semiconductor powders. FEMS Microbiol. Lett. 1985;29:211-214.
[14] Byrne JA, Eggins BR, Brown NMD, McKinnery B, Rouse M. Immobilisation of
TiO2 powder for the treatment of polluted water. Appl. Catal. B Environ. 1998;17:25-
36.
[15] Liu HL, Yang TCK. Photocatalytic inactivation of Escherichia coli and
Lactobacillus helveticus by ZnO and TiO2 activated with ultraviolet light. Process
Biochem. 2003;39:475-481.
[16] Gaswami DY, Trivedi DM, Block SS. Photocatalytic disinfection of indoor air. J.
Sol. Energ.-T. Asme 1997;119: 92-96.
[17] Maneerat C, Hayata Y. Antifungal activity of TiO2 photocatalysis against
Penicillium expansum in vitro and in fruit tests. Int. J. Food Microbiol. 2006;107:99-
103.
[18] Nonami T, Hase H, Funakoshi K. Apatite-coated titanium dioxide photocatalyst for
air purification. Catalysis Today 2004;96:113-8.
[19] Kim B, Kim D, Cho D, Cho S. Bactericidal effect of TiO2 photocatalyst on
selected food-borne pathogenic bacteria. Chemosphere 2003;52:277-81.
148
[20] Cho M, Cheng H, Choi W, Yoon J. Different inactivation behaviours of MS-2
phase and Escherichia coli in TiO2 photocatalytic disinfection. Appl. Environ. Microb.
2005;71:270-275.
[21] Madrid PA, Moorillon GVN, Borunda EO, Yoshida MM. Photoinduced
bactericidal activity against Pseudomonas aeruginosa by TiO2 based thin films. FEMS
Microbiol. Lett. 2002;211:183-8.
[22] Prasad GK, Agarwal GS, Singh B, Rai GP, Vijayaraghavan R. Photocatalytic
inactivation of Bacillus anthracis by titania nanomaterials. J. Hazard. Mater. 2009;165:
506-610.
[23] Kangwansupanonkon W, Lauruengtana V, Surassmo S, Ruktanonchai U.
Antibacterial effect of apatite-coated titanium dioxide for textile applications.
Nanomedicine 2009;5:240-249.
[24] Cai R., Hashimoto K., Itoh K., Fujishima A, Kubota Y. Photocatalytic Effect on
Tumor Cells. Photomed. Photobiol. 1988;10:253-255.
[25] Cai R, Hashimoto K, Itoh K, Kubota Y, Fujishima A. Photokilling of malignant
cells with ultrafine TiO2 powder. Bull. Chem. Soc. Jpn. 1991;64:1268-1273.
[26] Choi JY, Kim KH, Choy KC, Oh KT, Kim KN. Photocatalytic Antibacterial Effect
of TiO2 Film Formed on Ti and TiAg exposed to Lactobacillus acidophilus. J. Biomed.
Mater. Res. B 2007;80:353-359.
[27] Choi JY, Chung CJ, Oh KT, Choi YJ, Kim KH. Photocatalytic antibacterial effect
of TiO2 film on TiAg on Streptococcus mutans. Angle Orthod. 2009;79:528-532.
[28] Riley DJ, Bavastrello V, Covani U, Barone A, Nicolini C. An in-vitro study of the
sterilization of titanium dental implants using low intensity UV-radiation. Dent. Mater.
2005;21:756-760.
[29] Musk P, Campbell R, Staples J, Moss DJ, Parsons PG. Solar and UVC-induced
mutation in human cells and inhibition by deoxynucleosides. Mutation Res. Lett.
1989;227:25-30.
149
[30] Cheng CL, Sun CS, Chu WC, Tseng YH, Ho HC, Wang JB, et al. The effects of
the bacterial interaction with visible-light responsive titania photocatalyst on the
bactericidal performance. J. Biomed. Sci. 2009;16:7-17.
[31] Cushnie TPT, Robertson PKJ, Officer S, Pollard PM, McCullagh C, Robertson
JMC. Variables to be considered when assessing the photocatalytic destruction of
bacterial pathogens. Chemosphere 2009;74:1374-1378.
[32] Shiraishi K, Koseki H, Tsurumoto T, Baba K, Naito M, Nakayama K, et al.
Antibacterial metal implant with a TiO2-conferred photocatalytic bactericidal effect
against Staphylococcus aureus. Surf. Interface Anal. 2009;41:17-22.
[33] Gallardo-Moreno AM, Pacha-Olivenza MA, Saldaña L, Pérez-Giraldo C, Bruque
JM, Vilaboa N, et al. In vitro biocompatibility and bacterial adhesión of physico-
chemically modified Ti6Al4V surface by means of UV irradiation. Acta Biomater.
2009;5:181-192.
[34] Pacha-Olivenza MA, Gallardo-Moreno AM, Méndez-Vilas A, Bruque JM,
González-Carrasco JL, González-Martín ML. Effect of UV irradiation on the surface
Gibbs energy of Ti6Al4V and thermally oxidized Ti6Al4V. J. Colloid Interf. Sci.
2008;320:117-124.
[35] Gracia E, Fernandez A, Conchello P, Alabart JL, Perez M, Amorena B. In vitro
development of Staphylococcus aureus biofilms using slime-producing variants and
ATP-bioluminescence for automated bacterial quantification. Luminescence
1999;14:23-31.
[36] Gracia E, Fernandez A, Conchello P, Lacleriga A, Paniagua L, Seral F, et al.
Adherence of Staphylococcus aureus slime-producing strain variants to biomaterials
used in orthopaedic surgery. Int. Orthop. 1997;21:46-51.
[37] Chen CC, Lu CS, Fan HJ, Chung WH, Jan JL, Lin HD, Lin WY. Photocatalyzed
N-de-ethylation and degradation of brilliant green in TiO2 dispersions under UV
irradiation. Desalination 2008;219(1-3):89-100.
[38] Pilch P, Somerville R. Periodate oxidation of vicinal hydroxyls. J. Chem. Educ.
1977;54:449-
150
[39] Rodríguez EM, Mimbrero M, Masa FJ, Beltrán FJ. Homogeneous iron-catalyzed
photochemical degradation of muconic acid in water. Water Res. 2007;41:1325-1333.
[40] Shaw DJ. Introduction to colloid and surface chemistry. London: Butterworths;
1989.
[41] Maness PC, Smolinski S, Blake DM, Huang Z, Wolfrum EJ, Jacoby WA.
Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of its
killing mechanism. Appl. Environ. Microb. 1999;65(9):4094-4098.
[42] Crnigoj M, Kostanjsek R, Kaletunç G, Ulrih NP. Effect of different fluorescent
dyes on thermal stability of DNA and cell viability of the hyperthermophilic archaeon
Aeropyrum pernix. World J. Microb. Biot. 2008;24:2115-2123.
[43] Beck P, Huber R. Detection of cell viability in cultures of hyperthermophiles.
