Cellular Functionality on Nanotubes of Ti-30Ta Alloy
Patricia Capellato1,*,+, Barbara S. Smith2,+, Ketul C. Popat2,3,#, Ana P. R. Alves Claro1,#
1
Department of Materials, Faculty of Engineering Guaratinguetá, Sao Paulo State University-
UNESP, Av. Ariberto Pereira da Cunha, 333, Pedregulho, CEP 12516-410, Guaratinguetá, SP,
Brazil.
2
3
School of Biomedical Engineering, Colorado State University, Fort Collins CO 80523, USA
Department of Mechanical Engineering, Colorado State University, Fort Collins, CO 80523,
USA
+,#
Authors contributed equally to this article
*Author of correspondence: Patricia Capellato, Department of Materials, Faculty of Engineering
Guaratinguetá, Sao Paulo State University- UNESP, Av. Ariberto Pereira da Cunha, 333,
Pedregulho, CEP 12516-410, Guaratinguetá, SP, Brazil
E-mail: pat_capellato@yahoo.com.br
Abstract
Cellular Functionality on nanotubes of Ti-30Ta Alloy is the subject of this research. Recent
studies have identified strong correlations between anodized metals and the production of highly
biomimetic nanoscale topographies. These surfaces provide an interface of enhanced
biocompatibility that exhibits a high degree of oxidation and surface energy. In this study, the
mechanical substrate and topographical surface properties on nanotubes of Ti-30Ta alloy were
investigated using scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS)
and contact angle measurement. The anodization process was performed in an electrolyte
solution containing HF (48%) and H2SO4 (98%) in the volumetric ratios 1:9 with the addition of
5% dimethyl sulfoxide (DMSO) at 35V for 40 min. Human dermal fibroblasts (HDF, neonatal)
were utilized to evaluate the biocompatibility of Ti-30Ta nanotubes after 1 and 3 days of culture.
The results presented identify altered material properties and improved cellular interaction on Ti30Ta nanotubes as compared to the control substrates.
Key Words: Ti-30Ta nanotube arrays; Human dermal fibroblasts.
1. Introduction
In order to facilitate the long-term success of implantable devices, both the substrate [19]
and the surface [20] must be considered. The ideal implantable biomaterial should embody
mechanical properties similar to that of the natural tissue with which it will be interacting [21].
Clinical applications currently utilize materials such as titanium (Ti) and titanium alloys, steel,
cobalt alloys, and tantalum (Ta) [19, 27, 28] [29]. New developments in material science have
motivated alloy-directed research, showing a high correlation between improved mechanical
properties and alloyed titanium substrates with various non- allergic and non-toxic metals, such
as Ta, Zr, Nb, Hf, Mo, and Sn [13, 19, 29-42].
Of the alloys tested, 30% Ta with Ti exhibits
lower elastic modulus, improved strain-resistance and elongation to failure, and favorable
biocompatibility [29-33, 40, 47]. Current approaches used to modify material interfaces utilize
novel techniques such as annodization [49, 50], alkaline and heat treatments [34, 35], ion
implantation [51, 52], electrochemical etching [45, 53], simulated body fluid (SFB) [25, 54] and
plasma spray coatings [38, 55]. These surfaces have been shown to provide an interface that
exhibits a higher degree of oxidation and surface energy [61], as well as improved
biocompatibility.
Previous studies have shown the evidence supporting the use of anodized titanium for
implantable devices that interact with both hard and soft tissue [49, 50, 55, 56, 58, 59]. In this
the surface topographical properties of Ti-30Ta nanotubes were investigated using scanning
electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and contact angle
measurement [62]. The biocompatibility was evaluated using human dermal fibroblasts (HDF,
neonatal). The cell adhesion, proliferation, viability, cytoskeleton organization, and morphology
were investigated using fluorescence microscope imaging, biochemical assay and SEM imaging
respectively. The results presented identify altered mechanical properties, and improved cellular
interaction on Ti-30Ta nanotube as compared to the control substrates.
2. Methods and Materials
2.1 Anodization of Ti-30Ta Alloy
The anodization process was performed using platinum as the counter electrode and the
Ti-30Ta alloy substrate as the working electrode connected to a power supply (Fisher Scientific
FB300 Electrophoresis). The electrolyte solution contained HF concentrate (48%) and H2SO4
(98%) in the volumetric ratios 1:9 with the addition of 5% dimethyl sulfoxide (DMSO) [49]. The
experiment was performed at room temperature. In addition, the annealing of the Ti-30Ta
nanotube was performed in an oxygen ambient furnace at 530ºC, with a ramping rate of 1º C/min
for 3 hrs. Following annealing, all substrates were stored in a dissector until further
characterization.The surface topography of the anodized substrates was characterized by SEM
imaging (JEOL JSM 6100).
In order to obtain cross-sectional images, the surface was
mechanically scratched with metallic tool and imaged by tilting the chamber to 70°. The
elemental surface composition was determined using EDS (JOEL JSM 6100).
The
hydrophilicity of the substrate surfaces was investigated by a sessile drop method (2 ml) using a
contact angle goniometer (Kruss DSA 10) equipped with video capture.
