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Back to Journal »International Journal of Nanomedicine» Volume 16

Biological effects of a novel mulberry surface on titanium characterized by micro/nanopores and plasma-based graphene oxide deposition

Author Kim HS, Ji MK, Jang WH, Alam K, Kim HS, Cho HS, Lim HP

Published on October 28, 2021, the 2021 volume: 16 pages 7307-7317

DOI https://doi.org/10.2147/IJN.S311872

Single anonymous peer review

Editor approved for publication: Prof. Dr. Anderson Oliveira Lobo

Hee-Seon Kim1 *, Min-Kyung Ji2 *, Woo-Hyung Jang1 *, Khurshed Alam,3 Hyun-Seung Kim,4 Hoon-Sung Cho,3 Hyun-Pil Lim1 1 Department of Prosthodontics, School of Dentistry, Chonnam University, Gwangju, Republic of Korea ; 2 Chonnam University Optoelectronic Fusion Research Center, Gwangju, 61186, Republic of Korea; 3 Department of Materials Science and Engineering, Chonnam University, Gwangju, 61186, Republic of Korea; 4 New Project Department of KJ Meditech Co, Ltd, Gwangju, 61009, South Korea *These authors Made equal contributions to this work Republic of Korea Tel 82-10-2645-7528 Fax 82-62-530-5577 Email [email protected] Hoon-Sung Cho Department of Materials Science and Engineering, Chonnam National University, Gwangju, 61186 , Republic of Korea Tel 82-10-8545-2816 Fax 82-62-530-1717 E-mail [email protected] Purpose: This article introduces a technology that uses a combination of nitriding and anodizing to develop new surfaces for dental implants. Then the graphene oxide using atmospheric plasma is deposited. The effects of various surface treatments on bacterial adhesion and osteoblast activation were also evaluated. Method: Process CP titanium (control) into disc-shaped specimens. Vacuum nitriding is used for nitriding, and then a DC power supply is used for anodic oxidation in the electrolyte to form a new type of "mulberry surface". Graphene oxide deposition is performed using atmospheric plasma with an inflow of a carbon source. After analyzing the sample surface, Streptococcus mutans and Porphyromonas gingivalis were used to evaluate the antibacterial activity. The viability, adhesion, proliferation and differentiation of osteoblasts were also evaluated. The analysis of variance (ANOVA) using Tukey's post-hoc test was used to calculate statistical differences. Result: We observed that mulberry bark was formed on the surface of the samples after nitriding and anodizing. These samples showed more effective antibacterial activity than the control samples. We also found that samples with additional graphene oxide deposition showed better biocompatibility, which was verified by osteoblast adhesion, proliferation, and differentiation. Conclusion: The development of the surface of mulberry tree and the deposition of graphene oxide inhibited the adhesion of bacteria to the implant, and enhanced the adhesion, proliferation and differentiation of osteoblasts. These results indicate that the mulberry surface and graphene oxide deposition together can inhibit peri-implant inflammation and promote osseointegration. Keywords: nitridation, anodization, atmospheric plasma, biofilm formation, osteoblasts

Due to its high biocompatibility and osseointegration, titanium is widely used as dental implant material to replace missing teeth. 1 However, the failure of dental implant treatment is related to the lack of stability of the implant abutment interface, the occurrence of bacterial infection and the failure of restoration. 2 In addition, it is reported that changes in the chemical composition and surface roughness of titanium implants affect osseointegration and antibacterial activity. 3,4

Nitriding treatment of titanium surface has high biocompatibility; therefore, this kind of surface treatment of biomaterials has received great attention. In addition, in some of our previous studies, we reported that the adhesion of Streptococcus mutans to the titanium surface was reduced due to the presence of the TiN layer. 5-7 On the other hand, anodic oxidation can form a thin film composed of amorphous TiO2 that is electrochemically porous. The surface area of ​​the film can be increased by changing the underlying crystal structure or adjusting the thickness or surface roughness of the film, thereby improving cell viability, adhesion, proliferation and differentiation. 8,9 In addition, with microporous surfaces, it is reported that nanopores and nanopores are beneficial to the viability, adhesion and proliferation of osteoblasts. 10,11 In this research, we have developed a new type of titanium surface, which we call "mulberry surface", which contains both micropores and nanopores, and is obtained through titanium nitride and then anodized.

