Dental implants are increasingly important for the restoration of tooth defects and for replacements. The surface morphology and properties of dental implants will determine the adhesion, proliferation, and differentiation of cells, as well as the initial fixation and long-term survival of the implants.1,2 To improve biocompatibility, bioceramic coatings have been used on dental implants; however, there have been major concerns about their application. A promising alternative approach is surface modification by ion implantation-deposition.3 The present study investigated the implantation of zinc (Zn) ions into commercially pure titanium (cp-Ti) by plasma immersion ion implantation-deposition (PIIID). The chemistry of the modified surface and the effects of this modification on the focal adhesion plaque formation of human osteoblast-like cells were characterized.
Twenty-four disk-shaped titanium (Ti) specimens was fabricated into disc-shaped samples measuring10 mm in diameter and 1mm in thickness, and discs were machined from Commercial class-4 pure Ti (Baoji, Shanxi Province, China) rods (cp-Ti, TA3 purity 99.99%; Baoji Nonferrous Metal Processing Factory, Baoji, Shaanxi, China). They were then ground and polished on a grinding machine to an average surface roughness (Ra) of 0.4 μm, and then sonicated sequentially in acetone and anhydrous ethanol (20 minutes each). Twelve specimens (referred to without Zn-ion implantation or deposition as “cp-Ti”) were stored in a sealed container for subsequent use. The other twelve specimens (referred to as “Zn-Ti”) were subjected to Zn implantation in vacuum using a fourth-generation PIIID device developed by the State Key Laboratory of Advanced Welding Production Technology (Harbin Institute of Technology, Harbin, Heilongjiang Province, China). Each Zn-Ti specimen was cleaned by argon ion (Ar+) sputtering for 10 minutes, followed by Zn implantation for 80 minutes using the implantation source included a pulsed cathodic arccplasma with the following parameters: The implantation voltage (V) was 20 kV, the implantation pulse width (τ) was 300 μs, the Zn cathodic arc pulse width was 300 μs, and the operating pressure (P) was 0.1 Pa. All specimens were sterilized with ethylene oxide before use.
X-ray photoelectron spectroscopy (XPS)
XPS was performed with 300 W Al Kα radiation under 3×10–9 mbar vacuum (VG ScientESCALab220i-XL, Thermo VG Scientific, Hastings, UK). The excitation source was an Al Kα X-ray with approximately 300W of power. The basic vacuum degree for the analysis was 3×10–7 Pa.The electron binding energy was calibrated against the C1s peak (284.8 eV) from trace hydrocarbon contamination.
Focal adhesion plaque formation
The cp-Ti and Zn-Ti specimens were placed in a 24-well culture dish. A human osteoblast-like (MG-63; Chinese Academy of Medical Sciences, Beijing, China) cell suspension (2×104 cells/ml) was dispensed (1 ml) on each specimen. After a short period, 1 ml of minimum essential medium (α-MEM) was slowly added to each well along the sidewall and the dish was incubated under standard conditions (37°C, 100% relative humidity, and 5% CO2).
Focal adhesion plaque characterization
After incubation for 6, 24, and 48 hours, respectively, four cp-Ti and four Zn-Ti specimens were removed from the dish. Each specimen was rinsed with phosphatebuffered saline, fixed in 4% formaldehyde for 20 minutes, and immersed in 0.1% Triton X-100 for 20 minutes to increase cell permeability. After blocking nonspecific sites by immersion in 1% bovine serum albumin for 40 minutes, each specimen_was sequentially incubated with mouse monoclonal IgG, IgG recognizing human vinculin (1:50 diluted; 4°C in the dark overnight) and fluorescein isothiocyanate, FITC-labeled goat anti-mouse IgG (1:200 diluted; 37°C in the dark for 2 hours). Each specimen was then mounted in an antifade medium and examined for focal adhesion plaques under a laser confocal microscope. Twenty cells were randomly selected on each specimen for the purpose of counting the number of adhesion plaques by image analysis (Leica confocal software 4.0, Ernst Benz Company Lai, Germany).
Data were presented as mean ± standard deviation (SD) and analyzed using SPSS16.0 statistical software (SPSS Inc., USA). One-way analyses of variance followed by a Tukey test were employed to determine the statistical significance, which was accepted when P <0.05. All experiments were carried out in triplicate.
