Assessment of Inhibition of Biofilm Formation on Non-Thermal Plasma-Treated TiO2 Nanotubes

Peri-implantitis is an inflammatory disease similar to periodontitis, caused by biofilms formed on the surface of dental implants. This inflammation can spread to bone tissues and result in bone loss. Therefore, it is essential to inhibit the formation of biofilms on the surface of dental implants. Thus, this study examined the inhibition of biofilm formation by treating TiO2 nanotubes with heat and plasma. Commercially pure titanium specimens were anodized to form TiO2 nanotubes. Heat treatment was performed at 400 and 600 °C, and atmospheric pressure plasma was applied using a plasma generator (PGS-200, Expantech, Suwon, Republic of Korea). Contact angles, surface roughness, surface structure, crystal structure, and chemical compositions were measured to analyze the surface properties of the specimens. The inhibition of biofilm formation was assessed using two methods. The results of this study showed that the heat treatment of TiO2 nanotubes at 400 °C inhibited the adhesion of Streptococcus mutans (S. mutans), associated with initial biofilm formation, and that heat treatment of TiO2 nanotubes at 600 °C inhibited the adhesion of Porphyromonas gingivalis (P. gingivalis), which causes peri-implantitis. Applying plasma to the TiO2 nanotubes heat-treated at 600 °C inhibited the adhesion of S. mutans and P. gingivalis.


Introduction
Bacterial colonies that cause periodontitis are formed on the surface of dental implants within two weeks after being placed and exposed to an oral environment. Subgingival bacterial flora is established within four weeks, resulting in tissue inflammation and, thus, dental implant failure [1]. Since dental implants, unlike natural teeth, do not have any blood vessels or nervous tissues, patients do not feel any particular pain associated with peri-implantitis, and the inflammation is highly likely to spread to bone tissues and result in bone loss [2]. Therefore, it is essential to increase the bioactivity of the surface of the implants and suppress the formation of biofilms in order to maintain dental implants.
Various attempts have been made to modify the surface of dental implants to suppress bacterial growth and improve the adhesion of osteocytes [3]. Anodization is one method of modifying the titanium surface [4]. A thin, rough, porous amorphous TiO 2 oxide film is electrochemically created on the titanium surface to modify its physical properties. Once amorphous TiO 2 nanotubes are formed on the surface of the titanium implants via anodization, their surface area is expanded, and cell adhesion, proliferation, and differentiation are improved, which was reported to increase bone-implant integration, accelerate bone formation and reduce treatment times [5][6][7]. Peng et al. [8] reported that the initial adhesion and colonization of Staphylococcus epidermidis on TiO 2 nanotubes were significantly reduced when compared with polished or acid-etched titanium. In addition, heat treatment of the titanium surface changes the crystal structure of the TiO 2 films and suppresses the adhesion of bacteria without inhibiting the activity of cells [9,10]. Del Curto et al. [11] reported that the adhesion of bacteria was inhibited on the titanium surface by modifying its amorphous oxide film into an anatase crystal structure through heat treatment without inhibiting the activity of cells. However, in another study, osseointegration was further improved on a rutile crystal structure surface [12]. Furthermore, opinions are divided on whether anatase or rutile is a better TiO 2 oxide film for titanium implants. The adhesion of bacteria and the activity of cells are affected not only by the physical properties of dental implants, such as surface structure and roughness, but also by chemical properties, such as surface-free energy and hydrophilicity [5,6,13].
The chemical properties of dental implants, such as surface-free energy and hydrophilicity, play a critical role in the interaction between osteocytes and proteins and thus improve the treatment of cuts in the early stage of treatment [14,15]. Sawase et al. [16] found that the surface of dental implants with improved hydrophilicity showed better osseointegration than that of general implants. These results indicated that the chemical properties of the surface of dental implants are more important factors for the activity of cells than their surface structure.
Plasma is a gas with an energy that is loosely and partially ionized [17] and is used to sterilize the surfaces of dental equipment and biomaterials and to modify the surfaces of implants [18,19]. By applying plasma, the oxide layer on the surface of implants is modified due to the chemical properties of the applied gas without any change in the surface structure, which results in chemical changes, such as hydrophilicity [20]. Yoo et al. [21] reported that the shape of bacterial colonies cultured on the surface of titanium treated with plasma was changed and that over 80% of bacteria were killed. In addition, Duske et al. [13] found that plasma decreased contact angles and thus increased hydrophilicity, improving the early adhesion of osteoblasts and osseointegration.
Even though the chemical properties of the surface of dental implants are as important as their surface structure in inhibiting the adhesion of bacteria and the activity of cells, studies have been conducted only to evaluate changes in the surface structure and roughness of dental implants. Authors have been mostly focused on changes in the activity of cells and osseointegration after applying atmospheric pressure plasma to the surface of implants. However, there has been no study published on the inhibition of peri-implantitis. Moreover, previous studies on biofilm inhibition of TiO 2 nanostructures use antimicrobial agents (e.g., Ag, Au) on TiO 2 nanostructures [22,23]. This study created TiO 2 nanotubes on the titanium surface to change the physical properties of the surface of dental implants. In addition, heat treatment and atmospheric pressure plasma were applied to change the chemical properties of the surface of dental implants.
This study examined the effects of heat and plasma treatment on the surface of TiO 2 nanotubes on biofilm formation.