Graphene oxide is an allotrope with hexagonal symmetric carbon. As a nanomaterial with large surface area and excellent physical, chemical and biological properties, it has attracted attention in various fields. 12 Graphene oxide usually uses chemical vapor deposition and Hummer's method. 13-15 However, it requires additional treatment before it can be applied to the surface of the material. Therefore, in this study, we developed an innovative method of depositing graphene oxide on the surface of titanium using atmospheric plasma. 16 Please note that atmospheric plasma decomposes carbon molecules into carbon atoms and generates graphene oxide when flowing through the plasma flame. It is well known that plasma-based graphene oxide deposition on titanium can enhance biocompatibility and the viability and differentiation of osteoblasts. 15

In this study, the titanium surface was first nitrided and then anodized to form the surface of the mulberry tree. Next, graphene oxide is deposited on the surface. Finally, we evaluated the properties, antibacterial properties, and osteoblast viability of the new surface.

Commercial pure titanium (ASTM grade IV, Kobe Steel) was cleaned and processed into discs (diameter: 15 mm, thickness: 3 mm) and surface treated to generate our experimental samples, as shown in Table 1. Table 1 Treatment groups in the experiment

Table 1 Treatment groups in the experiment

The nitride layer on the titanium surface is formed by vacuum nitridation (Mirae-2VF600, Thermotec, Korea). The disc is heat-treated in the temperature range of 600-800°C, and injected with N2 gas at 1020°C under vacuum for 4 hours. Next, the disc was cooled under a pressure of 6 bar to form a nitride layer.

A DC power supply (Fine Power F-3005, South Korea) was used to anodize the porous surface. We prepared the electrolyte used in this study by adding 1 M phosphoric acid solution (Sigma-Aldrich, USA) and 1.5 wt% hydrofluoric acid solution (Sigma-Aldrich) to distilled water. The titanium and platinum disks are connected to the cathode and anode, respectively, and the sample is placed about 10 mm away from the platinum disk. A voltage of 20 V was applied, and the connected titanium and platinum discs were deposited in the electrolyte for 1 minute. After 60 minutes, the sample was removed from the electrolyte and washed under running water for about 30 minutes. Next, deposit the sample in distilled water for 1 hour and then dry it.

An atmospheric pressure plasma generator (PGS-300 Expantect, South Korea) was used to deposit graphene oxide on the titanium sample. Argon gas (4 L/min) and methane gas (3.5 mL/min) are mixed and introduced into the quartz tube, and graphene oxide is coated on the sample using a high-frequency (900 MHz) plasma generator with a power of 300 W . The distance between the plasma flame and the sample is maintained at 15 mm. In addition, the plasma flame was uniformly applied to the sample for 6 minutes, while the sample was rotated at a speed of 180 rpm, and the flame was rotated left and right (see Table 2 for details). Table 2 Parameters of atmospheric plasma generator

Table 2 Parameters of atmospheric plasma generator

Use a vernier caliper (MT-500-181-300, Mitutoyo, Korea) to measure the thickness of each sample at 3 points before and after the surface treatment to obtain an average value. A field emission scanning electron microscope (FE-SEM; S-4700, Hitachi, Japan) was used to analyze the formation of micropores and nanopores and the surface structure of samples with graphene oxide deposition. A laser Raman spectrophotometer (NRS-5100, JASCO, South Korea) was used to perform Raman spectroscopy on the sample under laser excitation at 532.13 nm to determine the state of graphene oxide deposition. X-ray photoelectron spectroscopy (XPS; MultiLab 2000, Thermo Electron Corporation, England) was used to analyze the chemical composition and bond energy of graphene oxide. A non-contact nano surface 3D optical profiler (OP; NV-E1000, Nano System, Korea) was used to observe the surface roughness of various processed samples. In each sample, measure three areas and calculate their average roughness or Ra value. Wettability is a key factor of biocompatibility, which is determined by contact angle measurement using a video contact angle measuring device (Phoenix 300, SEO, Korea). Drop distilled water (4 μL) on the surface of the sample. After 10 s, measure the angle between the surface and the solution and calculate the average value (Surfaceware 9 softwareⓇ, SEO).

