Electophoretically Deposition of Ti3C2 on Titanium Surface for Hard Tissue Implant Applications

As a metallic biomaterial, titanium (Ti) exhibits excellent biocompatibility, but its osteoinductivity is limited. Therefore, to improve this property, an electrophoretic deposition (EPD) technique was used to coat the Ti surface with Ti3C2 MXene (Ti3C2), a new class of two-dimensional nanomaterial. Ti3C2 is known to have good biocompatibility and better osteoinductivity than graphene oxide. The coating layer was characterized by a particulate microstructure and exhibited X-ray diffraction and Raman spectroscopy peaks corresponding to the Ti3C2 phase. In vitro cell tests using human mesenchymal stem cells confirmed that the cell attachment and proliferation on Ti3C2-coated Ti were similar to that of bare Ti, and that the osteoinductivity was significantly enhanced compared with bare Ti.


Introduction
Titanium (Ti) and its alloys have been extensively studied and used for hard tissue implants, primarily due to their excellent biocompatibility and mechanical properties [1][2][3]. Naturally formed thin oxide layers (thickness: few nanometers) occur on the surface of Tibased alloys. The inertness of this passive layer protects the metallic implant from corrosive environments, but limits interaction between the implant and surrounding bone tissue [4]. Osseointegration of metallic implants can be promoted in implant surfaces with micro or nanotopology or bioactive compositions. Several techniques (such as plasma spraying, anodic oxidation, physical vapor deposition, and electrophoretic deposition (EPD)) have been employed in an attempt to achieve these surface properties [5][6][7][8]. Although the EPD process is a conventional coating method, it is still useful because it is simple, cost-effective, and useful for coating materials that are charged in the electrolyte. In the EPD technique, electrically charged particles are deposited onto an anode or a cathode, under an electric field. Using this method, the surface of a Ti-based implant system has been modified with various substances, including bioactive inorganic, organic, and composites [9][10][11].
MXene (M n+1 X n or M n+1 X n T z ) is a new class of two-dimensional nanomaterials that can be obtained by selectively etching the A phase from the MAX phase (M n+1 AX n ; M: early transition metal, A: A-group element, X: C or N, n: 1-3). MXenes have a large specific surface area, exhibit good electrical conductivity, and contain hydrophilic surface groups such as -OH, -O, and -F [12]. Due to these properties, studies on these materials have focused mainly on the fields of energy storage, catalysts, and sensors [13][14][15]. In the biomedical field, MXenes have mainly been studied for use in cancer treatments, owing to their intrinsic photothermal properties and drug-loading ability [16]. MXenes are also used as filler materials and exhibit improved biocompatibility of polymer-based implant systems [17].
The aim of this work was to enhance the osteoinductivity of Ti by coating the Ti surface with Ti 3 C 2 MXene using an EPD technique. As far as we know, this is the first work that reports the effect of a Ti 3 C 2 coating layer on the behaviors of cells cultured on an implant surface. Negatively charged Ti 3 C 2 can be readily used in the EPD process without any pre-treatment. The coating layer was characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), and Raman spectroscopy. Moreover, the effect of the Ti 3 C 2 coating layer on the biological properties of the Ti surface was evaluated via in vitro cell tests using human mesenchymal stem cells (hMSCs).

Electrophoretic Deposition of Ti 3 C 2
A raw Ti 3 C 2 MXene powder was purchased from Invisible Co. (Invisible, Suwon, Korea) and used without further treatment. The basic characterization of Ti 3 C 2 was summarized in Figure S1. A suspension of Ti 3 C 2 in an EtOH-water co-solvent (80% EtOH) solution with a 3 mg/mL concentration was prepared as an electrolyte for the EPD process. The pH of the suspension was fixed at a value of 1.2 using 1 M HCl solution. A commercially pure Ti specimen (CP grade 2, diameter: 20 mm and thickness: 2 mm, Titanart, Incheon, Korea) was prepared and ground with a 1200-grit SiC abrasive paper. Owing to the positively charged nature of Ti 3 C 2 particles, a cathodic EPD process was performed by applying 1 A of direct current (DC) for 1 min using a DC power supply (TPM-1001D, Toyotech, Incheon, Korea). The Ti plate was used as a counter electrode (anode) and a distance of 1 cm was maintained during the process. After the coating process, Ti 3 C 2 -coated MXene was dried overnight in a drying oven and then ultrasonically cleaned.

