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Anode Glow Discharge Plasma Treatment Enhances Calcium Phosphate Adsorption onto Titanium PlatesDepartment of Oral Biomaterials and Technology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Correspondence: *corresponding author, yookun{at}dent.showa-u.ac.jp
Glow discharge plasma (GDP) supplied to a cathode (GDP-) has been applied for cleaning, surface activating, and sterilizing biomaterials, because the kinetic energy in the case of the GDP- is larger than that supplied to the anode (GDP+). However, no comparison between GDP+ and GDP- has been reported. In this study, a titanium surface pre-treated with GDP+ and GDP- was characterized by x-ray photoelectron spectroscopy (XPS). In addition, the wettability of the titanium surface was measured with and without GDP. Furthermore, XPS characterized the adsorption of inorganic ions on titanium surfaces with and without GDP and immersed in an electrolyte solution. The findings suggested that GDP+ enhances calcium phosphate nucleation, due to the accumulation of electrons. In addition, calcium phosphate was not nucleated on the specimen with GDP-. We conclude that GDP+ is more suitable for biomineralization on titanium.
Key Words: glow discharge plasma • XPS • titanium • biocompatibility
Glow discharge plasma (GDP) treatment is a method for cleaning, surface activating, and sterilizing biomaterials (Aronsson et al., 1997; Swart et al., 1992). We also reported that GDP pre-treatment of a titanium surface by means of an originally developed device increased wettability and promoted initial cell adhesion and differentiation (Shibata and Fujimori, 2000). It is well-known that the effect of GDP depends on parameters such as plasma current, voltage, gas composition, and pressure. The majority of impinging particles are electrons at the anode and positive ions and neutrals at the cathode. Therefore, a variety of processes may occur at the surfaces of the treated specimens, depending on the impinging particle (Aronsson et al., 1997). The impinging particles at the cathode can have three significant effects on the surface: (1) emission of atoms, molecules, clusters, and electrons from the outermost surface; (2) breaking of internal molecular bonds of molecules adsorbed at the surface; and (3) atomic mixing and ion implantation. The effects of impinging electrons at the anode have not been examined in earlier studies. Aronsson et al. also indicated that the GDP of biomaterials supplied to cathodes (GDP-) is more suitable than that supplied to anodes (GDP+), because the kinetic energy of positive ions and neutral GDP- is larger than that of electron GDP+. They also indicated that the increase in surface wettability was strongly dependent on the degree of elimination of contaminated particles (Aronsson et al., 1997). However, as reported in our previous study, since the wettability of titanium surfaces increased with increasing plasma current, we hypothesized that the surface wettability might be more influenced by the electrical charging on the surface than by the elimination of contamination (Shibata, 2000). In contrast, titanium, which can spontaneously nucleate a calcium phosphate layer similar to bone, like apatite in body fluids, has been used for dental implant systems (Hanawa and Ota, 1991; Wu and Nancollas, 1997). The biocompatibility of titanium strongly depends on this process, known as biomineralization (do Serro et al., 2000). However, details of these processes have yet to be identified. Hanawa reported that, in body fluids, a calcium phosphate layer was nucleated on titanium surfaces by the initial adsorption of phosphate ions, followed by that of the calcium ions (Hanawa et al., 1991). However, because the titanium surface is negatively charged in a solution with a pH of near 7, it seems that phosphate ions (PO4-) would initially hardly adhere to the titanium surface. Therefore, further surface analysis was needed for better identification of the biomineralization process on titanium. In this study, we examined the surface characterization of specimens with GDP, as determined by XPS. The surface wettabilities of both GDP+ and GDP- were measured. The inorganic ions' adsorption onto titanium surfaces, both with and without GDP, immersed in artificial body fluid was investigated by XPS to confirm our hypothesis, and to clarify the mechanisms of biomineralization on titanium, with and without GDP.
Specimen Preparation The material used was JIS grade 2 titanium plate (KS-50, Kobe Steel, Japan). Specimens were prepared with a disk of 8.0-mm diameter and 1.00-mm thickness, with a wire-type electric-discharge machining device. The surfaces of the specimens were gradually ground by means of waterproof polishing papers from #500 to #1200 under running water, then polished with alumina particles of 0.3 µm average diameter.
Cleaning of Specimens
GDP GDP+ was performed following the methods introduced in our previous study (Shibata, 2000). After the specimens were set in the holder in the chamber under argon gas replacement, GDP+ was processed under a vacuum of 8 x 10–3 Torr for 1 min. We chose an ion gun connected to XPS equipment for the GDP device since it is well-known that this device completely eliminates surface contamination by impinging Ar+ ions to the sample by almost the same principle as the GDP- device used previously. The GDP- (ion gun) was also processed under a vacuum of 2 x 10–4 Torr for 1 sec, by means of an ion gun connected to the XPS device. The surface characterization of the specimens treated with GDP- was then immediately analyzed by XPS. Specimens with GDP were used in the following tests after being processed.
Surface Characterization
Wettability
Immersion Protocol Specimens with and without GDP were immersed immediately after GDP treatment in HBSS at 37°C for 5, 15, 30 min, and 1 hr.
After immersion, specimens were washed in pure distilled water and stored in a desiccator for 24 hrs in 50% humidity and at a temperature of 23°C. The surface of each specimen was then analyzed by XPS. High-resolution spectra of Ti2p, O1s, C1s, Na1s, P2p, Ca2p, Cl2p, and K2p were analyzed by Mg K After being analyzed, specimens immersed for 1 hr were sputtered by an ion gun for 1 sec and supplied again for XPS analysis. Results of the tests were expressed as the mean + standard deviation (SD) of six specimens (n = 6). The time-course of each specimen and the findings of individual groups were analyzed statistically by ANOVA. Significant differences were considered to exist when p < 0.01.