FEMS Microbiol. Lett. 1997;147:11-14.
[44] Huang Z, Maness PC, Blake DM, Wolfrum EJ, Smolinski SL, Jacoby WA.
Bactericidal mode of titanium dioxide photocatalysis. J. Photoch. Photobio. A
2000;130:163-170.
[45] Busscher HJ, Ploeg RJ, van der Mei HC. Biofilms and biomaterials; mechanisms
of medical device related infections. Biomaterials 2009;30:4047-4048.
[46] Siemon U, Bahnemann D, Testa JJ, Rodríguez D, Litter MI, Bruno N.
Heterogeneous photocatalytic reactions comparing TiO2 and Pt/TiO2. J. Photoch.
Photobio. A 2002;148:247-255.
[47] Yatskiv VI, Korzhak AV, Granchak VM, Kovalenko AS, Kuchmii SY.
Peculiarities of the behaviour of porous titanium dioxide in the photocatalytic evolution
of molecular hydrogen from aqueous ethanolic solutions. Theor. Exp. Chem.
2003;39:172-176.
[48] Jagger J. Introduction to Research in ultraviolet Photobiology. Chicago, IL:
Prentice Hall; 1967.
[49] Blake DM, Maness PC, Huang Z, Wolfrum EJ, Huang J. Application of the
photocatalytic chemistry of titanium dioxide to disinfection and the killing of cancer
cells. Sep. Purif. Method 1999;28:1-50.
151
[50] Harm W. Biological effects of ultraviolet irradiation. New York: Cambridge
University Press; 1980.
[51] Sang C, Cheung LM, Ho CM, Zeng M. Repression of photoreactivation and dark
repair of coliform bacteria by TiO2-modified UV-C disinfection. Appl. Catal. B-
Environ. 2009;89:536-542.
[52] Sousa FJ, Lima LM, Pacheco AB, Oliveira CL, Torriani I, Almeida DF, et al.
Tetramerization of the LexA repressor in solution: implications for gene regulation of
the E. coli SOS system at acidic pH. J. Mol. Biol. 2006;359:1059-1074.
[53] Aertsen A, Van Houdt R, Vanoirbeek K, Michiels CW. An SOS response induced
by high pressure in Escherichia coli. J. Bacteriol. 2004;186:6133-6141.
[54] Mesak LR, Miao V, Davies J. Effects of subinhibitory concentrations of antibiotics
on SOS and DNA repair gene expression in Staphylococcus aureus. Antimicrob. Agents
Ch. 2008;52(9):3394-3397.
[55] Úbeda C, Maiques E, Knecht E, Lasa I, Novick P, Penadés JR. Antibiotic-induced
SOS response promotes horizontal dissemination of pathogenicity island-encoded
virulence factors in staphylococci. Mol. Microbiol. 2005;56(3):836-844.
[56] Crowley DJ, Courcelle J. Answering the call: coping with DNA damage at the
most inopportune time. J. Biomed. Biotechnol. 2002;2(2):67-74.
[57] Erill I, Campoy S, Barbé J. Aeons of distress: an evolutionary perspective on the
bacterial SOS response. FEMS Microbiol. Rev. 2007;31:637-656.
[58] Van der Borden AJ, van der Werf H, van der Mei H, Busscher HJ. Electric current-
induced detachment of Staphylococcus epidermidis biofilms from surgical stainless
steel. App. Environ. Microb. 2004;70:6871-6874.
152
CAPÍTULO 7
Explaining the bactericidal behavior of the
UV-treated Ti6Al4V substrata through
electrochemical surface analysis
Este capítulo es una reproducción de la publicación enviada recientemente a "Surface
and Coating Technology" realizada por: M.A. Pacha Olivenza, A.M. Gallardo-Moreno,
V. Vadillo-Rodríguez, M.L. González-Martín, C. Pérez-Giraldo, J.C. Galván, en
colaboración con el grupo del Dr. J.C. Galván del Centro Nacional de Investigaciones
Metalúrgicas, CENIM-CSIC (Madrid).
153
ABSTRACT
This research gets insight into the reasons behind the bactericidal effect exhibited by the
Ti6Al4V surface after being irradiated with UV-C light. It arises as a continuation of a
previous published work in Biomaterials in which we hypothesized, based on a range of
qualitative tests, that small surface currents occurring as a consequence of the electron-
hole pairs recombination that takes place after the excitation process were behind the
bactericidal properties of this UV-treated material. To corroborate this hypothesis we
have now used different electrochemical techniques such as electrochemical impedance
spectroscopy (EIS), potentiodynamic polarization curves and Mott-Schottky plots. EIS
and Mott-Schottky plots have shown that UV-C treatment causes an initial increase on
the superficial electrical conduction of this material. In addition, EIS and anodic
polarization curves have detected higher corrosion currents for the UV treated against
the non-irradiated samples. Despite this increase in the corrosion currents, EIS has also
shown that such currents are not likely affect the good stability of this material oxide
film since the irradiated samples completely recovered the control values after being
stored in dark conditions for a period not longer than 24 h. These results agree with the
already-published in vitro transitory behavior of the bactericidal effect, which was
shown to be present at initial times after the biomaterial implantation, a crucial moment
to avoid a large number of biomaterial associated infections.
7.1. INTRODUCTION
Titanium and its alloys are the most commonly used materials in modern orthopedic and
dental applications. This is due to their excellent mechanical properties and resistance to
corrosion, which derive from a compact, thin and chemically stable oxide film that
spontaneously develops on these materials surface under ambient conditions, such as air
or water [1,2]. This oxide film, further able to minimize ion release from the implants to
the surrounding tissues and hence inflammatory reactions in the body, is of a few
nanometers thickness ( 5 nm) and mainly composed of amorphous or poorly
crystallized TiO2 [3,4]. The presence of other oxides has also been detected in titanium
alloys; in particular, Milosev et al [5] identified small traces of TiO, Ti2O3 and Al2O3 on
the naturally occurring oxide film of Ti6Al4V through XPS experimentation.
TiO2 is a n type photoactive oxide semiconductor [6,7], which properties temporarily
change after exposure to UV radiation. Its photoactivation under UV light provokes
154
photocatalytic surface reactions that degrade organic material, conferring bactericidal
properties to this particular oxide [8-13]. Exploiting these properties in the biomedical
implant field has been limited by the impossibility of irradiating the material in situ. It is
for this reason that little is known about how clinically relevant bacterial strains, such as
Staphylococcus aureus and Staphylococcus epidermis, are affected by the photocatalytic
activity of TiO2. Interestingly, using these gram-positive and other clinically relevant
gram-negative strains [14], our research group has recently shown that the Ti6Al4V
alloy exhibits antibacterial adhesive properties after being subjected to UV radiation. In
particular, microbial cells adhered with a lower force to the UV treated than to the
untreated surface, an observation that has been related with the decrease of the material
surface hydrophobicity that originates from the irradiation [15]. Most interestingly, a
post-radiation bactericidal effect has been also shown by the Ti6Al4V surface [16].