Contact Angles
The contact angle of the non-heat-treated (H0), 400 • C heat-treated (H400), and 600 • C heat-treated (H600) samples was 19.5 ± 2 • , 14.2 ± 2 • , and 18.0 ± 2 • , respectively. After the plasma treatment, the contact angle of all groups was reduced. The contact angle of the non-heat-treated plasma-treated samples (H0P) was 3.3 ± 6 • , and the contact angle of the 400 • C heat-treated plasma-treated samples (H400P) and the 600 • C heat-treated plasma-treated samples (H600P) was reduced to the point that could not be measured ( Figure 1). the non-heat-treated plasma-treated samples (H0P) was 3.3 ± 6°, and the contact angle of the 400 °C heat-treated plasma-treated samples (H400P) and the 600 °C heat-treated plasma-treated samples (H600P) was reduced to the point that could not be measured ( Figure 1).

H0 H400 H600
No treatment Plasma treatment

Surface Structures
There were no changes in the height and surface structures of the TiO2 nanotubes after the heat treatment at 400 °C. However, when the samples were heat-treated at 600 °C, the diameter was decreased to 91.2 ± 1 nm, and their height was reduced to 498.0 ± 7 nm ( Figure 2, Table 1). There was no change in the surface structure of the samples before or after the plasma treatment.

Surface Structures
There were no changes in the height and surface structures of the TiO 2 nanotubes after the heat treatment at 400 • C. However, when the samples were heat-treated at 600 • C, the diameter was decreased to 91.2 ± 1 nm, and their height was reduced to 498.0 ± 7 nm ( Figure 2, Table 1). There was no change in the surface structure of the samples before or after the plasma treatment. the non-heat-treated plasma-treated samples (H0P) was 3.3 ± 6°, and the contact angle of the 400 °C heat-treated plasma-treated samples (H400P) and the 600 °C heat-treated plasma-treated samples (H600P) was reduced to the point that could not be measured ( Figure 1).

H0 H400 H600
No treatment Plasma treatment

X-ray Diffraction (XRD) Analysis
After plasma treatment, the C1s peaks were reduced, and the low peaks between 292.1 eV and 281.2 eV were slightly shifted to a higher bonding energy. Another C1s peak was observed in 283.6 eV and 281.9 eV, which are associated with C=C and C-Ti. Two additional O1s peaks (528.6 eV and 527.2 eV) were observed, which represent O 2− . The Ti2p1/2 and Ti2p3/2 peaks were observed at 461.7 eV and 456.1 eV, respectively, which are associated with TiO2 and Ti2O3. Another C1s peak in the H400P samples was observed at 289.7 eV, which is associated with O-C=O. Moreover, another Ti2p peak in the H600P samples was observed at 460.9 eV, which is associated with TiOx, a titanium compound. After heat treatment, the O1s peaks (530.0 eV, 531.5 eV, and 532.6 eV) and the Ti2p peaks (464.5 eV and 458.7 eV) of the H400 samples, as well as the O1s peaks (530.1 eV, 531.6 eV, and 532.7 eV) and the Ti2p peaks (464.7 eV and 458.9 eV) of the H600 samples were slightly shifted to a higher bonding energy.
After plasma treatment, the C1s peaks were reduced, and the low peaks between 292.1 eV and 281.2 eV were slightly shifted to a higher bonding energy. Another C1s peak was observed in 283.6 eV and 281.9 eV, which are associated with C=C and C-Ti. Two additional O1s peaks (528.6 eV and 527.2 eV) were observed, which represent O 2− . The Ti2p1/2 and Ti2p3/2 peaks were observed at 461.7 eV and 456.1 eV, respectively, which are associated with TiO 2 and Ti 2 O 3 . Another C1s peak in the H400P samples was observed at 289.7 eV, which is associated with O-C=O. Moreover, another Ti2p peak in the H600P samples was observed at 460.9 eV, which is associated with TiOx, a titanium compound.