Calibration of Spectra
Surface Characterization The shape and energy position of the Ti2p (Fig. 1A  ) and O1s (Fig. 1B) peaks revealed no difference between specimens. The shape of the C1s peak on the GDP+ specimens was unchanged compared with those without GDP (Fig. 1C). After GDP-, the C1s peak completely disappeared (Fig. 1C). Since the C1s peak was completely eliminated by GDP-, it is obvious that this peak observed in the specimens with GDP+ was produced by surface contamination, in a manner similar to those without GDP, and that the degree of contamination was not eliminated by GDP+.
The shape of the Ar2p was observed on the specimens with GDP- and scarcely observed on the specimens with GDP+ (not shown), indicating that argon accumulated on the surface of the GDP- specimen.
Wettability of the Specimens
Inorganic Ion Adsorption on Specimens The concentrations of titanium (Fig. 3A ) and oxygen (Fig. 3B) decreased with increasing immersion time for the GDP specimens (p < 0.01). However, the concentration of carbon increased on the specimens with GDP, particularly those with GDP- (Fig. 3C) (p < 0.01). Sodium, on the specimens with GDP+ but without GDP, increased in accordance with increases in immersion times (p < 0.01) (Fig. 3F). On the specimen with GDP+, the relative concentration of phosphorus increased with between 5 and 15 min of immersion (Fig. 3D) (p < 0.01), and then reduced gradually with increasing calcium adsorption (Fig. 3E) (p < 0.01). The adsorption of sodium (Fig. 3F) and calcium (Fig. 3E) on the specimen with GDP+ was higher than on that without GDP (p < 0.01).
After 1 sec of argon sputtering, the shape of the P2p was completely eliminated, and Na1s was slightly reduced on specimens with GDP+ and on those without GDP after 1 hr of immersion (not shown). Since the shape of the Na1s remained on the specimens after argon sputtering, sodium must have adsorbed first onto those specimens with GDP+ and without GDP.
In contrast, the relative concentrations of calcium (Fig. 3E
The three peaks of the curve fit of the C1s spectra on the specimens with GDP after 1 hr of immersion are shown (Fig. 4
The surface energy and the surface wettability of implants have significant influences on the cell responses on their surfaces (Hallab et al., 2001). In particular, increasing the wettability enhances the interaction between the implant surface and the biologic environment (Kilpadi and Lemons, 1994). Conventionally, GDP has been applied to clean biomaterials and implants by the use of DC power supply systems and connecting samples to the cathode (Swart et al., 1992). Aronsson et al. indicated that GDP- is more suitable than GDP+ for cleaning biomaterials, since positive ions and neutrals are more effective than electrons in eliminating surface contamination (Aronsson et al., 1997). However, in this study, the contact angles of the GDP+ specimens were reduced significantly compared with GDP- (p < 0.01), despite the fact that the surface contamination was not eliminated. These results suggest that an increase in surface wettability strongly depends on the accumulated electrons, and not on the surface cleaning effect of the impinging positive ions and neutrals. Subsequently, the electrical charging on a GDP+ specimen decreased with increasing adsorption of the inorganic ions and calcium phosphate nucleation. The physical and chemical characteristics between titanium and tissue surfaces determine tissue response to titanium implants (Healy and Ducheyne, 1992). Biocompatibility is determined by the host tissue's sensitivity to specific ions, corrosion properties, and the wettability of the implant surface (Kilpadi and Lemons, 1994). Our immersion testing results indicated that the sodium concentration increased in accordance with the immersion time on the GDP+ specimens, without GDP (p < 0.01), and that this was followed by phosphorus adsorption. In addition, the adsorption of sodium onto the GDP+ specimen was significantly (p < 0.01) higher than that onto those without GDP. Therefore, we suggest that the negative charging of the titanium surface by electrons accumulated during GDP+ processing influenced the process of biomineralization on the titanium surface. In addition, because the relative concentration of the calcium on the specimen increased with GDP+, compared with that on those without GDP (p < 0.01), it is able to form a calcium phosphate layer rapidly on the titanium surface, even with a short immersion time. In contrast, no specific adsorption of sodium or phosphorus was observed, and CaCO3 formed on the GDP- specimen. The titanium surface was neutralized or positively charged by the accumulation of argon (Ar+) positive ions during GDP- processing. The change in electrical charging in this processing induced CO- ions to the surface, and formed CaCO3, followed by Ca adsorption. Since the GDP+ was effective for the initial nucleation of calcium phosphate compared with the GDP-, we suggest that GDP+ is superior to the GDP- proposed in earlier studies.
This study has been conducted by the Department of Oral Biomaterials and Technology, Showa University School of Dentistry. The authors gratefully acknowledge the financial support received by a Grant-in-Aid for Scientific Research (B) of The Ministry of Education, Culture, Sports, Science and Technology. Received for publication July 20, 2001. Revision received August 12, 2002. Accepted for publication September 23, 2002.
Journal of Dental Research, Vol. 81, No. 12,
841-844 (2002)
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ABSTRACT
INTRODUCTION
radiation before the following experiments were carried out. A 20-mA emission current and an 8-kV accelerated voltage were deemed appropriate in this analysis. 
