Abstract
This study was performed to investigate the effects of argon plasma treatment under atmospheric pressure at room temperature on the cytotoxicity and antimicrobial effects of the graphene oxide layer on titanium. Plasma treatment of the graphene oxide coating on a nonthermal atmospheric-pressure plasma device was performed. Raman spectrum analysis confirmed that graphene oxide was successfully coated on the surface and AFM analysis confirmed that this coating affected the surface roughness. X-ray photoelectron spectroscopy (XPS), Alizarin Red S staining for cell differentiation, and Raman and atomic force microscopy (AFM) analyses were performed for the deposited surface. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to confirm the biocompatibility of the plasma-treated and bare titanium specimens. The biofilm formation test using Streptococcus mutans (S. mutans) was performed to examine potential antimicrobial effects. XPS analysis showed that with increasing plasma coating time, the carbon content of the surface decreased while that of oxygen increased. Alizarin Red S staining showed that the cell differentiation was promoted by the deposition of the graphene oxide. The graphene oxide on Ti significantly affected an osteoblast cell differentiation for bone growth, and no significant differences in antimicrobial effects were observed.
1. Introduction
In dentistry, titanium is widely used especially in dental implants [1]. The biocompatible nature of titanium shows a very high success rate when used as a dental implant in the oral cavity [2]. However, many scientists are working on various attempts to produce effects beyond the nature of titanium [3]. One such attempt is the improvement of titanium surface properties to increase the bonding strength to the bone.
Changes in the titanium surface have been tried variously [4]. Calcium, hydroxyapatite, etc. have been used to induce chemical adhesion [5, 6]. So far, many attempts have been made to develop dental implants, but the initial fixation force there is a need for a more innovative material to dramatically increase. Here, we are noticing the effect of graphene, which has been widely used in existing biomaterial parts. Graphene is one of the carbon-based materials [7, 8]. It consists of a single layer of sp2-hybridized carbon atoms and is known as the basic component of fullerenes and carbon nanotubes [9–11]. In addition to the physical properties, they have been shown to be highly biocompatible [12–14].
There are many ways to apply graphene to titanium surfaces [10–22]. Among them is atmospheric-pressure plasma at room temperature [23, 24]. Plasma is the state of the fourth substance and is different from solid, liquid, or gas [25]. Plasma is a state of matter separated by negatively charged electrons and positively charged ions [26]. Plasma is often generated when a gas is charged with high energy, but various attempts have been made to utilize it by generating plasma at room temperature and atmospheric pressure for applications in the human body [27, 28].
A method in which various gases passing through plasma decompose carbon-containing molecules into atoms to form graphene [29, 30]. The quality of graphene depends on the velocity of gas and the power of plasma [31]. However, there is a lack of research on plasma-based graphene generation. Therefore, in this study, we produced graphene using nonthermal atmospheric-pressure plasma, which was then coated on titanium and experimented variously. In the experiment, we attempted to confirm the feasibility of titanium coating using plasma and analyze the various cytotoxic and antimicrobial effects. The null hypothesis of this experiment is that plasma-based graphene does not affect the cytotoxic and antimicrobial effects of titanium.
2. Materials and Methods
2.1. Preparation of the Specimen and Plasma Treatment
The study materials are commercially available pure grade 2 titanium (thickness: 3 mm, diameter: 15 mm). Each specimen was sequentially polished with #400 to #2000 grit silicon carbide sandpaper, 1 μm alumina powder, and a polishing cloth. Deposition of the graphene oxide on the titanium substrate was performed by a nonthermal atmospheric plasma generator PGS-300 (Expantech Co., Suwon, Korea). Figure 1(a) shows the atmospheric plasma system for the graphene oxide on Ti substrate. Argon gas was used as a plasma carrier and mixed with methane gas for the carbon source. To generate plasma, 4 l/min of argon gas was carried through the resonator and the plasma was generated by the high radio frequency (900 MHz) resonance plasma generator (300 W). Once the plasma stabilized, 60 SCCM of the mixture gas (10% methane) was introduced into a quartz tube, and the deposition of the graphene oxide initiated. Figure 1(b) shows the deposition process of the plasma-based graphene oxide. The high-densed plasma induced the decomposition of methane into hydrogen and carbon. During the decomposed carbon flows down through the tube, the carbon-carbon bond is formed and the two-dimensional layers of the graphene oxide are deposited on a Ti substrate. The plasma was processed for a total of 60 s.