Streptococcus mutans
The changes in the thickness of S. mutans biofilms after the heat and plasma treatments to the TiO2 nanotubes are shown in Table 2 and Figure 5.
Compared with the H400 samples, the H0 and H600 samples showed a significantly smaller thickness of S. mutans biofilms (p < 0.017). There were statistically significant decreases in the thickness of S. mutans biofilms in all the groups after plasma treatment (p < 0.05).

Streptococcus mutans
The changes in the thickness of S. mutans biofilms after the heat and plasma treatments to the TiO 2 nanotubes are shown in Table 2 and Figure 5.
Compared with the H400 samples, the H0 and H600 samples showed a significantly smaller thickness of S. mutans biofilms (p < 0.017). There were statistically significant decreases in the thickness of S. mutans biofilms in all the groups after plasma treatment (p < 0.05).

Porphyromonas gingivalis
The changes in the thickness of P. gingivalis biofilms after the heat and plasma treatments to the TiO2 nanotubes are shown in Table 3 and Figure 6.
There were no statistically significant differences in the thickness of P. gingivalis biofilms after the heat treatment (p > 0.017). There were also no statistically significant differences in the thickness of P. gingivalis biofilms after the plasma treatment (p > 0.05).

Porphyromonas gingivalis
The changes in the thickness of P. gingivalis biofilms after the heat and plasma treatments to the TiO 2 nanotubes are shown in Table 3 and Figure 6.

Porphyromonas gingivalis
The changes in the thickness of P. gingivalis biofilms after the heat and plasma treatments to the TiO2 nanotubes are shown in Table 3 and Figure 6.
There were no statistically significant differences in the thickness of P. gingivalis biofilms after the heat treatment (p > 0.017). There were also no statistically significant differences in the thickness of P. gingivalis biofilms after the plasma treatment (p > 0.05).  There were no statistically significant differences in the thickness of P. gingivalis biofilms after the heat treatment (p > 0.017). There were also no statistically significant differences in the thickness of P. gingivalis biofilms after the plasma treatment (p > 0.05).

Discussion
It is essential to inhibit the adhesion of bacteria to the surface of dental implants to improve the adhesion between the surface and surrounding tissues. In this study, TiO 2 nanotubes were formed on the titanium surface to increase its surface area. The TiO 2 nanotubes were heat-treated under different conditions to modify the structure of the TiO 2 oxide films. In addition, atmospheric pressure plasma was applied to the TiO 2 nanotubes to change their surface energy and hydrophilicity and, thus, inhibit the formation of biofilms. Figure 7 shows a schematic of a summary of possible events on the plasma-treated TiO 2 nanotube surface. Recently it has been reported that the adhesion and proliferation of osteoblasts were increased on the surface of materials that had nanotube structures, and studies have been conducted to create nano-level structures on the surface of materials [24][25][26]. Electrochemical anodization can be used to create nano-level tubes on the surface of titanium, and the structure of nanotubes can be changed by controlling certain variables, such as the composition of electrolytes, temperature, and current density [27][28][29]. In this study, TiO 2 nanotubes were created using the anodic oxidation method suggested by Kim et al. [30] to form a nano-level amorphous TiO 2 oxide film on the surface of the titanium.

