Surface properties and biocompatibility of nanostructured TiO2 film deposited by RF magnetron sputtering
© Majeed et al.; licensee Springer. 2015
Received: 26 November 2014
Accepted: 5 January 2015
Published: 11 February 2015
Nanostructured TiO2 films are deposited on a silicon substrate using 150-W power from the RF magnetron sputtering at working pressures of 3 to 5 Pa, with no substrate bias, and at 3 Pa with a substrate bias of −50 V. X-ray diffraction (XRD) analysis reveals that TiO2 films deposited on unbiased as well as biased substrates are all amorphous. Surface properties such as surface roughness and wettability of TiO2 films, grown in a plasma environment, under biased and unbiased substrate conditions are reported according to the said parameters of RF power and the working pressures. Primary rat osteoblasts (MC3T3-E1) cells have been cultured on nanostructured TiO2 films fabricated at different conditions of substrate bias and working pressures. The effects of roughness and hydrophilicity of nanostructured TiO2 films on cell density and cell spreading have been discussed.
KeywordsTiO2 Working pressure Substrate bias Roughness Wettability Magnetron sputtering Cell culturing
The use of biomaterials dates far back to ancient civilizations. Artificial eyes, ears, teeth, and noses were found on Egyptian mummies [1,2]. Waxes, glues, and tissues for the restoration of the missing or malfunctioning parts of the body were used by the Chinese and Indians. Over the centuries, developments in synthetic materials, surgical techniques, and sterilization methods have permitted the use of biomaterials in many ways [2,3]. Nowadays, a large number of devices and implants are used as a medical practice. Biomaterials in the form of implants (ligaments, vascular grafts, heart valves, intraocular lenses, dental implants, etc.) and medical devices (pacemakers, biosensors, artificial hearts, etc.) are extensively used to replace and/or restore the function of disturbed or deteriorated tissues or organs, and thus improve the quality of life of the patients.
Commercially pure titanium (CP-Ti) and its alloys are chosen and extensively used as biomaterials, i.e., for dental and orthopedic implants or prosthesis, because of their better corrosion resistance, lower modulus, superior biocompatibility, durability, and strength [2,4,5]. However, being bioinert, it is assumed that the integration of such implants in bone is not good enough. The biocompatibility of titanium used as an implant material is accredited to surface oxides naturally formed in air and/or physiological fluids [6,7]. The surface properties, including composition, roughness, hydrophilicity, texture, and morphology of the oxide on titanium, greatly influence the cellular behaviors, e.g., adhesion, morphologic change, functional alteration, proliferation, and differentiation [4,7-9]. Among surface properties, surface roughness and composition have been considered the most essential parameters for altering cell activity . Titanium and its alloys, owing to their lower elastic modulus, are widely used as hard tissue replacements in artificial bones, joints, and dental implants, which, in general, are regarded as a biomechanical advantage on account of their smaller elastic modulus that can result in a smaller stress shielding [5,11].
The purpose of this work is to study the in vitro behavior of osteoblast cells cultured on nanostructured TiO2 film and investigate the effect of the nanostructured surface of TiO2 film on osteoblast cell density and cell spreading. Such accelerated cell density and cell spreading are beneficial for faster cure of dental and orthopedic patients, as well as for a variety of biomedical diagnostic and therapeutic applications .
Over the past few decades, implant coating has documented a wide range of applications. Thin-film coating of implant surfaces has been studied by several methods, including plasma spraying, dipping, electro-chemical deposition, pulsed laser deposition, ion beam dynamic mixing, and ion beam deposition . Some of these methods have severe limitations such as poor adhesion, microcrack formation and phase changes at high temperature, non-uniformity, and improper microstructural control, all of which make them inadequate for implant systems [14,15].
Most recently, magnetron sputtering deposition has been proposed by many researchers as a flexible deposition technique that offers many advantages including high-deposition rates; ease of sputtering any metal, alloy or compound; the formation of high-purity films; extremely high adhesion to films; and the ability to form dense coatings [16-18].
In this letter, we report the effect of working pressure and substrate bias on the surface roughness of a TiO2 film deposited by RF magnetron sputtering and see the influence of surface roughness in nanoscale  on surface hydrophilicity and cell behavior over the surface.
Fabrication of nanostructured TiO2 films
Summary of deposition conditions of TiO 2 films
1.4 ×10−3 Pa
3 Pa at a substrate bias of 0 V,
5 Pa at a substrate bias of 0 V,
3 Pa at a substrate bias of −50 V
5 Hrs for each sample
Argon flow rate
Oxygen flow rate
Target to substrate distance
Diameter of silicon (100) substrate
Diameter of the titanium target
The phase characterization of the TiO2 films deposited on silicon substrates at different working pressures with RF power of 150 W, with and without bias, was carried out by X-ray diffraction (XRD), using CuKα radiation (λ = 0.154056 nm) for 2θ values ranging from 20° to 80°. The diffractometer (XRD, Rigaku D/max 2550 VB/PC, Rigaku, Tokyo, Japan) was operated at 40 kV and 200 mA with a scanning speed of 8°/min at 2θ steps of 0.020°. The angle of the incident beam was 0.9°. The surface topography of the said TiO2 films, deposited under the same conditions, was characterized by atomic force microscopy (AFM, Nanoscope 3A, DI, USA) and the root-mean-square (RMS) roughness was estimated by an image analysis software called Nanoscope®III. The wettability of the films' surface was observed through water contact angle measurements, using contact angle measurement equipment (OCA 20, Dataphysics, Germany).