Although this post-radiation bactericidal property is time-dependent, its time duration
appeared enough to affect or to kill bacteria that could contaminate the implant surface
during the surgery procedure, which is the crucial to avoid a large number of
biomaterials associated infections. It is important to mention as well that the excellent
biocompatibility of this alloy was found not to be affected by the UV treatments, i.e.
human cells proliferate equally on the control and irradiated Ti6Al4V samples [14].
Investigations aimed to uncover the underlying basis of such bactericidal effect revealed
that the photocatalytic activity of this material surface, absent once the irradiation was
stopped, could not be responsible. It was hypothesized, based on a range of qualitative
tests, that small currents whose origin lies on the electron-hole pairs recombination that
takes place after the excitation process were behind the bactericidal properties of the
irradiated Ti6Al4V material. Thus, treating this material with UV produces superficial
electronic conductions that potentially interfere with the viability of the adhered
bacteria.
For this reason, the goal of this work is to quantitatively analyze the electric properties
of the Ti6Al4V alloy oxide film after being excited with UV radiation using
electrochemical techniques such as: electrochemical impedance spectroscopy (EIS),
potentiodynamic polarization curves and Mott-Shottky plots. All of these are powerful
technique to study charge transfer processes that have been previously used to evaluate
recombination processes taking place at the Ti6Al4V-TiO2/electrolyte interface [7,17].
155
The findings presented in this work clarify the origin of the bactericidal properties of the
Ti6Al4V UV treated materials.
7.2. MATERIALS AND METHODS
7.2.1. Ti6Al4V
Disks of Ti6Al4V were cut from bars of 25 mm in diameter kindly supplied by
SURGIVAL S.A., Spain. A TIMETAL 6–4 ELI alloy was processed as a hot rolled
annealed bar at 700 ºC for 2 h, then air cooled. The disks were abraded on successively
finer silicon carbide papers, mechanically polished with diamond paste, and finished
with silica colloidal.
Prior to their use, the Ti6Al4V disks were carefully cleaned with DSF disinfectant
(DERQUIM DSF 11; Panreac Quimica S.A., Spain) and distilled water at 60 ºC 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 ºC for 1 h
and stored in a desiccator for no longer than 24 h. Samples, denoted as control, were not
subjected to any further treatment. A second set of samples, referred as UV, 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 [15]. 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 the users.
7.2.2. Solution electrolyte
In order to simulate the physiological conditions around the bone tissues, the
electrochemical assays were carried out by immersing the samples in Ringer’s solution
(Ringer Lactato Grifols; Laboratorios Grifols S.A., Barcelona, Spain) [18]. This
solution contains 600 mg of NaCl, 40 mg of KCl, 27 mg of CaCl2 and 305 mg of
NaHCO3 diluted in 100 ml of water.
156
7.2.3. Electrochemical characterization
The electrochemical characterization (EIS, potentiodynamic polarization studies and
Mott-Schottky analysis) was carried out in a conventional electrochemical cell filled
with the Ringer´s solution and using the investigated specimen as working electrode
(control or UV), a counter electrode of platinum, and a reference electrode of Ag/AgCl
saturated in a potassium chloride solution. Measurements were made with a
potentiostat/galvanostat AUTOLAB EcoChemie PGSTAT30 (Eco Chemie, Utrecht,
The Netherlands) equipped with a frequency analyzer module FRA2.
In all the experiments the working electrode was firstly conditioned with the Ringer’s
solution (15 ml) for 5 min in complete darkness. This time was enough for the
adsorption of the ionic species from solution and for reaching the stabilization of the
open circuit potential value (EOCP). Experiments were repeated at least three times using
always fresh Ringer’s solution.
7.2.4. Electrochemical Impedance Spectroscopy (EIS)
The EIS technique was used to evaluate the properties exhibited by the working
electrode under alter current (AC) polarization. It is a versatile technique and suitable
for studying the behavior of biomaterials under corrosive environments. In the
experiments, an electric potential of variable frequency is applied to the material inside
an electrochemical cell and then its current response is measured and analyzed [19].
The input signal was a sine wave perturbation with ±10 mV amplitude. The frequency
range selected was from 10 kHz to 1 mHz, measuring five impedance points per
frequency decade. The impedance spectrums were analyzed in terms of Nyquist and
Bode diagrams and the resistance and capacitance values were obtained through the
application of the software ZView 3.1c (Scribner Associates Inc, Southern Pines, NC,
EEUU). The goodness of the proposed fitting for the selected equivalent circuit was
given by the values of Chi-square and the relative error.
7.2.5. Potentiodynamic Polarization Curves
The polarization curves allow the determination of the corrosion potential and intensity
as well as the anodic and cathodic Tafel slopes. This experiment was done by applying
a range of direct current (DC) potentials between the working electrode and the
reference electrode. The potentials were scanned at a rate of 0.5 mVs-1
in anodic
direction between -0.5 and 2 V. Then the electric current between the sample (working
157
electrode) and the reference electrode was registered. The analysis of the curves was
performed with the option “Corrosion rate analysis” implemented on the software
General Purpose Electrochemical Systems (GPES). The “Fit Tafel Slope” and
“Levenberg/Marquardt” models were used.
7.2.6. Mott-Schottky Curves
The Mott-Schottky experiments allowed for the evaluation of the interfacial capacity
associated to the oxide layer in terms of the DC potential. The range of the applied DC
potential was the same than in the Potentiodynamic Polarization technique, with
potential steps of 0.1 V. In order to obtain the capacitance at each applied potential, it
was used a sine wave perturbation with ±10 mV of amplitude and constant frequency of
10 kHz.
7.3. RESULTS AND DISCUSSION
In a previous work we qualitatively observed that bacteria adhered not directly to the
irradiated surface but on a thin metallic film, which was previously deposited on the
irradiated sample, appeared dead, against those adhered to the same metallic thin film
but dropped on a non-irradiated Ti6Al4V surface (Fig. 7.1). This observation led to the
hypothesis that there should be some electrical effect on the irradiated surface
responsible of the degradation of the bacterial cells.
Fig. 7.1. Adhesion experiment in which bacteria were not deposited directly in contact with the
Ti6Al4V surface but on a thin metallic film dropped previously on the Ti6Al4V surface (a).
Images of green (live) or red-like (dead or damaged) stained adhered bacteria on the thin
metallic film dropped on the control (non-irradiated) (b) or UV (irradiated) (c) Ti6Al4V
surface.
