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RESEARCH REPORT |
Department of Oral Biomaterials and Technology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan;
*corresponding author, yookun{at}dent.showa-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: glow discharge plasma • XPS • titanium • biocompatibility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Cleaning of Specimens
The prepared specimens were ultrasonically cleaned in acetone, detergent solution (7X, ICN), and pure distilled water for 15 min each. Then, the specimens were dried and stored in a desiccator for 24 hrs in 50% humidity and at a temperature of 23°C. The surface characterization of the specimens was examined by x-ray photoelectron spectroscopy (XPS) (ESCA-3400, SHIMADZU) with Mg K
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.
GDP
Our newly developed GDP device is able to remove specimens from the chamber, delivered in a clean bench (VG842K, Iwaki Glass Co., Iwaki, Japan) and without exposing the specimens to the atmosphere after being processed. One may achieve a vacuum using this device by connecting a turbocharged molecular pump to the conventional rotary pump.
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
Specimens with GDP+ were set in a transfer vessel originally designed in a clean bench to prevent atmospheric exposure. After connecting the transfer vessel to the XPS device, we imported specimens to the analyzing room of the XPS device, without exposed them to the atmosphere. The surfaces of specimens with and without GDP were analyzed by XPS. High-resolution spectra of Ti2p, O1s, Ar2p, and C1s were analyzed by Mg K radiation.
Wettability
After GDP treatment, the contact angles of the specimens GDP+, GDP-, and without GDP relative to pure distilled water were immediately measured by means of a contact angle meter (KYOWA KAGAKU). The temperatures and humidity were kept at 50% and 23°C during this measurement, and the contact angles were measured by the sessile drop technique (Amaral et al., 2002). Results of the test were expressed as the mean + standard deviation (SD) of six specimens (n = 6). The findings were analyzed statistically by an analysis of variance (ANOVA). Significant differences were considered to exist when p < 0.01.
Immersion Protocol
Hanks' balanced salt solution without organic ions (HBSS) with ion concentrations of 142.0 mM Na+, 5.5 mM K+, 8 mM Mg2+, 1.26 mM Ca2+, 140.0 mM Cl-, 8.25 mM HPO42-, and 4.2 mM HCO3- was used in this study. This solution was buffered at a pH of 7.4 with adequate HCl for the immersion test.
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 radiation.
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
The binding energies for each spectrum were calibrated by C1s spectra of 285.0 eV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) 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+.
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Wettability of the Specimens
The contact angles of the specimens to pure distilled water with GDP were reduced significantly (p < 0.01) compared with those without GDP. However, the contact angles of the specimens with GDP+ were significantly (p < 0.01) reduced compared with those of the specimens with GDP- (Fig. 2). Therefore, the specimens with GDP+ showed excellent wettability.
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) 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).
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In contrast, the relative concentrations of calcium (Fig. 3E
) and carbon (Fig. 3C) on specimens with GDP- increased with increasing immersion time (p < 0.01). No specific adsorption of phosphorus was observed on the specimens with GDP- (Fig. 3D).
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). Peak 1 was set at 285.0 eV for -H2-, Peak 2 at 286.5 eV for -C-N-, and Peak 3 at 288 eV for -C=O- (Deligianni et al., 2001). Since Peak 3 of the carbon doubly bonded to oxygen increased (Fig. 4B), calcium increased without phosphate adsorption, and Ca2p3/2 was observed at 347.0 eV on the specimen with GDP- (not shown), it could be assumed that the CaCO3 calcium carbonate formed on the specimen with GDP- after a short immersion time.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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Received July 20, 2001; Last revision August 12, 2002; Accepted September 23, 2002
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Aronsson BO, Lausmaa J, Kasemo B (1997). Glow discharge plasma treatment for surface cleaning and modification of metallic biomaterials. J Biomed Mater Res 35:49–73.[Medline]
Deligianni DD, Katsala N, Ladas S, Sotiropoulou D, Amedee J, Missirlis YF (2001). Effect of surface roughness of the titanium alloy Ti-6Al-4V on human bone marrow cell response and on protein adsorption. Biomaterials 22:1241–1251.[Medline]
do Serro AP, Fernandes AC, de Jesus Vieira Saramago B (2000). Calcium phosphate deposition on titanium surfaces in the presence of fibronectin. J Biomed Mater Res 49:345–352.[Medline]
Hallab NJ, Bundy KJ, O'Connor K, Moses RL, Jacobs JJ (2001). Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion. Tissue Eng 7:55–71.[Medline]
Hanawa T, Ota M (1991). Calcium phosphate naturally formed on titanium in electrolyte solution. Biomaterials 12:767–774.[Medline]
Healy KE, Ducheyne P (1992). Hydration and preferential molecular adsorption on titanium in vitro. Biomaterials 13:553–561.[Medline]
Kilpadi DV, Lemons JE (1994). Surface energy characterization of unalloyed titanium implants. J Biomed Mater Res 28:1419–1425.[Medline]
Shibata Y (2000). Glow discharge treatment on titanium plate (in Japanese). J Jpn Soc Dent Mater Dev 19:84–91.
Shibata Y, Fujimori S (2000). Glow discharge treatment is effective for cell proliferation and differentiation of osteoblast-like cells on titanium surface (in Japanese). J Showa Univ Dent Soc 20:24–29.
Swart KM, Keller JC, Wightman JP, Draughn RA, Stanford CM, Michaels CM (1992). Short-term plasma-cleaning treatments enhance in vitro osteoblast attachment to titanium. J Oral Implantol 18:130–137.[Medline]
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  H. Yamamoto, Y. Shibata, and T. Miyazaki Anode Glow Discharge Plasma Treatment of Titanium Plates Facilitates Adsorption of Extracellular Matrix Proteins to the Plates J. Dent. Res., July 1, 2005; 84(7): 668 - 671. [Abstract] [Full Text] [PDF]
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