Characterization of Coating Layer
The surface morphology of the coating layer was assessed by means of field emission scanning electron microscopy (FE-SEM; Sigma300, Zeiss, Oberkochen, Germany) and atomic force microscopy (AFM; Park Systems, Suwon, Korea). The presence of Ti 3 C 2 in the coating layer was confirmed via X-ray diffraction (XRD; Ulima IV, Rigaku, Tokyo, Japan) and Raman spectroscopy (DXR2xi, Thermo, Waltham, MA, USA).

In Vitro Cell Tests
The effect of Ti 3 C 2 coating on the cellular behaviors of the hMSCs (A15652, Thermo Fischer, Waltham, MA, USA) were cultured in Minimum Essential Medium Eagle-alpha modification (alpha-MEM, LM 008-02, Welgene, Gyeongsan, Korea) supplemented with 1% (w/v) penicillin/streptomycin (ThermoFisher, Waltham, MA, USA) and 10% (v/v) fetal bovine serum (Gibco, Erie County, NY, USA) at 37 • C, with 5% CO 2 . The hMSCs were evaluated by considering in vitro cell morphology, proliferation, and osteogenic differentiation. The passage of as-received hMSC was 4. After 2 times of subculturing, we seeded the hMSCs (passage 6) onto the sterilized specimen surfaces with a concentration of 5 × 10 3 cells/mL. The sterilizing of the specimens was carried out by soaking the specimens into 70% EtOH for 30 min and drying it. The sterilized specimens were then rinsed with phosphate buffered saline (21-040-CV, Corning, Corning, NY, USA) solution 3 times. After 1 day of culturing, the hMSC-seeded specimens were observed by FE-SEM. The cell proliferation of hMSCs was examined after 1 and 3 days of culturing using an MTS assay kit (G3580, Promega, Madison, WI, USA), in accordance with the manufacturer's manual. The osteogenic differentiation of hMSCs was verified via an alkaline phosphatase (ALP, Anaspec, Fremont, CA, USA) activity test and Alizarin Red S (ARS, Sigma-Aldrich, St. Louis, MO, USA) staining test after 14 days of culturing. An osteogenic medium containing 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid was used to induce the differentiation.

Statistical Analysis
All in vitro tests were performed 3 times. The data was analyzed by analysis of variance and presented as the mean ± standard deviation (n > 3). Statistical significance was considered when the p value was less than 0.05. Figure 1a shows digital optical images of the bare and Ti 3 C 2 -coated Ti specimens. The coated surface became considerably darker than the bare one, owing mainly to the fact that the color of Ti 3 C 2 is intrinsically black. FE-SEM images showing the microstructures of the bare Ti surface and Ti 3 C 2 -coating layer are shown in Figure 1b,c, respectively. The groove structure created during mechanical grinding of the bare Ti surface (Figure 1b) was covered by a particulate layer after EPD of Ti 3 C 2 (see Figure 1c). AFM topography images revealed similar tendencies, as shown in Figure 1d,e. Mechanical grooves and particulate morphologies were observed on the bare and Ti 3 C 2 -coated Ti surfaces, respectively. A scratch made on the coating layer was used as a marker to investigate the cross-sectional structure of the layer (see Figure 1f). It was also assumed that the thickness of the coated layer was about 1 µm. The results suggested that the layer is composed of a MXene-like substance with a stacking structure of numerous two-dimensional flakes comprising the layer.

