The Influence of Titanium Dioxide on Diamond-Like Carbon Biocompatibility for Dental Applications
The physical and chemical characteristics of diamond-like carbon (DLC) films make them suitable for implantable medical and odontological interests. Despite their good interactions with biological environment, incorporated nanoparticles can significantly enhance DLC properties. This manuscript studies the potential of titanium dioxide (TiO2) incorporated-DLC films in dental applications. In this scene, both osteoblasts attachment and spreading on the coatings and their corrosion characteristics in artificial saliva were investigated. The films were grown on 304 stainless steel substrates using plasma enhanced chemical vapor deposition. Raman scattering spectroscopy characterized the film structure. As the concentration of TiO2 increased, the films increased the osteoblast viability (MTT assay), becoming more thermodynamically favorable to cell spreading ( values became more negative). The increasing number of osteoblast nuclei indicates a higher adhesion between the cells and the films. The potentiodynamic polarization test in artificial saliva shows an increase in corrosion protection when TiO2 are present. These results show the potential use of TiO2-DLC films in implantable surfaces.
Implantable surfaces probably have the main contribution for increasing the implant dentistry. Complex reactions at tissue-material interface settle the osseointegration and the long-term success of the implants . Therefore, a surface modification to improve biocompatibility is mostly necessary . In this scene, diamond-like carbon (DLC) coatings can reveal wear resistance, hardness, and corrosion resistance to a medical device surface [3–6]. These coatings consist of dense amorphous carbon or hydrocarbon and their mechanical properties fall between those of graphite and diamond [3, 4]. Some studies reported modified-DLC films improved biocompatibility, lubricity, stability, and cell adhesion [6, 7]. However, incorporated nanoparticles can change DLC performance according to the individual properties of each nanoparticle . Yun et al.  related these characteristics to structural bonds , surface roughness , and whether the film is hydrophobic or hydrophilic.
A thin native oxide layer formed spontaneously on titanium surface caused by air, water, or any other electrolyte assigns titanium biocompatibility . This layer is responsible for bone-bonding characteristics of titanium implants . The photocatalytic nature of titanium dioxide (TiO2) enables its use as antibacterial agent for decompose organisms [13–15]. These properties are strongly depending on the crystalline structure, morphology, and crystallite size [13, 15].
In the last recent years, previous manuscripts reported production and characterization of TiO2-DLC films for biological applications [15–17]. However, besides the cytotoxicity, odontological applications also depend on the corrosion resistance [1, 18, 19]. A hostile electrolytic environment, as the mouth, causes gradual degradation of metallic biomaterials by electrochemical attack . This manuscript studies the potential of TiO2-DLC films in dental applications. In this scene, both osteoblasts attachment and spreading on the coatings and their corrosion characteristics in artificial saliva were investigated.
2. Experimental Procedures
The 304 stainless steel (10 mm × 10 mm 1 mm) was the substrates. DLC and TiO2-DLC films were deposited using plasma enhanced chemical vapor deposition to a thickness of around 2.0 μm . Hexane was the feed gas to produce DLC films. Dispersions of TiO2 (Aeroxide® from Evonik), in anatase crystalline form (average particle size of 17 nm), in hexane (0.1 and 0.5 g/L), substitute hexane to produce TiO2-DLC films.
Raman scattering spectroscopy (Renishaw 2000 system with an Ar+-ion laser (λ = 514 nm) in backscattering geometry) analyzed the atomic arrangement of the films.
All animal procedures were in agreement with guidelines of the Research Ethics Committee of the School of Dentistry in São José dos Campos (027/2008-PA/CEP). Enzymatic digestion harvested cells from newborn (2–4 days) Wistar rat calvaria . The cells were plated on samples in 24-well polystyrene plates (density of 2 104 cells/well) using α-MEM (Gibco), supplemented with 10% fetal bovine serum (Gibco), 50 mg/mL gentamicin (Gibco), 5 μg/L ascorbic acid (Sigma), and 7 mM β-glycerophosphate (Sigma). During the experiments, cells were incubated (37°C) in a humidified atmosphere (5% CO2) and the medium was changed every three days. The inverted microscope (CK40 Olympus) examined the progression of cultures.
The cell viability assay monitors the response and health of cells quantifying the mitochondrial activity by analyzing formazan crystals formed by reducing the salt 3-[(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) (Sigma-Aldrich). After treatment, 0.5 mg/mL of MTT was added to the cultures, which were incubated for 1 h (37°C), followed by 30 min gentle agitation in 200 mL of dimethyl sulfoxide. ELISA Spectracount Reader (Packard Instrument) with a 570 nm filter read the plates.
Phosphate-buffered saline (PBS) washed the cells. They were incubated for 10 min at room temperature with 300 nM 4′,6-diamidino-2-phenylindole and dihydrochloride (DAPI; Molecular Probes). For fixing, 200 mL paraformaldehyde (Sigma-Aldrich) at 4% was added to the wells and the plate incubated at room temperature for 10 min. Fluorescence microscope (DMLB Leica) examined the cells and a digital video camera (Leica DFC 300 FX) took the images.