The cp-Ti specimen (Figure 1A) exhibited XPS peaks for Ti, oxygen (O), and carbon (C). The Ti2p3/2 peak at 458.5 eV (Figure 2A) is attributed to titanium dioxide (TiO2). The Zn-Ti (Figure 1) had additional 2p3/2 peaks at 532.0 eV and 530.2 eV. Gaussian fits of the spectra (Figure 2B) indicate that the two peaks are attributed to TiO2 and zinc oxide (ZnO), respectively.
Focal adhesion plaque formation
Both cp-Ti and Zn-Ti displayed focal adhesion plaques on their surfaces, which appeared as green fluorescence from the immunostained vinculin (Figure 3). On both surfaces, the densities of focal adhesion plaques increased with increasing incubation time (Figure 4). After incubation for 6, 24, and 48 hours, the Zn-Ti surface had 60.16±16.37, 70.31±16.16, and 75.43±13.01 focal adhesion plaques, respectively, while the cp-Ti surface had 46.13±13.15, 63.33±16.03, and 73.68±16.76 plaques, respectively (both counted from 20 cells, n=12). After 6 hours of incubation, the difference between the two groups was statistically significant (P=0.025). After 24 and 48 hours, both surfaces had large numbers of focal adhesion plaques and the difference between them was not statistically significant (P=0.23 and 0.44 respectively).
The PIIID technique modifies surface properties by accelerating ionized atoms or molecules into the solid substrate. It has been shown to dramatically improve the resistance of metallic materials to surface wear, corrosion, and fatigue.4 PIIID overcomes the line-of-sight effect of traditional ion-beam implantation techniques and works well for substrates with complex geometries. Recently, PIID has been used to modify the surfaces of biomaterials.5,6 It can form a well-adhered gradient thin film with improved biocompatibility, without affecting the bulk properties of the substrate. In the present study, Gaussian fits of the XPS Ti2p peak suggested that the cp-Ti surface consisted primarily of TiO2, and that the Zn-Ti surface contained TiO2 and ZnO, thus confirming that PIIID successfully implanted Zn into the Ti to form a modified surface.
Cell attachment to a biomaterial surface is a complicated process involving multiple steps such as initial adhesion, spreading, cytoskeleton reassembly, focal adhesion plaque formation, and extracellular matrix (ECM) production and reorganization. Cellular adhesion is related to intracellular signaling pathways, whereas focal adhesion plaques and cytoskeletons are prerequisites for intracellular signaling in mature cells. The focal adhesion plaque is a complex of cytoplasmic proteins. Integrins bind to the ECM and associate with adhesion plaques to form an ECMintegrin- adhesion plaque complex. This complex transmits extracellular signals (e.g., from growth factors) into cells to induce changes in cytoskeletons, and subsequently a series of changes in cell morphologies and functions.7 Studies have shown that differences in ECM composition, structure, and physical properties result in different focal adhesion plaques. These differences allow the ECMintegrin- adhesion plaque complex to send different signals and thereby activate different cell functions.8 The formation of focal adhesion plaques can be visualized by fluorescent staining of vinculin, an adhesion plaque protein involved in the linkage of integrin to the cytoskeleton. Vinculin maintains the integrity of cell membranes contacting extracellular materials, and the lack of positive vinculin staining indicates the absence of favorable cell-substrate adhesion.9
After culturing for 6 hours, MG-63 cells in the current study formed significantly more focal adhesion plaques on the Zn-Ti surface than on the cp-Ti surface. This increase in early focal adhesion plaque formation indicates that Zn implantation improved the biocompatibility and bioactivity of Ti similar to the positive effects of fluoride modification reported by Cooper et al.10
The positive effects of Zn implantation may be attributable to the formation of ZnO on the modified surface. Under neutral pH conditions,11 ZnO may be adsorbed by hydroxyapatite and thereby liberate ions (e.g., calcium and phosphate). These ions participate in cellular metabolism and enhance cell adhesion and focal adhesion plaque formation. After culture for 24 hours, however, the Zn-Ti and cp-Ti surfaces had similar densities of focal adhesion plaques. This diminished difference over time may be related to the effects of Zn on intracellular signaling in MG- 63 cells via the mitogen-activated protein kinases, MAPK pathway.12–14 This possibility should be investigated in the future.
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Keywords:© 2013 Chinese Medical Association
titanium; plasma immersion ion implantation-deposition; zinc, osteoblast; focal adhesion plaque