Discussion
It is essential to inhibit the adhesion of bacteria to the surface of dental implants to improve the adhesion between the surface and surrounding tissues. In this study, TiO2 nanotubes were formed on the titanium surface to increase its surface area. The TiO2 nanotubes were heat-treated under different conditions to modify the structure of the TiO2 oxide films. In addition, atmospheric pressure plasma was applied to the TiO2 nanotubes to change their surface energy and hydrophilicity and, thus, inhibit the formation of biofilms. Figure 7 shows a schematic of a summary of possible events on the plasma-treated TiO2 nanotube surface. Recently it has been reported that the adhesion and proliferation of osteoblasts were increased on the surface of materials that had nanotube structures, and studies have been conducted to create nano-level structures on the surface of materials [24][25][26]. Electrochemical anodization can be used to create nano-level tubes on the surface of titanium, and the structure of nanotubes can be changed by controlling certain variables, such as the composition of electrolytes, temperature, and current density [27][28][29]. In this study, TiO2 nanotubes were created using the anodic oxidation method suggested by Kim et al. [30] to form a nano-level amorphous TiO2 oxide film on the surface of the titanium. Heat treatment of titanium can modify the crystal structure of the TiO2 oxide film. Park et al. [31] reported that heat treatment of 400 °C changed the phase of the amorphous Heat treatment of titanium can modify the crystal structure of the TiO 2 oxide film. Park et al. [31] reported that heat treatment of 400 • C changed the phase of the amorphous TiO 2 into anatase TiO 2 and that temperatures over 600 • C changed the phase to an intermixed population of anatase and rutile phases with the TiO 2 rutile phase forming a major X-ray peak. Therefore, in this study, the temperature of the heat treatment was set at 400 • C and 600 • C to assess the adhesion of bacteria to amorphous TiO 2 , anatase TiO 2 , and anatase/rutile mixed-phase TiO 2 .
When TiO 2 , which has photocatalytic properties, is combined with light, it reacts with H 2 O and O 2 and generates photo-generated holes (h+) that have a high oxidizing power, such as hydroxyl radicals (OH) and superoxide free radicals (O 2− ) [32]. These photogenerated holes (h+) interact with proteins, nucleic acids, and bacterial cell membranes and can damage the structures of bacteria [33,34]. However, the adhesion of bacteria after heat treatment showed different results with the two species of bacteria. The lowest adhesion of S. mutans was seen in the anatase TiO 2 (H400), and the lowest adhesion of P. gingivalis was seen in the anatase/rutile mixed TiO 2 (H600). The relative photocatalytic activity differed depending on the crystal structure. Zhang et al. [35] reported that anatase TiO 2 usually exhibited higher photocatalytic activity than rutile TiO 2 . In addition to the crystal structure, the metabolism of bacteria is associated with complex factors that control the biointerface and bacterial behaviors. In this study, the adhesion of S. mutans onto anatase TiO 2 seemed to be inhibited as the photocatalytic activities that affected the metabolism of S. mutans were more strongly dependent on the crystal structure.
Fluoride ions seemed to be detected on every surface due to the anodic oxidation of electrolytes since fluorine in the anodized electrolytes remained on the surface of the TiO 2 nanotubes. The heat treatment reduced the amount of residual fluorine, and the two species of bacteria observed in this study showed different results depending on the fluorine content P. gingivalis showed the lowest adhesion in the H0 samples where the fluorine content was the highest and statistically more adhesion in the H400 samples, where the fluorine content was the lowest. In comparison, S. mutans showed statistically less adhesion in the H400 samples, where the fluorine content was the lowest. Yoshinari et al. [36] reported that fluorine has two mechanisms that affect bacteria. One is that fluoride ions act as an enzyme inhibitor, affecting the metabolism of bacteria, and the other is that metal-fluoride complexes suppress proton-translocating F-ATPases. In P. gingivalis, the enzyme inhibitor mechanism of fluoride ions seemed to work more strongly.
Chemical changes were observed on the surface of samples treated with plasma due to the composition of process gases [37]. The results of this study did not show any change in the surface structure of TiO 2 nanotubes after the plasma treatment. Although there was no change in the surface structure, specimens treated with plasma showed a lower contact angle than those that were not treated. XPS analysis was performed to identify the functional groups that determine surface hydrophilicity and electric charge. The results showed that C-H and C-C peaks after plasma treatment were reduced and that O-H, O1s, and TiO 2 peaks were increased. Previous studies have found that plasma treatment formed various oxygen functional groups on the surface of materials through chemical oxidation, which breaks C-C and C-H bonds on the surface and removes carbohydrates connected to C-H bonds [38,39]. It was also reported that oxygen, mixed for plasma treatments, attaches a hydroxy group (O-H) on the surface of titanium, and generates reactive oxygen species (ROS), which improves hydrophilicity [40].
In this study, a fluorescent nucleic acid staining method was used to assess the adhesion of bacteria after the plasma treatment, and the two species of bacteria showed different results. In S. mutans, the biofilm thickness on all surfaces was significantly reduced after plasma treatment regardless of heat treatment, while P. gingivalis did not show any statistically significant differences. It has been reported that bacteria adhere better to hydrophobic or nonpolar surfaces than to hydrophilic surfaces [41]. Hydrophobic surfaces increase the adhesion of hydrophobic bacteria [42], and hydrophilic surfaces increase the adhesion of hydrophilic bacteria [43]. S. mutans, used to test the changes in biofilm formation in this study, is a hydrophobic bacterial species [44], while P. gingivalis is hydrophobic and hydrophilic [45]. Therefore, the increase in the hydrophilicity of the TiO 2 nanotubes after applying atmospheric pressure plasma can be attributed to the decrease in the adhesion of S. mutans, a hydrophobic species.
This study assessed the adhesion of bacteria based on the thickness of their biofilms. If bacteria that adhere to the surfaces can be quantified, the adhesion of bacteria can be more accurately assessed. Only the reactions of two species of bacteria were observed in this study. Since various bacteria cause peri-implantitis, it is necessary to conduct further research on peri-implantitis using various species of bacteria or mixed bacterial colonies. In addition, this study was carried out using a single gas plasma and one treatment time; thus, it is also necessary to apply different gas processes and treatment times in future studies. The results of this study showed that heat and plasma treatments inhibited the adhesion of bacteria. However, it will be necessary to perform additional in vitro and in vivo experiments to verify the biostability of TiO 2 nanotubes on the surfaces of dental implants.