Primary rat osteoblasts (MC3T3-E1) were cultured for 7 days in a humidified atmosphere of 5% CO2 –95% air at 37°C in 25 cm2 flasks until confluent. Cells were then detached using trypsin/EDTA (0.25% w/v trypsin/0.02% EDTA of pH 7.2). Subsequently, cells were re-suspended in the supplemented culture medium as described above and seeded with the density of 2 × 104 cells/cm2 on the specimen surfaces for the biocompatibility study. After being fixed in 4% paraformaldehyde (Sigma, USA), cells were stained with acridine orange (AO, Sigma, USA), and the cells' behavior for 12 h over the specimen surface was examined with a fluorescent microscope (×200).
Results and discussions
X-Ray diffraction analysis
When a negative bias is applied to the substrate, it can lead to an increase in the energy of the surface atoms resulting in an enhanced surface diffusion, which can cause better adhesion, nucleation, and crystal structure . When the substrate is biased to −50 V, more energy is transferred from ions driven by the substrate bias to the growing film, which can make the film more compact. Nevertheless, the transferred energy is not high enough to make the film crystallinized; thus, the TiO2 film, obtained under the substrate bias condition, shows an amorphous structure .
Surface wettability analysis
This theoretical relation is true only for ideally smooth and homogeneous solid surfaces.
For more authenticity and reliability of the results, the contact angle measurements were also carried out dynamically. If the three-phase contact line is in actual motion, the contact angle produced is called a ‘dynamic’ contact angle. In particular, the contact angles formed by expanding and contracting the liquid are referred to as the advancing contact angle \( \theta \)a and the receding contact angle \( \theta \)r, respectively.
Usually the hysteresis is greater for the rough surfaces, but it is dominated by chemical interactions and heterogeneities rather than roughness itself . Nonetheless, to the best of our knowledge and studies, no one has reported the effect of contact angle hysteresis on the cell behavior over surface. So further investigation is needed to analyze the contact angle hysteresis on the cell behavior over the surface.
The results might be well elaborated in terms of surface energy, which could play a very vital role on the surface wettability, and the surfaces with high surface energy (greater surface roughness) are usually regarded as hydrophilic. According to the illustration of the film deposition at different working pressures, a higher working pressure leads to a rougher surface with larger surface defects and greater surface energy, which results in a more hydrophilic surface as shown in Figure 4. Moreover, Figures 4 and 5 reveal the comparison of static as well as the dynamic (both advancing and receding) contact angles, indicating that the corresponding static as well as dynamic angles are showing the same increasing/decreasing trend. This clearly justifies that the experimental results are well consistent with each other.
Cell density and cell spreading on TiO2 samples
The number of adherent cells was determined by means of nuclei quantification on the TiO2-nano, Ti-nano, and Ti-micro substrates . The interaction between the outermost surface of a biomaterial and its environment was a highly dynamic process, in which direct and indirect cell adhesions induced by protein previously adsorbed onto the surface were two competing processes. In general, the direct cell adhesion tends to occur efficiently on hydrophilic surfaces but inefficiently on hydrophobic surfaces, whereas indirect cell adhesion dominates over smooth and hydrophobic surfaces .
In comparison with samples 1 and 3, sample 1 with a rough surface promotes direct cell adhesion; on the contrary, sample 3 with a smooth surface encourages indirect cell adhesion through adsorption of proteins on its surface. That is probably the main reason that there is no big difference in cell densities over the samples 1 and 3. As regards sample 1 and 2, both of them having rough surfaces are dominated by direct cell adhesion, since sample 2 is rougher and more hydrophilic than sample 1, so cell density over sample 2 is relatively higher.
On the other hand, most of the studies [36,37] indicate that the cell tends to spread on the hydrophilic surfaces rather than hydrophobic surfaces, which is consistent with our experiment. More studies are still underway to investigate the effect of surface roughness and wettability on the biocompatibility of the TiO2 films including cell adhesion, proliferation, and differentiation.
In this article, TiO2 films, with different surface roughnesses measured in nanometer scale, were deposited on a silicon substrate using a power level of 150 W from the RF magnetron sputtering at different working pressures under varying bias conditions for biocompatibility analysis. The XRD analysis of the TiO2 film revealed its amorphous nature. Water contact angle measurements clarified that the rough surface was more hydrophilic than the smooth surface, which was well elaborated in terms of surface energy that could play a very vital role on the surface wettability.
It was concluded that the surface could be designed to influence cell density and cell spreading, and RF reactive magnetron sputtering method could be a potential method to improve the cytocompatibility of titanium-based implants by depositing a layer of TiO2 with suitable roughness.
The work was financially supported by the National Natural Science Foundation of China (grant no. 11275127, 90923005, 31400859) and Biomedical Engineering Cross Research Foundation of Shanghai Jiao Tong University (YG2013MS60). Asif Majeed acknowledges the financial assistance provided by the Chinese Scholarship Council for providing him the Distinguished Chinese Scholarship for pursuing his PhD studies in Shanghai Jiao Tong University. The authors would like to thank Mrs. H.Q. Li (Analysis and Measurement Center of Shanghai Jiao Tong University) for AFM measurements.
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