Streptococcus mutans (KCOM 1054, Gwangju, South Korea), a gram-positive bacteria known to be involved in the early stages of biofilm formation, and Porphyromonas gingivalis (KCOM 2804, Gwangju, South Korea), a species known to cause surrounding The gram-negative anaerobic bacteria-implantitis formed by biofilm was obtained from the Korean Collection of Oral Microbiology (KCOM). Strains of Streptococcus mutans were cultured in a culture room (LIB-150M, DAIHAN Labtech Co., Namyangju, Korea) using brain-heart infusion (BHI; Becton, Dickinson and Company, Sparks, MD, USA) medium at 37°C. Porphyromonas gingivalis strains are also used in anaerobic culture chambers (Forma Anaerobic System 1029; Thermo Fisher Scientific, Waltham, MA, USA) in tryptic soy broth (Becton, Dickinson and Company, Sparks, MD, USA) Incubate at 37°C.

All samples were sterilized in an autoclave (HS-3460SD, Hanshin Medical Co., Korea) for 2 hours. Each set of samples was prepared and fixed on a 24-well plate (SPL Life Sciences Co., Ltd., Korea). Each sample was inoculated with Streptococcus mutans and Porphyromonas gingivalis (1.5 × 107 CFU/mL) and cultured for 24 and 48 hours, respectively.

The degree of bacterial adhesion was analyzed by crystal violet staining. After the bacteria are cultured, the medium is removed and the sample is washed twice with a phosphate buffered saline (PBS) solution. Stain the bacteria adhering to the sample by dispensing 500 μL of 0.3% crystal violet solution. After 10 minutes, the crystal violet solution was removed, and the remaining samples were washed three times with PBS. The sample is then dried for 15 minutes, and then 500 μL of demineralization solution (80% ethanol 20% acetone) is dispensed. To ensure that the solution does not evaporate, it is sealed and stirred for 1 hour. Next, distribute 200 μL of each sample into a 96-well plate (SPL Life Sciences Co., Ltd), and measure it at a wavelength of 595 nm using a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA) Absorbance).

MC3T3-E1 (MC3T3-E1 subclone 4, ATCC CRL2593, USA) is an osteoblast cell line cultured in a 5% CO2 incubator (Forma Series II3111 Water Jacketed CO2 Incubator, Thermo Fisher Scientific) at 37°C . α-Minimum Essential Medium (Dulbecco's Modified Eagle Medium, Gibco-BRL, Grand Island, NY, USA) is used with 10% Fetal Bovine Serum and 1% Penicillin.

To evaluate cell adhesion, we fixed 10 samples from each group on a 24-well plate. Dispense the cultured cell solution with a concentration of 4×104 cells/mL onto the sample. The cells were cultured at 37°C in a 5% CO2 incubator. Cell adhesion was assessed after 24 hours of incubation, and cell proliferation was assessed on the fifth day after cell distribution. The WST-8 detection kit (EZ-Cytox, Itsbio, Inc., Korea) was used to analyze cell adhesion and proliferation. Add 100 μL of WST-8 solution to each well of the plate and incubate again in a 37°C 5% CO2 incubator. After 1 h, when the color of the sample turns orange, distribute the suspension (100 μL) in each well to a 96-well plate, using the VersaMax ELISA microplate reader (Molecular equipment).

LIVE/DEAD™ Viability/Cytotoxicity Kit for visual assessment of cell adhesion and proliferation in mammalian cells (US, catalog number L3224). Distribute cultured cells at a concentration of 4×104 cells/mL to each sample and culture them in a CO2 incubator set at 5% CO2 and 37°C for 24 and 48 hours. Then use the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher Scientific) to stain the cells at 37°C for 30 minutes. At each time point, the cells were washed three times with PBS, and then stained with 200 µL of fluorescent reagent (AM: ethidium homodimer-1: dH2O = 0.5 µL: 2 µL: 1 mL) for 30 minutes. Then, after washing the sample three times with PBS, a confocal laser scanning microscope (Leica TCS SP5 AOBS/tandem, Leica Microsystems, Bensheim, Germany) was used to analyze cell adhesion and proliferation.

The cell differentiation was evaluated using 10 samples per group fixed on a 24-well plate. Dispense the cultured cell solution with a concentration of 1×104 cells/mL on the sample (cells are cultured in a 5% CO2 incubator at 37°C).