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2.2. Surface Analysis
The surface characteristics of the deposited plasma-based graphene film were analyzed by X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and atomic force microscopy (AFM). XPS (VG Multilab 2000, ThermoVG Scientific, Southend-on-Sea, Essex, UK) confirmed the surface argon atomic composition appearance and the other compositions. Raman spectroscopy (NRS-5100, JASCO, Tokyo, Japan) was performed with a 532 nm laser to confirm the presence of graphene oxide on the titanium surface. AFM (Nanoscope-III, Digital Instruments, Santa Barbara, CA, USA) was used to confirm the plasma effect on the average surface roughness ().
2.3. In Vitro Testing
Cytotoxicity and antibacterial activity of the graphene oxide-deposited titanium were evaluated by MTT assay and Alizarin Red S stain and compared with pure titanium (). The L-929 mouse fibroblasts cells (NCTC clone 929, ATCC® CCL-1™) were used for cytotoxicity test. Eight specimens from each group were placed in a 24-well plate. L-929 cells were seeded at a density of and incubated for 3 days. Cytotoxicity was evaluated by the MTT assay(3-(4,5-dimethylthiazon-2-yl)-2,5-diphenyl tetrazolium bromide, M6494, Invitrogen, USA). MC3T3-E1 mouse osteoblast cells (MC3T3-E1 subclone 4, ATCC® CRL-2593™) were used for the cell differentiation test. Eight specimens from each group were plated in a 24-well plate and cells were incubated at a density of in each well. Osteoblast differentiation evaluated by Alizarin Red S stain (A5533, Sigma-Aldrich, St. Louis, MO, USA) after designated time periods of incubation. The antibacterial activity test was conducted using S. mutans (Korean Collection for Oral Microbiology, KCOM, Gwangju, Korea, KCOM 1504). S. mutans was maintained in brain heart infusion (BHI) broth (Becton, Dickinson and Co. Sparks, MD, USA). Eight specimens from each group were placed in a 24-well plate and incubated with 1 ml of a bacterial suspension for 24 hours at 37°C. After the incubation, one hundred microliters of the bacterial suspensions was transferred to 900 μl of phosphate-buffered saline (PBS) and diluted up to 10−6. A 100 μl aliquot of the diluted solution was plated on BHI agar (Agar, Becton, Dickinson and Company, Sparks, MD, USA) plates, applied evenly with a spreader, and cultured at 37°C for 2 days. At the end of the incubation period, the number of colonies (colony-forming units) was counted on BHI agar plates.
2.4. Statistical Analyses
All statistical analyses were analyzed by statistics software (SPSS version 20.0, IBM Korea Inc., Seoul, Korea). The cytotoxicity and antibacterial effects were evaluated by one-way analysis of variance, and post hoc comparisons were processed by Tukey’s test. A value of <0.05 was considered significant.
3. Results and Discussion
3.1. Surface Analysis
3.1.1. XPS
Comparison of XPS spectra survey scan in Figure 2 reveals the relative ratio of carbon decreased while that of oxygen increased with increasing plasma treatment time. The results show that hydrocarbon layers were removed by the plasma treatment, and the titanium oxide layer was revealed. Table 1 summarizes the surface atomic composition by the plasma treatment. The carbon content decreased from 26.86% to 20.14%, whereas the oxygen ratio increased from 60.77% to 63.90% after plasma treatment for 60 s. The titanium composition increased from 12.37% to 15.87% with increasing argon plasma treatment time.
3.1.2. Alizarin Red S Staining and MTT Assay
Figure 3 shows the growth of fibroblast cells after three days of incubation on the plasma-treated titanium surface with a none-treated Ti surface and on a well plate for comparison. The surface of the pure titanium specimen was less biocompatible than that of the cell culture plate and plasma-treated titanium. The argon plasma-treated titanium surface was more biocompatible than that of pure titanium. This figure does not include the results of the cell counting method, but the images clearly indicate the cell affinities of the various surfaces. The cell culture plate was coated with positively charged glucose, which was optimized for uniform distribution of cells bearing negative charge. The cells were well distributed on the cell culture plate and plasma-treated titanium surface. This result corresponds with the data of MTT assay (Figure 4) indicating that the plasma treatment enhanced the cell affinity on the surface by increasing hydrophilicity.
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3.1.3. Raman Spectroscopy
The Raman spectra were used to determine the characteristics of surface graphene oxide film and were effective for analyzing the plasma-based graphene film. Raman spectroscopy can distinguish between the presence of graphene or graphene oxide, evaluate crystallinity, and determine the defect levels of the surface carbon film.
The Raman spectra of graphene typically show three distinct peaks: D, G, and 2D (also called G) peaks. Typical Raman spectra of graphene oxides are characterized by G and D peaks at 1605 and 1353 cm-1, respectively, while graphene typically exhibits G and D peaks at 1580 and 1350 cm-1, respectively [32, 33].