One-way ANOVA (Graph Pad Prism 6®) analyzed the statistical differences. The populations from stainless steel, DLC, and TiO2-DLC films were obtained with normal distribution and independent to each experiment. Statistical differences pointed out values less than 0.05.
A sessile drop method with a Kruss EasyDrop (DSA 100) measured the contact angle () of the samples. According to Owens method , the contact angle values with two different liquids (distilled water and diiodomethane) calculated the surface energy. Thermodynamically, the adhesion and spreading of cells from a liquid suspension onto solid substrata can be described by , where is the interfacial free energy of adhesion, is the cell-solid interfacial free energy, the cell-liquid interfacial free energy, and is the solid-liquid interfacial free energy, respectively. If is negative, cell spreading is energetically favorable; while if is positive, cell spreading is thermodynamically unfavorable.
A conventional three-electrode cell performed the electrochemical tests. In this cell, a saturated Ag/AgCl electrode was the reference electrode, platinum wire was the counter electrode, and the stainless steel, DLC, and TiO2-DLC films were the working electrodes. The working electrode exposed area was 0.5 cm2. The electrolyte solution was a modified Fusayama artificial saliva , which consisted of NaCl (400 mg/L), KCl (400 mg/L), CaCl2·2H2O (795 mg/L), NaH2PO4·H2O (690 mg/L), KSCN (300 mg/L), Na2S·9H2O (5 mg/L), and urea (1000 mg/L). The electrolyte was adjusted to pH 6.3 by either lactic acid or sodium hydroxide and maintained at 37°C. Potentiodynamic tests were carried out by polarization of samples in the anodic direction, from −1.0 to +1.0 V, just after exposition to the electrolyte solution. The potential sweep rate was 1 mV/s. The electrochemical tests were performed at 37°C.
3. Results and Discussion
Raman scattering spectroscopy evaluated the chemical structure of the DLC films. Two broad bands compose the spectra for visible light, one centered in ~1330 cm−1 ( band) and other in ~1530 cm−1 ( band) . The and band positions were determined by subtracting a linear background and fitting a Gaussian function to the peak of the Raman spectrum (Figure 1). Table 1 summarizes the main parameters obtained through the spectra. The bond stretching of all pairs of sp2 atoms in both rings and chains causes the band [26, 27]. The breathing modes of sp2 atoms in rings caused the band and appears only in the presence of defects [26, 27]. The TiO2-DLC films presented a shift in and band positions towards higher wavenumbers. The full width at half maximum (FWHM) of band measures structural disorder and arises from bond length distortions . The presence of TiO2 nanoparticles in DLC films results in the increase of the intensity ratio of and area () and a shift of and bands towards higher wave numbers. These characteristics imply the increase of the graphite-like bonds in DLC matrix [26–28].
In this study, mouse osteoblastic cells were the cell culture system. Calvaria cells have a high proliferation capacity, being able to expand in vitro, which is appropriate to study interactions with biomaterials .
The MTT assay is a quantitative method for evaluating a cell response to external factors. It measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by NAD(P)H-dependent oxidoreductase enzymes, largely in the cytosolic compartment of the cell, specifically in mitochondrial compartment. The mitochondrial enzyme succinate-dehydrogenase within viable cells is able to cleave the tetrazolium salt into a blue product (formazan). The color produced is directly proportional to the number of viable cells .
Figure 2 shows the mitochondrial activity for all the studied samples. Stainless steel shows a significant difference when compared to control (osteoblast cells). The decrease number of viable cells on stainless steel characterizes cell death. There was no significant difference among DLC, TiO2-DLC (0.1 g/L), and the control. An increased concentration of TiO2 nanoparticles (0.5 g/L) in DLC films raised the mitochondrial activity on these samples, which are obviously nontoxic. TiO2-DLC (0.5 g/L) is significantly different () compared to DLC and extremely significant () when compared to stainless steel.
DAPI (blue) labeled the cell nuclei during 24 h (Figure 3). All the TiO2-DLC samples had a high cell attachment. The DAPI test showed a strong compatibility of the TiO2-DLC films. Figure 4 shows the counted osteoblast cells adhered on the samples. The number of cells on stainless steel is considered different from DLC films () and very different from TiO2-DLC films (). There was an increase in cells according to the increased concentration of TiO2 in DLC films. TiO2-DLC films are considered different from DLC films ().
The study of bone metabolism and biomaterial/cell, which is essential for bone tissue engineering, generally uses osteoblasts . However, not only cell adhesiveness and toxicity have to be investigated to design biomaterials. The existence of interface phenomena can affect the initial proliferation and cell recruitment at the biomaterial surface . The hydrophilicity tries to obtain correlation between cell response and the biomaterial surface .