2.2 Cell Culture
Human Dermal Fibroblast (HDF, Clonetics) cells, isolated from neonatal. The cell
density was determined by trypan blue dye exclusion, using a hemocytometer. The experiments
for this study were performed using fourth passage HDF cells.
HDF cells were seeded on polished Ti30Ta (control) and anodized Ti30Ta alloys
(substrate diameter: 3.0 mm) in a 24-well plate.
The substrates stained for DAPI were
concurrently stained F-actin to evaluate their cytoskeletal organization using fluorescence
microscope imaging.
The substrates were incubated in rhodamine-conjugated phalloidin
(dilution 1:40) for 20 min at room temperature to stain for F-actin on the cell membranes. The
substrates were imaged with a fluorescence microscope using DAPI BP 445/50 blue filter and
HQ Texas Red BP 560/40 red filter (Zeiss). The cell morphology was investigated using SEM
imaging to visualize the cellular interaction with the nanotube architecture, after 1 and 3 days of
culture.
Each experiment was reconfirmed on at least three different substrates from (nmin = 3).
All the quantitative results were analyzed using an analysis of variance (ANOVA). Statistical
significance was considered at p < 0.05. During the analysis, variances among each group were
not assumed to be equal and a two-sample t-test approach was used to test the significance
between the Ti-30Ta alloys and the Ti-30Ta nanotube. This analysis was conducted using the
Microsoft Office Excel data analysis software.
3. Results and Discussion
The anodized surface has been shown enhanced osseointegration in Ti cp, Ti-binary and
Ti-ternary alloys [16, 20, 50, 60, 65]. Thus, in this study Ti-30Ta substrates with a vertically
oriented array of nanotubes have been evaluated to determine their effect on HDF cells.
Fibroblast cells proliferate rapidly in vivo, producing a highly dense matrix of proteins and
growth factors through an organized network of cells with elongated morphologies [59]. In this
study, the functionality of fibroblast cells has been investigated after 1 and 3 days of culture on
Ti-30Ta nanotube as compared to the control substrates.
The surface charge of a biomaterial is among the most important properties in an
implantable device. This trait translates into the hydrophilicity or relative wettability of a
material, and plays a key role in directing cell-material interaction. In biomedical applications,
lower hydrophilicity or increased hydrophobicity is required for improved cellular interaction.
Previous studies have related the surface energy of a biomaterial with cellular functionality such
as protein adsorption, platelet adhesion and activation leading to blood coagulation, and bacterial
adhesion [52, 64, 76]. Thus, in this study, the hydrophilic behavior of anodized and unaltered
Ti-30Ta substrates was investigated by measuring their respective contact angles.
The cell cytoskeleton is a structural masterpiece, composed of a network of
microfilaments, microtubules and intermediate filaments; whose organization is indicative of
intra- and extra-cellular communication, integration, recruitment and differentiation. Cellular
health, reproduction, tissue mechanics and cell/tissue functionality are all dependent on the
ability of the cell cytoskeleton to reorganize itself. Thus, the substrates stained for DAPI were
concurrently stained F-actin to evaluate their cytoskeletal organization using fluorescence
microscope imaging, after 1 and 3 days of culture. The fluorescence images confirmed the
presence of cytoskeletal elongation on both substrates; once again identifying the Ti-30Ta alloy
as promoting positive cellular proliferation, communication and integration.
However, the
definitive presence of spherical cells on the untreated Ti-30Ta substrate indicates reduced
cellular integration as compared to the nanotube Ti-30Ta substrates. This heightened cellular
interaction and integration seen on the nanotube surface will likely lead to increased protein
expression and extracellular matrix production on and around the biomaterial interface, further
improving biomaterial integration.
SEM imaging was utilized to identify the effects of the nanotube interface with respect to
fibroblast morphology after 1 and 3 days of culture. The results indicate a considerable increase
in short-term fibroblast-nanotube interaction. After 3 days of culture, the unaltered Ti-30Ta
substrate shows a clear mixture of both activated and unactivated cells on the material surface;
however high-magnification images of the nanotube Ti-30Ta substrates identify extracellular
matrix production, not present on the control substrates.
Cellular interactions with their
environment direct further tissue and ECM production. The results of the nanotube Ti-30Ta
substrates identify a promising material interface for improved tissue-biomaterial integration.
4. Conclusion
The surface properties of the Ti-30Ta nanotube show improved wettability properties as
compared to Ti-30Ta alloy. Surface elemental composition analysis confirmed reduction in the
amount of tantalum at the material interface. The water-drop method identified a contact angle
of 15.04˚ on the nanotube surface, indicating a hydrophilic interface, which is preferable for
eliciting a favorable environment for cellular interaction. Cellular analysis identified improved
fibroblast functionality on the nanotube surface, showing increased elongation, and extracellular
matrix production on the Ti-30Ta nanotubes. In conclusion, this is the first study on fabrication
of nanotubes on Ti-30Ta alloy surface and evaluating cellular interaction on the nanotube
architecture. Thus, the formation of the nanotube on Ti30Ta alloy may have potential application
as interface for implantable devices.
5. Acknowledgements
Partial funding support for this work was provided by Brazilian agencies CNPq via grant
Doctored sandwich, project number 201271/2010-9. The authors would like to thank Patrick
McCurdy for his assistance with scanning electron microscopy.
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