The cell differentiation was assessed on the 21st day after the cells were allocated and analyzed using ALP analysis. Add 200 μL of ALP assay solution to each well and incubate in a 37°C 5% CO2 incubator. After 1 h, transfer 80 μL of the suspension in each well to a 96-well plate, and then treat it with 50 μL of p-nitrophenyl phosphate (pNPP) solution. Next, the sample was incubated again for 1 hour at 37°C in a 5% CO2 chamber. Subsequently, the sample was treated with 20 μL of stop solution and its absorbance was measured at 405 nm using a VersaMax ELISA microplate reader (Molecular Devices).

Use SPSS software (version 21.0; SPSS Inc., Chicago, IL, USA) for statistical analysis. When the normality assumption is satisfied in the Shapiro-Wilk test, the Tukey post-hoc test is used to perform one-way analysis of variance (ANOVA). When the significance level is 95% and the p-value is less than 0.05, the result is considered statistically significant.

The thickness of each group of samples before surface treatment is constant at 3mm. In contrast, after surface treatment, the thickness of the sample without any treatment (control) is 3 mm; due to the deposition of the nitride layer, the thickness of the "N" group coated with the nitride layer has changed, which is confirmed to be 3.02 mm; in the "NA" group, the thickness is confirmed to be slightly higher due to the nitriding layer and 1 hour anodizing treatment, which is 3.05 mm; in the "NAG" group, it is confirmed that the thickness is due to the nitride layer coating, anodic oxidation and graphite oxide The thickness increases to 3.07 mm, and this group shows the deposition of the thickest layer of all groups. The nitride layer on the titanium surface was treated with an electrolyte composed of 1 M H2PO4 and 1.5 wt% HF, and anodized at 20 V for 60 minutes. This resulted in the formation of a new type of mulberry surface with micropores and nanopores. The FE-SEM analysis results (Figure 1) revealed the machined surface in the control group (Figure 1A); no micropores or nanopores were produced in the N group coated with a nitride layer (Figure 1B). The NA group after nitriding and anodizing (Figure 1C) showed a mulberry surface, combining micropores (average size: 50-100 µm) and nanopores (average size: 25-30 nm). In the NAG group (Figure 1D), additional graphene oxide nanolayers are deposited on the porous surface, and these nanolayers aggregate into clouds, as shown in the Raman spectrum. Figure 1 FE-SEM image of titanium sample: (A) control group; (B) group N containing only nitride layer; (C) NA group after nitriding and anodization; (D) NAG group after nitriding, Anodizing and graphene oxide deposition (10,000x FE-SEM mode). (E) control group; (F) group N with only nitride layer; (G) group NA after nitridation and anodization; (H) group NAG after nitridation, anodization and graphene oxide deposition (100,000x FE-SEM mode).

Figure 1 FE-SEM image of titanium sample: (A) control group; (B) group N containing only nitride layer; (C) NA group after nitriding and anodization; (D) NAG group after nitriding, Anodizing and graphene oxide deposition (10,000x FE-SEM mode). (E) control group; (F) group N with only nitride layer; (G) group NA after nitridation and anodization; (H) group NAG after nitridation, anodization and graphene oxide deposition (100,000x FE-SEM mode).

Raman spectroscopy reveals the unique peaks of graphene oxide in the NAG group deposited by graphene oxide (D band at ~1350 cm-1, G band at ~1590 cm-1 and 2D band at ~2580 cm-1 ) (See Figure 2). This confirms that graphene oxide is deposited on the titanium surface. Figure 2 Raman spectrum of the graphene oxide layer formed on the titanium surface, confirming that the deposited carbon film is composed of graphene oxide.

Figure 2 Raman spectrum of the graphene oxide layer formed on the titanium surface, confirming that the deposited carbon film is composed of graphene oxide.

In XPS analysis, Ti (2p1), O (1s), and C (1s) electrons were detected in all samples; in addition, N (1s) electrons were detected in the N, NA, and NAG groups. In the N group, the intensity of the N(1s) peak increases after nitriding. In the NA group, the intensity of the N(1s) peak decreased after anodization, while the intensity of the C(1s) peak increased. In the NAG group, due to the deposition of graphene oxide, the intensity of the C (1s) peak is three times that of the NA group (Figure 3). In the XPS analysis of the anodized NA group and the anodized and graphene oxide deposited NAG group, we could not check the nitride layer due to the thin nitride layer and the resulting weak TiN peak. Figure 3 XPS spectra of titanium samples: (A) control group; (B) N group; (C) NA group; (D) NAG group.