Table 2 shows the contact angles of the samples formed with distilled water and diiodomethane. As the concentration of TiO2 nanoparticles in DLC films increased, the water contact angle decreased from 82 to ~53°, and in the case of diiodomethane, the contact angle had a slight increase. Usually, a hydrophobic surface has a contact angle higher than 70°, while a hydrophilic surface has a contact angle lower than 70° . These results indicate that TiO2-DLC is hydrophilic. This characteristic may be derivate from the amorphous TiO2 surfaces .
Table 2 also lists the surface energy components according to the Owens method . Dispersive ( mN/m) and polar ( mN/m) components gave the total surface energy (γ) of 40.0 mN/m for the as-deposited DLC. The interfacial free energy settles the wetting and so the wall shear stress when the liquid touches the surface . As the concentration of TiO2 in DLC increased, the total surface energy had a slight increase. The increasing in the polar component (a quantitative indicator of hydrophilicity) increases the total surface energy of TiO2-DLC. Oxide particles cause a higher polar component on TiO2-DLC surface . The polar component attracts the electric dipole of water, which minimizes the interfacial energy and the water contact angle . The water contact angle decreased as the polar component in the surface energy increased. The polar component attracts electric dipole of water molecule, which reduces the interfacial energy between the surface and the water and, thus, the wetting angle of water .
A thermodynamic approach offers a powerful tool to predict cell spreading to solids [36, 37]. For this, the surface free energy values of the cells () calculated by Schakenraad et al.  were used. Table 2 lists the average values of work of adhesion () for the studied samples. All the calculated values for for cell adhesion on DLC and TiO2-DLC films are negative. point out favorable conditions for cell adhesion, as provided by Schakenraad et al. . In addition, as the TiO2 concentration increases, values became more negative. This result suggests that as the TiO2 content in DLC films increased, DLC films became more thermodynamically favorable to cell spreading.
Figure 5 shows the correlation between the number of cells on the samples (Figure 4) and the calculated work of adhesion (Table 2). Besides the fact that there are two independent results, this comparison corroborates that TiO2 content increased the cell adhesion on DLC films.
However, one of the physical characteristics that settle the implant corrosion is the thermodynamic force [38, 39]. It causes corrosion by either oxidation or reduction reaction [38, 39]. Oral liquids, like saliva, attacks the metallic implants causing the dissolution of them . Therefore, the release of metallic ions caused by corrosion also affects the biocompatibility of the implant. The potentiodynamic polarization test assessed the corrosion susceptibility of the metallic implant in artificial saliva. Figure 6 shows the potentiodynamic curves for stainless steel, DLC, and TiO2-DLC films. Table 3 summarizes the electrochemical parameters obtained from the potentiodynamic polarization curves (Figure 6).
The penetration of the test solution causes the negative open circuit potential values . TiO2-DLC (0.1 g/L) films presented the greatest value (−0.146 mV) in potentiodynamic polarization test. The corrosion current density () reduced from 1.72 to 0.054 nA/cm2 and the protection efficiency  increased from 98.5 to 100.0% with the increase of TiO2 content. Lower current density and higher potential generally points out better corrosion resistance. The shift in the polarization curve towards the region of lower current density and higher potential is an indicative of the increased corrosion protection with the increased TiO2 concentration.
The difference in the film microstructure assigns different corrosion behaviors . The comparison between electrochemical parameters (Table 3) and chemical structure (Table 1) shows a tendency to reduce the corrosion current density of the samples as the film disorder increase. Robertson  states sp3 bonding of DLC confers on it many of the valuable properties of diamond itself, such as its chemical and electrochemical inertness. Generally, the higher the sp3/sp2 ratio, the higher the electrochemical corrosion resistance .
In this paper, the osteoblast attachment and spreading on DLC coatings was studied when TiO2 nanoparticles are incorporated. The presence of TiO2 nanoparticles increases the graphite-like bonds and decreases the DLC disorder. As TiO2 increased, the films increased the osteoblast viability (MTT assay), becoming more thermodynamically favorable to cell spreading ( values became more negative). This was evidenced through the increasing number of osteoblast nuclei suggesting a higher adhesion between the cells and the films. The potentiodynamic polarization test in artificial saliva shows an increase in corrosion protection with the increased TiO2. These results show the potential use of TiO2-DLC films in implantable surfaces for dental applications.
The authors declare that they have no competing interests.
The authors thank Professor Dr. Rosilene Fernandes da Rocha and Dr. Isabel Chaves Silva Carvalho from School of Dentistry, Paulista State University, São José dos Campos, Brazil, for kindly providing the cells from newborn Wistar rat calvaria and Sao Paulo Research Foundation, FAPESP (2011/17877-7), (2011/20345-7), (2012/15857-1), and (2013/20054-8) for the financial support.
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