Figure 5 shows two lines taken from the different treatment times for the deposition of graphene oxides. The graph of 5 min deposition time shows the G and D peaks at 1608 and 1351 cm-1, and the graph of 10 min deposition time also shows the peaks at 1608 and 1352 cm-1 with increase of the intensity, respectively. Slight shifts from the ideal positions can be generated by defects such as vacancies, grain boundaries, and amorphous carbon species [34–36]. From these results, we can conclude that the surface film deposited on the titanium surface was graphene oxide.
3.1.4. AFM Analysis
Figure 6 shows the 3-dimentional atomic force microscope (AFM) images over a scanned area, which confirmed the surface carbon deposition. In Figure 6, the as-polished titanium showed a smooth surface (Figure 6(a)) with of 0.589 nm and the plasma-treated surface (Figure 6(b)) with of 0.857 compared with the carbon film-deposited specimen, which exhibited an of 9.258 nm (Figure 6(c)), which is 15.7 times higher than that of the polished titanium surface. The surface topographies also support the surface deposition of materials.
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3.1.5. Alizarin Red S Staining for the Osteoblast Cell Differentiation
Alizarin Red S staining shows amount of metal ions (calcium ions are the target in this experiment) made by osteoblast cell differentiation on the specimen surface. Figure 7(a) shows the activity of promoted MC3T3-E1 osteoblast cells on the graphene oxide-deposited Ti compared with the cell culture plate indicating the enhancement of the growth, attachment, and differentiation of the osteoblast cell on the graphene oxide-deposited Ti. Both results showed that the surface-treated titanium specimen is superior in cell activity to the cell culture plate. The carbon-deposited titanium can be a better substrate for MC3T3-E1 osteoblast cell to grow and differentiate for making bone flakes.
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3.1.6. Antibacterial Effects
Comparison of the observed CFUs shows the antibacterial properties of the graphene oxide plasma-treated titanium surface [37]. Because the starting bacterial concentration was identical, only the effects of the specimen influenced bacterial proliferation. Titanium coated with the graphene oxide film was expected to exhibit beneficial antibacterial properties and decreased bacterial growth. In Figure 7(b), the plasma-treated titanium specimen showed a 3.45% decrease in the amount of proliferated bacteria compared with the control (cell culture plate). Although the graphene oxide-deposited specimen showed antibacterial activity, the intensity of the effect was negligible, and this difference was not statistically significant. Compared with the as-polished titanium specimen, there was no significant change of antibacterial effect by the graphene oxide. Even though some antimicrobial effect was observed, it could not be determined whether this effect originated from the plasma or graphene.
The Raman spectra confirmed that our plasma system effectively deposited graphene oxide layers on the titanium surface [38]. The method developed herein is effective for examining the effect of plasma and the effects of the deposition of the graphene oxide. The changes in carbon and oxygen content as determined by XPS analysis were similar to those observed for conventional plasma treatment [39].
It is surprising that the antimicrobial effects of the plasma treatment and the graphene oxide were muted. These effects have also been associated with increased surface roughness, but in general, an increase in surface roughness results in increased bacterial attachment to the surface [40]. In our experiments, the deposition of the plasma-based graphene oxide resulted in a significant increase in surface roughness (Figure 6). Therefore, it is likely that the antimicrobial effect could be enhanced if the surface roughness could be controlled. The development of a deposition method that does not increase surface roughness would likely improve antibacterial activity.
Previous publications reported an enhancement of the osteoblast cell differentiation and antimicrobial effects of graphene oxide and the plasma treatment [17, 41–43]. In this study, no statistically significant differences of the antimicrobial effect between the experimental and control groups were observed; however, a solid increase in osteoblast cell differentiation was measured. This can be attributed to the good biocompatibility of titanium.
4. Conclusions
From the limited results of this study, we can conclude the following: (1)The graphene oxide prepared with nonthermal atmospheric-pressure plasma can be effectively applied to titanium surfaces(2)The argon plasma treatment enhanced biocompatibility of titanium equivalent to that of the cell culture plate(3)The deposition of graphene oxide using nonthermal atmospheric-pressure plasma would promote bone growth in the vicinity of the implant by enhancing osteoblast cell differentiation
Data Availability
The data obtained for this study are presented in the tables and figures herein. In addition, you may contact the corresponding authors to request for the original datasets at any time.
Conflicts of Interest
The authors declare no conflict of interest.
Authors’ Contributions
Keonho Rho and Chan Park contributed equally to this work.
Acknowledgments
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; No. 2017R1A2B4007769) and the Chonnam National University (2014-0615 and 2015-1830).