Ti6Al4V
Thin metallic film (a) (b) (c)
158
7.3.1. Electrochemical Impedance Spectroscopy (EIS)
7.3.1.1. EIS for Ti6Al4V before and after the UV irradiation treatment
The Nyquist diagrams for both samples (control and UV) show a unique capacitive loop
with a wide incomplete semicircle for all the frequency range studied (Fig. 7.2a). This
behavior is typical of thin oxide films [2,20-26]. The irradiation process alters the
electrochemical behavior of the interface Ti6Al4V-TiO2/electrolyte. The spectrum for
the control sample presents higher impedance values (imaginary part) than those
obtained for the irradiated sample.
The Bode diagrams (Fig. 7.2b) have been registered under the same conditions as the
previous Nyquist diagrams. The comparative analysis between control and UV shows
changes in the impedance module (|Z|) and phase versus frequency, which are due to
phenomena related to the passive film [21,27-29]. In both cases a single time constant
has been obtained. At high frequencies, |Z| and phase reach minimum and constant
values with a phase angle closes to 0º, this fact being related with a predominant
influence of the electrolyte resistance. In the region of medium and low frequencies the
|Z| diagram presents a lineal tendency with a slope close to -1 and a phase angle close to
80 º, especially in the case of the control sample. This is a typical capacitive response,
associated to the presence of passive films [30-32]. The high impedance values close to
106 ohms at the lowest frequencies indicate the good dielectric and protector properties
of the oxide film formed on the alloy surface [33].
In order to provide a full interpretation of the EIS results and a clearer understanding of
the electrocatalytic processes occurring at the interface Ti6Al4V-TiO2/ electrolyte the
establishment of a mathematical model is necessary [17]. The best model describing the
impedance spectrum was an electrical equivalent circuit with a single capacitive loop, as
shown in Fig. 7.3 [21,29,34,35]. This circuit consists of a resistance mainly operating at
high frequencies, related with the electrolyte resistance (Rs) [30,36,37] and a parallel
association of a constant phase element (CPE), to correct the non-ideal behaviour of the
capacitance, and a polarization resistance (Rp) that is inversely proportional to the
corrosion current [38].
159
Fig. 7.2. Impedance spectra for the Ti6Al4V alloy before (Control) and after being subjected to
UV radiation (UV). (a) Nyquist diagram representing the imaginary -jZimag vs. the real Zreal
parts of the impedance. (b) Bode diagram representing the modulus of the impedance (|Z|) in a
double-logarithmic scale and the phase angle in a semi-logarithmic scale vs. frequency.
The CPE is an electric element for which the impedance is a function of the angular
frequency, ω, but its phase does not depend on ω. This impedance is expressed as,
(7.1)
where Y0 is the constant phase element parameter of the CPE, j is the imaginary number
and = 2f, f being the frequency in Hz. The exponential n, known as the
exponent of the CPS, is related with a non-uniform current distribution due to the
surface roughness or other distributed properties. n can be related with the phase angle
of the CPE, , through the expression n = /(/2). When 0 < n< 0.5 the CPE is mainly
a resistance, when 0.5 <n <1 the CPE is mainly a capacitor [39] and if -1<n<0, the CPE
is mainly an inductor [35].
Mansfeld et al. reported a way concerning the conversion of the Constant Phase
Element Parameter Y0 into a capacitance C [40,41], by using the following equation:
(7.2)
where is the angular frequency at which the imaginary part of the impedance (Zimag)
has a maximum in the representation of Nyquist plots.
101
102
103
104
105
106
|Z| /
ohm
s·c
m2
10-3
10-2
10-1
100
101
102
103
104
0
-20
-40
-60
-80
Frequency / Hz
Ph
ase
/ d
eg
ree
s
0 1x106
2x106
3x106
0
1x106
2x106
3x106
-j·Z
ima
g /
oh
ms·c
m2
Zreal
/ ohms·cm2
Control1
Control3
Control4
UV1
UV2
UV4
FitResults_Control1
FitResults_Control3
FitResults_Control4
FitResults_UV1
FitResults_UV2
FitResults_UV4
(a) (b)
2 3
3
2 3
3
160
The CPE of the equivalent circuit of Fig. 7.3 represents two capacitors CCS and CH, CCS
being the space-charge capacitance and CH the capacitance of the Helmholtz layer. The
total capacitance C is expressed as C = (CSC-1
+ CH-1
)-1
.
Fig. 7.3. Equivalent circuit for the electrochemical film system Ti6Al4V-TiO2 / solution. Rs
represents the electrolyte resistance, Rp the polarization resistance and CPE a constant phase
element.
The results of numerical analysis via Complex Nonlinear Least Squares Fitting (CNLS)
of the spectra obtained in Fig. 7.2 by using this equivalent circuit are shown in Table
7.1. Comparing both samples, it is observed that Rp decreases after the UV treatment
which is likely related to the increase of electron-hole pairs generated during the
irradiation process [25,42]. Such increment is also related with the higher values of Y0-
CPE in irradiated samples
Table 7.1. Values obtained for the equivalent circuit parameters. Samples: Control (non-
irradiated Ti6Al4V) and UV (UV-irradiated Ti6Al4V). Rs: electrolyte resistance; Y0-CPE: CPE
parameter; n-CPE: CPE exponent; RP: polarization resistance. Inmersion time in Ringer´s
solution before EIS measurements: 5 min. The goodness of the fit, given by the statistical Chi-
square values, was always of the order of 10-4
for a sample control and the orden of 10-5
for UV,
and percentages of error were never more than 4 %.
Rs(Ω) Rp(MΩ) Y0-CPEx10-5
(sn/Ω) n-CPE
Ti6Al4V
(Control)
72.86±11 14.2±2.4 4.75±0.08 0.938±0.02
Ti6Al4V
(UV)
55.07±5.5 3.81±0,8 6.56±0.12 0.918±0.01
Rs
RP
CPE
Ringer TiO2
Ti6
Al4
V
Hel
mholt
z
161
The generation of electron-hole pairs increases the capacitance and, by consequence, the
surface conduction of the alloy [43]. The values obtained for n-CPE are similar in both
samples and close to 1. This indicated a near ideal capacitance of the passive film
formed on the Ti alloy [28]. However, it is always slightly lower for the UV sample.
Although it was not the scope of the manuscript, it is worth mentioning that we made a
parallel experience in which the sample was subjected to an extreme irradiation. In this
case the irradiation source was located to the minimum separation distance from the
sample (1 cm) and it was operated for the standard time of 15 h. The EIS results (data
not shown), fitted through the equivalent circuit of Fig. 7.3, presented a Rp of 0,021
MΩ. This value can provoke corrosion currents which are able to cause important
superficial deterioration of the sample with no possibility of recuperation.
7.3.1.2. EIS for Ti6Al4V after different post-radiation times
In the previous work [16] we showed that the bactericidal effect was transitory and
appeared highly suitable for avoiding the risk of early infections after implantation. It is
for this reason that EIS experiments were also carried out with samples that were
irradiated and keeping in complete darkness 5, 15, 30, 45, 60 min and 24 h before the
electrochemical assays.
The EIS diagrams for such experiments are presented in Fig. 7.4 and they show again
the typical shape of open and uncompleted semicircles, with a diameter depending on
the waiting time.