Anodic Oxidation
Anodic oxidation was performed using a DC power supplier (Fine Power F-3005, SG EMD, Anyang, Republic of Korea). An electrolyte mixture was prepared by adding 1 M phosphoric acid and 1.5 wt% hydrofluoric (HF) acid to distilled water. The titanium specimen was connected to the anode, and a platinum plate (3 mm × 4 mm × 0.1 mm) was connected to the cathode. The specimen and platinum plate were placed approximately 10 mm apart and dipped in the electrolyte mixture for one minute. After that, 20 V were applied to the specimen and platinum plate for anodic oxidation for 10 min.

Heat Treatment
The heat treatment was performed using a sintering furnace (DUOTRON PRO ex-6100, ADDIN Co., LTD, Suwon, Republic of Korea). Under an air atmosphere, the temperature was increased to 400 • C and 600 • C at a rate of 1 • C/min. The furnace was cooled after maintaining the temperature for one hour.

Non-Thermal Plasma Treatment
The non-thermal plasma treatment was performed using an atmospheric pressure plasma generator (PGS-200, Expantech Co., Suwon, Republic of Korea) (Table 4). Ar and O 2 gases were mixed in a ratio of 99% to 1%, and applied to the specimen at 300 W at a rate of 10 L/min. The distance between the plasma flame and the specimen was maintained at 5 mm. The specimen was rotated at 180 rpm, and the plasma flame was moved to the right and left ten times (12 s for one time) to evenly apply the atmospheric pressure plasma to the specimen. Thus, the plasma flame was applied for a total of 120 s ( Figure 2). Four microliters of distilled water were dropped onto the surfaces of the specimens to compare the changes in the hydrophilicity. The angle between the surface and the solution was measured after ten seconds using a video contact angle measuring device (Phoenix 300, SEO Co., Suwon, Republic of Korea). For each group, the angle of three specimens was measured and averaged.

Surface Structures
The changes in the surface structure of the specimens were observed using a scanning electron microscope (FE-SEM S-4700, Hitachi Horiba, Tokyo, Japan).

XRD Analysis
The changes in the crystal structure on the surface of the specimens were analyzed using an X-ray diffractometer (XRD) (X'Pert PRO Multi-Purpose X-ray Diffractometer, PANalytical, Almelo, The Netherlands). Diffraction was analyzed using X-rays of CuKα at a speed of 1.5 • /min at an angle of 2θ ranging from 20 • to 90 • .

XPS Analysis
The chemical compositions and bonds of specimens were analyzed using X-ray photoelectron spectroscopy (XPS, VG Mulrilab 2000, Thermo Scientific, Oxford, UK). The peak area values of each element detected were normalized and expressed as a quantitative ratio.