Figure 3 XPS spectra of titanium samples: (A) control group; (B) N group; (C) NA group; (D) NAG group.

The surface roughness of each treatment group was obtained from the 3D-OP analysis, and it was found that the surface roughness of the control group and the N, NA, and NAG groups were 0.03, 0.05, 3.2, and 4.2 µm, respectively. Therefore, the roughness of the control group was the lowest, while the roughness of the NAG group was the highest (Figure 4). The adjustment of surface roughness in samples with graphene oxide deposition has not been studied separately. We prioritize research on cell compatibility and focus on the best coating type for cell compatibility. Figure 4 Three-dimensional surface morphology and roughness of titanium samples: (A) control group; (B) N group; (C) NA group; (D) NAG group.

Figure 4 Three-dimensional surface morphology and roughness of titanium samples: (A) control group; (B) N group; (C) NA group; (D) NAG group.

The water contact angles of the samples of the control group and the N, NA and NAG groups were 53.27°, 60.72°, 62.93° and 125.48°, respectively. Therefore, the control group, the N group, and NA showed a hydrophilic trend, while the NAG group showed a hydrophobic trend (Figure 5). Figure 5 Water contact angle measurement results of titanium samples: (A) control group; (B) N group; (C) NA group; (D) NAG group.

Figure 5 Water contact angle measurement results of titanium samples: (A) control group; (B) N group; (C) NA group; (D) NAG group.

The result of crystal violet determination showed that compared with the control group, the adhesion of Streptococcus mutans in NA and NAG groups to the sample was significantly reduced; in addition, compared with the N group, the adhesion of NA and NAG groups was reduced. Compared with the adhesion of the control group, the N group and the NA group, the adhesion of Porphyromonas gingivalis to the samples in the NAG group was also significantly reduced (Figure 6). These results indicate that by inhibiting the adhesion of Streptococcus mutans and Porphyromonas gingivalis, the NA and NAG groups exhibited stronger antibacterial activity than the control group. Considering that the antibacterial activity is significantly affected by the surface shape and roughness, the porous oxide film formed on the surface of the mulberry tree in the 17 NA group increased the surface area of ​​the sample, and the graphene oxide deposition in the NAG group further increased the antibacterial activity. Figure 6 The results of one-way analysis of variance analysis of the adhesion of Streptococcus mutans (SM) and Porphyromonas gingivalis (PG) to the control group and the titanium samples in the N, NA, and NAG groups. **Marginal significance at p <0.001.

Figure 6 The results of one-way analysis of variance analysis of the adhesion of Streptococcus mutans (SM) and Porphyromonas gingivalis (PG) to the control group and the titanium samples in the N, NA, and NAG groups. **Marginal significance at p <0.001.

After the cells were cultured for 24 hours, the adhesion of MC3T3-E1 cells was analyzed using the WST assay. The adhesion of cells to the surface of the N, NA, and NAG groups was significantly higher than that of the control group, indicating that the cell viability of the three experimental groups was higher than that of the control group. After the cells were cultured for five days, the cell proliferation was analyzed using the WST assay. Compared with the control group, the cell proliferation of the N group and the NA group increased significantly, while the cell activity of the NAG group was the highest (Figure 7). The image of fluorescently stained cells attached to the surface is shown in (Figure 8). After 1 day of incubation, the cells were evenly dispersed on each support. Especially after 5 days of culture, the cell density showed a well-dispersed form. With the increase of the culture time, the cell density increased, indicating that all the surface-treated experimental groups provided a good environment for cell adhesion and proliferation. Figure 7 One-way analysis of variance results of WST detection on cells cultured for 24 hours or 5 days in titanium samples in the control group and the N, NA, and NAG groups. **Marginal significance at p <0.001. Figure 8 Fluorescence staining image of cells cultured for 24 hours (64x laser confocal scanning microscope mode). (1) Control group; (B) N group; (C) NA group; (D) NAG group. Fluorescence staining image of cells cultured for 5 days (128x laser confocal scanning microscope mode). (E) Control group; (F) N group; (G) NA group; (H) NAG group. Green fluorescence indicates live cells, and red fluorescence indicates dead cells.