The fitted parameters for the equivalent circuit of the system Ti6Al4V-TiO2/electrolyte
at the different waiting times are summarized in Table 7.2. The lowest Rp is observed at
the lowest waiting time, 5 min, and it increases as the waiting time becomes longer,
reaching a similar value to that of the control sample at 24 h, within experimental error.
The opposite behavior is observed for Y0-CPE and for the capacitance of the passive
film calculated from equation (7.2) of Mansfeld. The highest values belong to the
lowest waiting time of 5 min and then they decrease to values similar to control. The n-
CPE suffers small changes and its value increases as the waiting times increase which
denote a higher capacitive behavior of the system. All the RS values are similar, within
the experimental error.
162
Fig. 7.4. Impedance spectra for the Ti6Al4V alloy before (Control) and after being subjected to
UV radiation (UV). (a) Nyquist diagram representing the imaginary -jZimag vs. the real Zreal
parts of the impedance. (b) Bode diagram representing the modulus of the impedance (|Z|) in a
double-logarithmic scale and the phase angle in a semi-logarithmic scale vs. frequency for
different waiting times: 5, 30, 45, 60 min and 24 h.
Table 7.2. Values obtained for the equivalent circuit parameters by modelling the experimental
impedance at different waiting times after irradiating a Ti6Al4V sample with UV.
Waiting
times
(minutes)
Rs(Ω)
Rp(MΩ)
Y0-CPEx10-5
(sn/Ω)
n-CPE
C(µF)
5 50±3 3.74±0.2 7.62±1.2 0.897±0,03 145.8±2.3
15 67±12 4.40±0.1 5.20±0.3 0.933±0,01 76.8±1.4
30 57±9 4.43±0.1 4.89±0.1 0.935±0,02 71.2±1.1
45 56±8 7.87±0.08 4.86±0.3 0.937±0,01 72.4±1.7
60 53±7 9.63±0.1 4.81±0.1 0.936±0,02 73.1±1.5
1440 (24h) 51±3 12.3±2.1 4.02±0.3 0.940±0,02 59.73±1.1
These results indicate that although after irradiation the surfaces present high corrosion
rates due to the lowest Rp values, the process is controlled and the recovery of the
sample is reached without affecting the good functionality of the biomaterial.
This recovery is in agreement with the short-term bactericidal effect. However, it is
clearly appreciated in the Niquist diagram a mostly total recovery after 60 min while at
101
102
103
104
105
106
107
Control 4
PostRadiation, 5 min
PostRadiation, 15 min
PostRadiation, 30 min
PostRadiation, 45 min
PostRadiation, 60 min
PostRadiation, 24 h
|Z| /
ohm
s·c
m2
10-3
10-2
10-1
100
101
102
103
104
0
-20
-40
-60
-80
Phase / d
egre
es
Frequency / Hz
0 1x106
2x106
3x106
0
1x106
2x106
3x106
-j
·Zim
ag/ oh
ms·c
m2
Zreal / ohms·cm2
Control 4
PostRadiation, 5 min
PostRadiation, 15 min
PostRadiation, 30 min
PostRadiation, 45 min
PostRadiation, 60 min
PostRadiation, 24 h
(a) (b)
163
this time bacterial viability still remained compromised. This agrees with the previously
proposed bactericidal mechanism in which it was suggested that the combination of
electrical effects and radiation emission from the surface after irradiation should be
considered [16].
7.3.2. Potentiodynamic polarization curves
This technique allows the evaluation of the reactions that occur on the working
electrode when operating near to its EOCP and enables to determine the effect of
different electrochemical variables on the total oxidation current. This is a great
advantage because the intrinsic kinetic parameters of the reaction can be estimated,
particularly when the process is controlled by electron transfer such as in the present
study [7].
As an example, Fig.7.5 shows the anionic and cationic polarization behavior of a
control and UV sample recorded in Ringer solution with a scan speed of 0.5 mVs-1
.
Fig. 7.5. Potentiodynamic polarization curves for the Ti6Al4V alloy before (Control)
and after being subjected during 15 h to UV radiation (UV).
The shape of the curves is typical of Ti6Al4V alloys [20,27-29,33,44,45]. Using the “Fit
Tafel Slope” model, Ecorr and Icorr were determined by lineal extrapolation of the anionic
(ba) and cationic (bc) Tafel lines. These values were used as initial parameters in the
“Levenberg/Marquardt” model to determine the best fit values through an iterative
method in which each step involves a least square fit.
-0.5 0.0 0.5 1.0 1.5 2.010
-12
10-11
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
UV irradiated samples
Cu
rre
nt
inte
nsi
ty,
I (A
)
Potential, E vs Ag/AgCl (V)
Control samples
Potential, E (V) vs. Ag/AgCl, sat. KCl
164
Table 7.3 shows that the UV treated samples exhibit Icorr values of one order of
magnitude larger than those obtained for the control samples (2.78×10-9
Acm-2
vs.
21.6×10-9
Acm-2
), revealing for the treated samples a higher susceptibility to corrosion
in Ringer solution. However, a shift of the curve to more positive potentials is also
observed, Ecorr taking values of -412 mV and -156 mV for the control and treated
samples, respectively. This is a clear indication that a more protected potential corrosion
has been developed on the material film after UV treatment [25]. In the passivation
region of both samples, the current intensity is independent of the applied potential,
suggesting that the samples do not suffer any kind of localized attacked and that the
TiO2 film remains stable during the immersion time in Ringer solution. This is a
characteristic behavior of titanium, known as valve metals [46].
Table 7.3. Results obtained through the Tafel Slope model for the control and UV Ti6Al4V
samples. Ecorr/SCE is the corrosion potential with respect to the reference potential, Icorr the
corrosion density current, and ba and bc the anionic and cationic slopes. The goodness of the fit,
given by the Chi-square values, was always of the order of 10-13
.
Even if the UV treatment increases the values of Icorr, resulting in material surfaces with
larger susceptibility for oxidation, the protecting potentials also found to be higher. The
latter guarantee the good stability of the biomaterial in Ringer solution after UV
treatment, in agreement with the complete recovery observed in the EIS experiments.
7.3.3. Mott-Schottky experiments
The Mott-Schottky analysis was carry out to evaluate the effect of the DC potential on
the capacitance behavior of the Ti6Al4V alloy after being subjected to UV radiation in a
control manner. This model predict that if the oxide film that develops on a metal
surface behaves as a semiconductor, there will be a relation between the film space-
charge capacitance and the superimposed potential, according to equation (7.3) and
(7.4) for n and p type semiconductors, respectively:
Ecorr /SCE(mV) icorr x10-9
(Acm-2
)
ba(V/dec) bc(V/dec)
Ti6Al4V
(Control)
-412±9 2.78±0.4 0.09±0.02 0.08±0.02
Ti6Al4V
(UV)
-156±15 21.6±5 0.29±0.03 0.35±0.05
165
(7.3)
(7.4)
where CSC is the space-charge capacitance, q the electric charge of the carriers, ND and
NA the number of donor and acceptor charge carriers, respectively, the film relative
permittivity, 0 the vacuum permittivity, V the superimposed potential, VFB the flatband
potential, k the Boltzmann constant and T the temperature [35].