Bacterial Culture
In this study, Streptococcus mutans (KCOM 1504), a Gram-positive aerobic bacterium associated with initial biofilm formation, and Porphyromonas gingivalis (KCOM 2804), a Gram-negative anaerobic bacterium that causes peri-implantitis, were used. The two species of bacteria were obtained from the Korean Collection for Oral Microbiology (KCOM, Gwangju, Republic of Korea). S. mutans was cultured in a Brain Heart Infusion broth (BHI, Becton, Dickinson and Company, Sparks, MD, USA), and P. gingivalis was cultured in a Tryptic Soy Broth (TSB, Becton, Dickinson and Company, Sparks, MD, USA). Single colonies cultured on a solid medium were cultured in a liquid medium and grown to a concentration of 1.5 × 10 7 CFU/mL.

Artificial Saliva Coating
Specimens were coated with artificial saliva to create an oral environment. The artificial saliva used in this study was prepared by adding 1% mucin (Mucin from porcine stomach, M1778; Sigma-Aldrich, St. Louis, MO, USA) to an adhesion buffer [46]. Three specimens per group were put in a 24-well plate, and the prepared artificial saliva was applied. The specimens were slowly stirred in the culture medium at 37 • C for two hours to coat the specimens with the artificial saliva. S. mutans was cultured in BHI at 37 • C (LIB-150M, DAIHAN Labtech Co., Namyangju, Republic of Korea), and P. gingivalis was cultured in anaerobic TSB at 37 • C (Forma Anaerobic System 1029; Thermo Fisher Scientific, Waltham, MA, USA).

Bacterial Inoculation
After two hours, the artificial saliva was removed, and the specimens were dried for 15 min. Bacteria were inoculated on the surface of the specimens at a concentration of 1.5 × 10 7 CFU/mL. S. mutans was cultured for 24 h, and P. gingivalis was cultured for 48 h.

Fluorescent Nucleic Acid Staining Assessment
The biofilm formation was assessed using a green-fluorescent nucleic acid stain (SYTO 9 ® , Molecular Probes Europe BV, Leiden, The Netherlands). After culturing bacteria, the culture medium was removed. The specimens were carefully cleaned two times using phosphate-buffered saline (PBS) to remove bacteria that did not adhere. Afterwards, 200 µL of fluorescent dye (SYTO 9 ® :dH 2 O = 1.5 µL:1 mL) was injected onto the specimens. The plate was sealed with aluminum foil to block out light and was placed at room temperature for 15 min. After the reaction, the residual staining solution was carefully cleaned using PBS, and the surfaces of the specimens were observed using a laser confocal scanning microscope (Leica TCS SP5 AOBS/tandem, Leica, Bensheim, Germany). The thickness of the biofilms formed on the specimens was measured using an analysis program (Leica LAS AF software, Leica Microsystems, Bensheim, Germany).

Results of Heat Treatment
When the assumption of normality was met, the significance of the heat treatment results was tested using a parametric one-way analysis of variance (ANOVA) and Tukey's post-hoc test. The significance level was set at a p-value less than 0.05. When the assumption of normality was not met, the results were statistically analyzed using a non-parametric Kruskal-Wallis test. The two groups were paired and tested using a Mann-Whitney U-test. Type I errors were corrected using Bonferroni's method. The significance level was set at a p-value less than 0.017.

Results of Plasma Treatment
When the assumption of normality was met, the significance of the plasma treatment results was statistically analyzed using a parametric independent t-test. When the assumption of normality was not met, the two groups were compared using a non-parametric Mann-Whitney U-test. The significance level was set at a p-value less than 0.05.

Conclusions
In this study, TiO 2 nanotubes were formed on the titanium surface of dental implants to increase the surface area. TiO 2 nanotubes were heat-treated under different conditions to modify the structure of the TiO 2 oxide films. In addition, atmospheric pressure plasma was applied to the TiO 2 nanotubes to change their surface energy and hydrophilicity. This study found that the TiO 2 nanotubes heat-treated at 400 • C inhibited the adhesion of S. mutans, associated with early biofilm formation, and the TiO 2 nanotubes heat-treated at 600 • C inhibited the adhesion of P. gingivalis, which causes peri-implantitis. Applying plasma to TiO 2 nanotubes heat-treated at 600 • C can also inhibit the adhesion of S. mutans and P. gingivalis.