Figure 7 One-way analysis of variance results of WST detection on cells cultured for 24 hours or 5 days in titanium samples in the control group and the N, NA, and NAG groups. **Marginal significance at p <0.001.

Figure 8 Fluorescence staining image of cells cultured for 24 hours (64x laser confocal scanning microscope mode). (1) Control group; (B) N group; (C) NA group; (D) NAG group. Fluorescence staining image of cells cultured for 5 days (128x laser confocal scanning microscope mode). (E) Control group; (F) N group; (G) NA group; (H) NAG group. Green fluorescence indicates live cells, and red fluorescence indicates dead cells.

The differentiation of MC3T3-E1 cells was analyzed using the ALP assay after 7 days of culturing the cells. Compared with the control group and the N group, a significant increase in cell differentiation was observed in the NAG group (Figure 9). Figure 10 compares the cell viability and antibacterial effects of different titanium surfaces. With the addition of surface treatment, cell viability gradually increased, while the antibacterial effect showed the opposite trend. Compared with the control group (that is, without any surface treatment), the cell viability of the NAG group was increased by 45%, while the bacterial activity was reduced by 60%. Figure 9 Results of one-way analysis of variance on cells cultured for 7 days on titanium samples in the control group and the N, NA, and NAG groups and measured using the ALP assay. *Slightly significant at p <0.05, ** Slightly significant at p <0.001. Figure 10 Comparison of cell viability and antibacterial effect between the titanium control group and the N, NA and NAG groups.

Figure 9 Results of one-way analysis of variance on cells cultured for 7 days on titanium samples in the control group and the N, NA, and NAG groups and measured using the ALP assay. *Slightly significant at p <0.05, ** Slightly significant at p <0.001.

Figure 10 Comparison of cell viability and antibacterial effect between the titanium control group and the N, NA and NAG groups.

Titanium is widely used as a successful implant material due to its high biocompatibility. 19 In addition, it is reported that a surface with micropores and nanopores can maximize the contact between bone tissue and implants, improve osseointegration, and inhibit bacterial adhesion. 20-22 Taking these two factors into account, Electrochemical treatment (such as anodization) can form a titanium surface with both micropores and nanopores, and the shape of the surface can be changed by controlling process variables, such as electrolyte composition, voltage, and temperature. twenty three

Graphene oxide is a kind of nanomaterials with biological properties. Because of its antibacterial activity and properties related to cell adhesion, proliferation and differentiation, it is also used in dental implants. However, its production also has several disadvantages, such as low accessibility, high cost, and the generation of harmful gases that cause pollution. 13-15 These problems can be solved by using an atmospheric plasma-based method, which is easy to implement and can continuously produce graphene oxide in large quantities. 24 Therefore, in this study, we developed a new method of using atmospheric plasma to deposit graphene oxide and found that it significantly promotes cell differentiation. 16 In this study, both nitriding and anodic oxidation performed titanium surface on the cells to achieve the roughness of the mulberry surface that contains both micropores and nanopores. In addition, graphene oxide is deposited on the surface to take advantage of its properties related to the inhibition of bacterial adhesion and properties related to the adhesion, proliferation and differentiation of osteoblasts.

Using 3D-OP analysis to measure the surface roughness, it was observed that the roughness values ​​of the NA and NAG groups were higher than those of the control and N groups. It is reported that the higher the surface roughness, the sharper the surface, and therefore, in this case, the effect of inhibiting bacterial adhesion is better. 25 In addition, studies on antibacterial mechanisms have shown that graphene oxide inhibits bacterial adhesion mainly through oxidative stress and direct contact of bacteria with their sharp surfaces. 26-28 Consistent with the above findings, in this study, we also observed that the titanium surface treated with anodization and graphene oxide deposition became rougher and sharper, which in turn increased the inhibition of bacterial adhesion. By measuring the water contact angle to test the wettability of the surface, we observed that the control group and the N and NA groups showed a hydrophilic tendency. In contrast, the NAG group showed a hydrophobic trend. Many studies have been conducted on the correlation between the inhibition of bacterial adhesion and surface properties. For example, Agarwalla et al.29 studied the inhibitory effect of surface treated with graphene oxide deposition on Streptococcus mutans. They report that the resulting hydrophobic surface increases the inhibition of bacterial adhesion. Our findings are consistent with these results.