In Fig. 7.6 the Mott-Schottky plot for a control and irradiated Ti6Al4V samples is
shown together with the lineal correlation coefficient R2 for each straight line. The
positive sign of the slope of the lines indicates a capacity decreases as the anionic
potential increases over the flatband potential. This denotes that the oxide film covering
our samples is a n type semiconductor, in agreement with the published literature for
films grown on pure Ti and its alloys [47,48].
Fig. 7.6. Mott-Schottky diagram for the Ti6Al4V alloy before (Control) and after being
subjected during 15 h to UV radiation (UV) obtained in Ringer solution. A 10 kHz AC
signal with a 10 mV amplitude was used.
It is worth noting that the equivalent circuit proposed in Fig.7.3 for EIS contains two
different capacitors, i.e. the capacitor of the space-charge region (CSC) and the
Helmholtz capacitor (CH), but the Mott-Schottky curves shows only one (CSC).
However, considering that C CSC [35,49], a direct comparison between these results
-0,5 0,0 0,5 1,0 1,5 2,0
1x1010
2x1010
3x1010
4x1010
Frequency = 10 kHz
(-
·Zim
ag)2
/ (
1/F
)2
Potential, E (V) vs. Ag/AgCl, sat. KCl
Control
UV
-0.250 V
R2= 0.999
R2= 0.998 -0.604V
166
and the calculated from EIS can be done. The increase in the values of CSC for the UV
treated alloy with respect to the control (Fig. 7.6) is in agreement with the results found
in EIS.
According to the approximation of Mott-Schottky, the slope of the straight lineal regime
of the 1/C2
SC vs E representation corresponds to 2/ 0qND. If kT/q is negligible, the
flatband potential can be obtained by extrapolation of the linear regime of the graph to
1/C2
SC = 0. Thus, an accurate calculation of ND strongly depends of the correct
determination of the relation between CSC and E [35]. The density of free carriers
calculated through the lineal correlation gave values 30% higher for the Ti6Al4V-UV
electrode than for the control. The values obtained, of the order of 1020
cm-3
, are
relatively high but within reported values in the literature [50]. The flatband potential
calculated (see Fig. 7.6) is 55 % higher for the control than the Ti6Al4V-UV electrode.
It is difficult to establish a clear relation between the thin oxide film semiconductor
properties and its corrosion characteristics; however, some authors working with
stainless steel were able to relate the density of free carriers within the film with a high
concentration of superficial defects [51]. Lee et al. [52] also observed that the number of
metastable pits was linked to the density of donor carriers of the passive film of 316L
and 316 LN stainless steels. Ningshen et al. [53] using EIS were able to detect a
decrease in the polarization resistance for these materials with the increase of the oxide
film free carriers. This is in agreement with the results obtained in this work for the
Ti6Al4V alloy, for which EIS results on UV treated samples showed a decrease in RP,
Mott-Schottky an increase in ND and the polarization curves an increase in the corrosion
currents. These findings indicate that even if the good superficial properties of the TiO2
semiconductor are slightly compromised, they completely recover under the controlled
treatment at which they have been here subjected.
Different authors have already shown this phenomenon to occur during the irradiation
process [17,54,55]. The origin is not clear, but most authors have attributed it to the
capture of minority charge carriers located in superficial states. If the number of free
superficial states is high enough, a considerable amount of charge can be stored,
provoking thus a band position change [55].
167
7.4. CONCLUSIONS
It has been shown that electrochemical type experiments are a powerful tool to
investigate the surface phenomena that take place after the irradiation of the Ti6Al4V
alloy with UV light. EIS shows that this treatment causes an initial increases on the
superficial electrical conduction that fade out after storing the sample in dark conditions
for a period of 24 hours. Mott-Schottky curves further confirmed these results. Thus, the
hypothesis stated on our previous work [16] relating the bactericidal properties of these
substrata with an increase on electrical surface currents is here corroborated. In addition,
the anionic polarization curves detected higher corrosion currents for the UV treated
material, however, EIS confirmed that these did not affect the good stability of the
material oxide film.
7.5. REFERENCES
[1] Fonseca C, Barbosa MA. Corrosion behaviour of titanium in biofluids containing
H2O2 studied by electrochemical impedance spectroscopy. Corros. Sci. 2001;43:
547-559.
[2] Zhang XL, Jiang ZhH, Yao ZhP, Song Y, Wu ZhD. Effects of scan rate on the
potentiodynamic polarization curve obtained to determine the Tafel slopes and
corrosion current density. Corros. Sci. 2009;51:581-587.
[3] Bao Y, Wang W, He B, Wang M, Yin Y, Liang L et al. EIS analysis of
hydrophobic and hydrophilic TiO2 film. Electrochim. Acta 2008;54:611-615.
[4] Vörös J, Wieland N, Ruiz-Taylor L, Textor M, Brunette DM. in: Brunette DM,
Tengvall P, Textor M, Thomsen P (Eds.), Titanium in medicine: material science,
surface science, engineering, biological responses and medical applications,
Springer-Verlag, Berlin, 2001; p87.
[5] Milosev I, Metikos-Hukovic M, Strehblow H-H. Passive film on osthopaedic
TiAlV alloy formed in physiological solution investigated by X-ray photoelectron
spectroscopy. Biomaterials 2000;21:2103-2113.
[6] Textor M, Sittig C, Frauchiger V, Tosatti S, Brunette DM, in: Brunette DM,
Tengvall P, Textor M, Thomsen P (Eds.), Titanium in medicine: material science,
surface science, engineering, biological responses and medical applications,
Springer-Verlag, Berlin, 2001, p. 171.
168
[7] Pedraza Avella JA, Acevedo Peña P, Pedraza Rosas JE. Photocatalytic oxidation of
cyanide on TiO2: An electrochemical approach. Catal. Today 2008;133-135:611-
618.
[8] Byrne JA, Eggins BR, Brown NMD, McKinnery B, Rouse M. Immobilisation of
TiO2 powder for the treatment of polluted water. Appl. Catal. B Environ
1998;17:25-36.
[9] Liu HL, Yang TCK. Photocatalytic inactivation of Escherichia coli and
Lactobacillus helveticus by ZnO and TiO2 activated with ultraviolet light. Process
Biochem. 2003;39:475-481.
[10] Gaswami DY, Trivedi DM, Block SS. Photocatalytic disinfection of indoor air. J.
Sol. Energy. Eng. 1997;119:92-96.
[11] Maneerat C, Hayata Y. Antifungal activity of TiO2 photocatalysis against
Penicillium expansum in vitro and in fruit tests. Int. J. Food Microbiol.
2006;107:99-103.
[12] Nonami T, Hase H, Funakoshi K. Apatite-coated titanium dioxide photocatalyst for
air purification. Catal. Today 2004;96:113-118.
[13] Kim B, Kim D, Cho D, Cho S. Bactericidal effect of TiO2 photocatalyst on selected
food-borne pathogenic bacteria. Chemosphere 2003;52:277-81.
[14] Gallardo Moreno AM, Pacha Olivenza MA, Saldaña L, Pérez Giraldo C, Bruque
JM, Vilaboa N et al. In vitro biocompatibility and bacterial adhesion of physico-
chemically modified Ti6Al4V surface by means of UV irradiation. Acta Biomater.
2008;5:181-192.
[15] Pacha Olivenza MA, Gallardo Moreno AM, Méndez Vilas A, Bruque JM,
González Carrasco JL, González Martín ML. Effect of UV irradiation on the
surface Gibbs energy of Ti6Al4V and thermally oxidized Ti6Al4V. J. Colloid
Interf. Sci. 2008;320:117-124.
[16] Gallardo Moreno AM, Pacha Olivenza MA, Fernández Calderón MC, Pérez
Giraldo C, Bruque JM, González Martín ML. Bactericidal behaviour of Ti6Al4V
surfaces after exposure to UV-C light. Biomaterials 2010; 31:5159-5168.
[17] Leng WH, Zhang Z, Zhang JQ, Cao CN. Investigation of the kinetics of a TiO2
photoelectrocatalytic reaction involving charge transfer and recombination through
surface states by electrochemical impedance spectroscopy. J. Phys. Chem. B
2005;109:15008-15023.
169
[18] Long M, Rack HC. Titanium alloys in total joint replacement -a materials science
perspective. Biomaterials 1998;19:1621-1639.
[19] Princeton NJ. Basic Electrochemical Impedance Spectroscopy (EIS). Application
Note AC-1. EG&G Princeton Applied Research 1989.
[20] Grosgogeat B, Boinet M, Dalard F, Lissac M. Electrochemical studies of the
corrosion behaviour of titanium and the Ti-6Al-4V alloy using electrochemical
impedance spectroscopy. Biomed. Mater. Eng. 2004;14:323-331.
[21] Xu J, Linlin L, Lu X, Jiang S. Effect of carbon doping electrochemical behavior of
nanocrystalline Ti5Si3 film in NaCl solution. Electrochem. Commun. 2011;13:102-
105.
[22] Wang M, Wang W, He BL, Sun ML, Bao YS, Yin YS et al. Corrosion behaviour of
hydrophobic titanium oxide film pre-treated in hydrogen peroxide solution. Mater.
Corros. 2010;62:320-325.
[23] Bao Y, Wang W, He B, Wang M, Yin Y, Liang L et al. EIS analysis of
hydrophobic and hydrophilicTiO2 film. Electrochim. Acta 2008;54:611-615.
[24] Mindroiu M, Pirvu C, Ion R, Demetrescu I. Comparing performance of
nanoarchitectures fabricated by Ti6Al7Nb anodizing in two kinds of electrolytes.
Electrochim. Acta 2010;56:193-202.
[25] Li M, Lou S, Wu P, Shen J. Photocathodic protection effect of TiO2 films for
carbon steel in 3% NaCl solutions. Electrochim. Acta 2005;50:3401-3406.
[26] Huang H-H. Surface characterization of passive film on NiCr-based dental casting
alloys. Biomaterials 2003;24:1575-1582.
[27] Shukla AK, Balasubramaniam R, Bhargava S. Properties of passive film formed on
CP titanium, Ti-6Al-4V and Ti-13.4Al-29Nb alloys in simulated human body
conditions. Intermetallics 2005;13:631-637.
[28] Tamilselvi S, Raman V, Rajendran N. Corrosion behaviour of Ti-6Al-7Nb and Ti-
6Al-4V ELI alloys in the simulated body fluid solution by electrochemical
impedance spectroscopy. Electrochim. Acta 2006;52:839-846.
[29] Alves VA, Reis RQ, Santos ICB, Souza DG, Goncalves TF, Preira-da-Silva MA et
al. In situ impedance spectroscopy study of the electrochemical corrosión of Ti and
Ti-6Al-4V in simulated body fluid at 25 ºC and 37 ºC. Corros. Sci. 2009;51:2473-
2482.
[30] Baron A, Simka W, Chrzanowski W. EIS tests of electrochemical behaviour of
Ti6Al4V and Ti6Al7Nb alloys. J. Achiev. Mat. Eng. 2007;21:23-26.
170
[31] Aparicio C, Gil FJ, Fonseca C, Barbosa M, Planell JA. Corrosion behaviour of
commercially pure titanium shot blasted with different materials and sizes of shot
particles for dental implant applications. Biomaterials 2003;24:263-273.
[32] Vasilescu C, Drob P, Vasilescu E, Demetrescu I, Ionita D, Prodana M et al.
Characterisation and corrosion resistance of the electrodeposited hydroxyapatite
and bovine serum albumin/hydroxyapatite films on Ti–6Al–4V–1Zr alloy surface.
Corros. Sci. 2011;53:992-999.
[33] Paszenda Z, Walke W, Jadacka S. Electrochemical investigations of Ti6Al4V and
Ti6Al7Nb alloys used on implants in bone surgery. J. Achiev. Mat. Eng.
2010;38:24-32.
[34] González JEG, Mirza-Rosca JC. Study of the corrosion behavior of titanium and
some of its alloys for biomedical and dental implant applications. J. Electroanal.
Chem. 1999;471:109-115.
[35] Harrington SP, Devine TM. Analysis of electrodes displaying frequency dispersion
in Mott-Schottky tests. J. Electrochem. Soc. 2008;155(8):381-386.
[36] Bozzini B, Carlino P, Durzo L, Pepe V, Mele C, Venturo F. An electrochemical
impedance investigation of the behavior of anodically oxidised titanium in human
plasma and cognate fluids, relevant to dental applications. J. Mater. Sci–Mater. M.
2008;19:3443-3453.
[37] Mogoda AS, Ahmad YH, Badawy WA. Corrosion behaviour of Ti–6Al–4V alloy
in concentrated hydrochloric and sulphuric acids. J. Appl. Electrochem.
2004;34:873-878.
[38] Stern M, Geary AL. Electrochemical Polarization: I. A theoretical analysis of the
shape of polarization curves. J. Electrochem. Soc. 1957;104(1):56-53.
[39] Wang D, Liu Y, Hu H, Zeng Z, Zhou F, Liu W. Electrochemical characterization of
the solution accessibility of CaTiO3 microstructures and improved
biomineralization. J. Phys. Chem. 2008;112:16123-16129.