Several recent studies have reported an increase in the antibacterial activity of graphene oxide deposits. 26,30,31 A report showed that graphene oxide deposits reduce the thickness of the biofilm formed by Pseudomonas putida and increase the ability to separate the biofilm from the surface. 32 The antibacterial activity mechanism of graphene oxide deposition is not yet clear, and research is still in progress. It is reported that there are two reasons for the known antibacterial mechanism of graphene oxide deposition: oxidative stress of graphene oxide deposition and inhibition of bacterial adhesion caused by direct contact with the sharp edges of graphene oxide deposition. 33-35 In other words, it is believed that the surface of the application of anodic oxidation and graphene oxide deposition becomes rougher and the edges become sharper, thereby increasing the ability to inhibit bacterial adhesion. However, contrary to these arguments, some studies conducted related in vitro experiments, which indicated that the ROS mechanism is not the main mechanism of the antibacterial effect of graphene oxide deposition. 36, 37 Another antibacterial process involves the dispersion and retention of oxygen. -Graphene oxide deposits contain functional groups. 38,39 It is reported that graphene oxide deposits with hydrophobic functional groups exert antibacterial properties by forming a stable dispersion and providing a large contact area for interaction with bacterial cells. Experiments and discussions are ongoing to study the antibacterial effect of graphene oxide deposition and the role of functional groups. These effects should be studied and examined more carefully in the future.

In terms of cell activity, Sagirkaya et al.33 reported that the level of cell adhesion varies according to the surface roughness of the materials, their chemical composition and the size of the pores. Zhang et al. 34 reported that a porous oxide film containing micropores and nanopores on the titanium surface has a positive effect on cell adhesion and stability. In addition, Park et al.35 reported that the surface of nanopores with a size range of 15-30 nm facilitates the adhesion of various types of cells, including osteoblasts. Several studies, including Ekaterina,40,41, have shown that osteoblasts recognize surface roughness through the interaction of proteins in the extracellular matrix, and they respond to grooves of 100 nm or smaller. It is worth noting that the size of the integrin molecules present in osteoblasts is similar to the size of the nano-scale grooves formed on the surface of the implant. Therefore, the NA group with nanopores appeared to have increased osteoblast adhesion compared to the control group in this study.

Previous studies reported that graphene oxide has good biocompatibility and plays a biological role in promoting osteogenic adhesion, proliferation, differentiation, and calcium mineral deposition. 42-44 The deposited graphene oxide sheet has many negatively charged oxygen-containing functional groups, which can interact with cell membrane phospholipids and proteins through electrostatic interactions, hydrogen bonds, etc. This interaction causes osteoblasts to attach to the deposited graphene oxide sheets and then proliferate. 45 The excellent surface properties of graphene oxide deposits cause some biologically active ions and proteins to be adsorbed on it, which promotes the diffusion of cytoskeletal actin filaments into the matrix and further stimulates osteogenic differentiation. 46 It was found that the integration of the micro-nano structure surface and the graphene oxide deposition system provides a microenvironment suitable for the osteogenic differentiation of cells. Therefore, due to the deposition of graphene oxide, the surface properties have undergone significant changes, further promoting osteogenic differentiation.

The adhesion, proliferation, and differentiation of osteoblasts in the NAG group treated with graphene oxide also increased, and these results can be attributed to the above-mentioned properties of graphene oxide. 47

This study confirmed that titanium implants with a mulberry surface (that is, both micropores and nanopores) and a graphene oxide layer can effectively inhibit bacterial adhesion and increase the activity of osteoblasts.

The nitridation and anodization to form a mulberry surface on a titanium implant and the deposition of graphene oxide on the surface were studied. We have observed that bacterial adhesion is inhibited, and the adhesion, proliferation and differentiation of osteoblasts are enhanced on this surface. Therefore, we concluded that titanium implants with mulberry surface and graphene oxide deposition can reduce peri-implant inflammation and improve osseointegration.

This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government (MSIP) (No. 2020R1F1A1076982 and 2018R1A6A1A03024334).

Hee-Seon Kim, Min-Kyung Ji and Woo-Hyung Jang are the co-first authors. Ms. Hee-Sun Kim reported on the grant from the National Research Foundation of Korea (2020R1F1A1076982, 2018R1A6A1A030243340) during the research period. The author reports that there is no conflict of interest in this work.

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