[40] Hsu CH, Mansfeld F. Technical Note: Concerning the conversion of the constant
phase element parameter Y0 into a capacitance. Corrosion 2001;57:747-748.
[41] Huang Y, Shih H, Mansfeld F. Concerning the use of constant phase elements
(CPEs) in the analysis of impedance data. Mater. Corros. 2010;61:302-305.
[42] Baram N, Ein-Eli Y. Electrochemical impedance spectroscopy of porous TiO2 for
photocatalytic applications. J. Phys. Chem. C 2010;114:9781-9790.
171
[43] Nelson BP, Candal R, Corn RM, Anderson M. Control of surface and ξ potentials
on nanoporous TiO2 films by potential-determining and specifically adsorbed ions.
Langmuir 2000;16:6094-6101.
[44] Schiff N, Grosgogeat B, Lissac M, Dalard F. Influence of fluoridated mouthwashes
on corrosion resistance of orthodontics wires. Biomaterials 2004;25:4535-4542.
[45] Paul S, Yadav K. Corrosion Behaviour of surface-trated implant Ti-6Al-4V by
electrochemical polarization and impedance studies. J. Mater. Eng. Perform.
2011;20:422-435.
[46] Pan J, Leygraf C, Thierry D, Ektessabi AM. Corrosion resistance for biomaterial
applications of TiO2 films deposited on titanium and stainless steel by ion-beam-
assisted sputtering. J. Biomed. Mater. Res. 1997;35:309-318.
[47] Tsuchiya H, Macak JM, Ghicov A, Rader AS, Taveira L, Schmuki P.
Characterization of electronic properties of TiO2 nanotube films. Corros. Sci.
2007;49:203-210.
[48] Taveira LV, Sagues AA, Macak JM, Schmuki P. Impedance behaviour of TiO2
nanotubes formed by anodization in NaF electrolytes. J. Electrochem. Soc.
2008;155:C293-C302.
[49] Antunes RA, Oliveira MCL, Costa I. Study of the correlation between corrosión
resistance and semi-conducting properties of the passive film of AISI 316L
stainless steel in physiological solution. Mater. Corros. 2011;62:1-7.
[50] Baumanis C, Bahnemann DW. TiO2 thin film electrodes: correlation between
photocatalytic activity and electrochemical properties. J. Phys. Chem. C
2008;112:19097-19101.
[51] Schmuki P, Bohni H, Mansfeld F. A photoelectrochemical investigation of passive
films formed by alternating voltage passivation. J. Electrochem. Soc. 1993;
140:L119-L121.
[52] Lee JB, Yoon SI. Effect of nitrogen alloying on the semiconducting properties of
passive films and metastable pitting susceptibility of 316L and 316LN stainless
steels. Mater. Chem. Phys. 2010;122:194-199.
[53] Ningshen S, Mudali UK. Hydrogen effects on pitting corrosion and semiconducting
properties of nitrogen-containing type 316L stainless steel. Electrochim. Acta
2009;54:6374-6382.
[54] Spagnol V, Sutter E, Chouvy CD, Cachet H, Baroux B. EIS study of photo-induced
modifications of nano-columnar TiO2 films. Electrochim. Acta 2009;54:1228-1232.
172
[55] Hagfeldt A, Bjorksten U, Gratzel M. Photocapacitance of nanocrystalline oxide
semiconductor films: band-edge movement in mesoporous TiO2 electrodes during
UV illimination. J. Phys. Chem. 1996;100:8045-8048.
173
CAPÍTULO 8
Conclusiones generales
174
1.- La implantación iónica con silicio provoca ligeros cambios en la hidrofobicidad
superficial del Ti6Al4V y es capaz de reducir la acumulación bacteriana sobre su
superficie en presencia de fuerzas de cizalladura.
2.- La irradiación con luz UV-C provoca cambios significativos en la hidrofobicidad y
energía de Gibbs superficial sobre la superficie del Ti6Al4V cuya magnitud viene
determinada no sólo por el tiempo de radiación sino por las características de la capa de
TiO2 formada sobre su superficie. La hidrofilización de la superficie es más rápida en la
superficie oxidada espontáneamente que en aquella oxidada térmicamente, sin embargo,
tras 15 h de radiación se consigue una superficie completamente hidrófila en ambos
casos. Este hecho fundamentalmente está relacionado con el significativo aumento del
parámetro electrón-donador de la componente ácido-base de la tensión superficial,
aspecto que indica una alta adsorción de grupos hidroxilos y agua orientada sobre la
superficie. En cualquier caso, los dos procesos de oxidación superficial en la aleación
deben compartir un mecanismo similar de hidrofilización debido a las relaciones
análogas mostradas por los cosenos de los líquidos prueba.
3.- Los gradientes térmicos durante la fabricación de barras anchas de Ti6Al4V
provocan ligeros incrementos en la fracción en volumen de la fase β al desplazarnos
desde el exterior al interior de las probetas. Este hecho hace que aparezca una
dependencia radial de la hidrofobicidad y energía de Gibbs superficial tras irradiar las
muestras con luz UV-C, la cual se minimiza aumentando el tiempo de exposición a la
radiación.
4.- La completa hidrofilización de la superficie del Ti6Al4V tras ser irradiada con luz
UV-C disminuye de forma significativa la adhesión de diferentes cepas de S.
epidermidis en condiciones de flujo así como el número de bacterias retenidas a dicha
superficie tras ser sometida a fuerzas de cizalladura simulando condiciones in vivo. El
modelo teórico de adhesión basado en la aproximación X-DLVO ha permitido
relacionar directamente los cambios físico-químicos en la superficie antes y después de
su irradiación con las pendientes iniciales de adhesión a través de la energía de Gibbs de
interacción evaluada especialmente a distancias correspondientes al mínimo secundario
de energía.
5.- Tras el cese de la radiación UV-C la superficie de la aleación Ti6Al4V exhibe un
novedoso comportamiento bactericida residual cuyo origen no parece estar relacionado
175
con reacciones fotocatalíticas sino con procesos de emisión de energía y fenómenos
eléctricos superficiales. Se ha comprobado que dicho efecto presenta una duración
temporal garantizado, en cualquier caso, en las primeros momentos de formación de la
biocapa, período crucial para el éxito o fracaso del implante.
6.- A través de análisis electroquímicos se han podido detectar cambios en propiedades
eléctricas superficiales del Ti6Al4V tras ser irradiado con luz UV-C. La espectroscopía
de impedancia electroquímica y los diagramas Mott-Schottky han demostrado que
después de la radiación se incrementa la conducción eléctrica superficial como
consecuencia de la recombinación de pares electrón-hueco en el TiO2 presente en la
superficie. Este hecho también está relacionado con un aumento en las corrientes de
corrosión sobre la muestra irradiada que parecen no afectar la buena estabilidad de la
aleación, ya que ésta se recupera completamente en condiciones de oscuridad, hecho
que está de acuerdo con la transitoriedad del efecto bactericida.