Statistical Analysis
All in vitro tests were performed 3 times. The data was analyzed by analysis of variance and presented as the mean ± standard deviation (n > 3). Statistical significance was considered when the p value was less than 0.05. Figure 1a shows digital optical images of the bare and Ti3C2-coated Ti specimens. The coated surface became considerably darker than the bare one, owing mainly to the fact that the color of Ti3C2 is intrinsically black. FE-SEM images showing the microstructures of the bare Ti surface and Ti3C2-coating layer are shown in Figure 1b,c, respectively. The groove structure created during mechanical grinding of the bare Ti surface (Figure 1b) was covered by a particulate layer after EPD of Ti3C2 (see Figure 1c). AFM topography images revealed similar tendencies, as shown in Figure 1d,e. Mechanical grooves and particulate morphologies were observed on the bare and Ti3C2-coated Ti surfaces, respectively. A scratch made on the coating layer was used as a marker to investigate the crosssectional structure of the layer (see Figure 1f). It was also assumed that the thickness of the coated layer was about 1 μm. The results suggested that the layer is composed of a MXene-like substance with a stacking structure of numerous two-dimensional flakes comprising the layer. Further characterization of the coated substance was performed using XRD and Raman spectroscopy (see Figure 2). As shown in the figure, characteristic peaks of the Ti substrate [18] occurred in the XRD patterns of both the bare and the Ti3C2-coated samples. The intensities of the major Ti-phase peaks in the coated sample were, however, lower than those of the bare sample. Furthermore, additional peaks at 2θ = 8°, 18°, 28°, and 60° appeared after the Ti3C2 coating. These peaks correspond to the (002), (006), (008), and (110) planes of Ti3C2, respectively, and are the major peaks of Ti3C2 [19] (Figure 2a). Figure 2b shows the Raman spectra of the bare and Ti3C2-coated Ti. Ti3C2 characteristic peaks at 215, Further characterization of the coated substance was performed using XRD and Raman spectroscopy (see Figure 2). As shown in the figure, characteristic peaks of the Ti substrate [18] occurred in the XRD patterns of both the bare and the Ti 3 C 2 -coated samples. The intensities of the major Ti-phase peaks in the coated sample were, however, lower than those of the bare sample. Furthermore, additional peaks at 2θ = 8 • , 18 • , 28 • , and 60 • appeared after the Ti 3 C 2 coating. These peaks correspond to the (002), (006), (008), and (110) planes of Ti 3 C 2 , respectively, and are the major peaks of Ti 3 C 2 [19] (Figure 2a). Figure 2b shows the Raman spectra of the bare and Ti 3 C 2 -coated Ti. Ti 3 C 2 characteristic peaks at 215, 387, 620, and 705 cm −1 resulted from the vibration of functional groups, and the peak at 120 cm −1 was generated by oxidezed Ti 3 C 2 [20]. These characteristic peaks occurred for the Ti 3 C 2 -coated Ti but were absent for the bare Ti. The surface characterization results confirmed that, by using a simple EPD technique, Ti 3 C 2 can be easily coated onto a metallic surface such as Ti. 387, 620, and 705 cm −1 resulted from the vibration of functional groups, and the peak at 120 cm −1 was generated by oxidezed Ti3C2 [20]. These characteristic peaks occurred for the Ti3C2-coated Ti but were absent for the bare Ti. The surface characterization results confirmed that, by using a simple EPD technique, Ti3C2 can be easily coated onto a metallic surface such as Ti. For use as biomedical implants, materials should exhibit both biocompatibility and biofunctionality. Biocompatibility was investigated using a simple cell attachment, as shown in Figure 3a,b. The hMSCs attached to the bare Ti (one of the most biocompatible metallic implant materials) exhibited a widely stretched morphology. This stretched morphology of cells generally means that the cells are actively attached to the substrate [21]. The number and morphology of hMSCs attached to the Ti3C2-coated Ti surface were similar to those of the hMSCs attached to the bare Ti. The biocompatibility of the Ti3C2-coated layer was also evaluated in terms of cell proliferation. The MTS results, shown in Figure  3c, revealed that the cell viability varied only slightly between the two groups. In other words, the biocompatibility of the Ti3C2 coating layer is equivalent to that of the bare Ti. Although there was no statistical significance, the mean value of cell viability decreased after Ti3C2 coating. In some cases, under bioactive circumstance, stem cells showed more differentiating properties rather than proliferating [22,23].

Results and Discussion
The biofuctionality of Ti was investigated by considering the osteogenic differentiation of hMSCs. The ALP activity of hMSCs after 14 days of culturing the bare and Ti3C2coated Ti substrate is shown in Figure 3d. As shown in the figure, the osteogenic differentiation of the coated Ti group is significantly higher than that of the bare Ti sample group. Moreover, the effect of the Ti3C2 coating on the osteogenic differentiation of hMSCs is also studied using an ARS staining method. The level of hMSC absorbance after staining is shown in Figure 3e. The ARS staining method can be used to quantitatively evaluate the amount of calcium deposited by cells. The optical absorbance of hMSCs gathered from the Ti3C2-coated Ti layer was considerably higher than that of the hMSCs collected from the bare Ti substrate. Cellular behaviors are affected by various surface properties, such as surface chemistry, morphology, and wettability [24]. Several studies have reported that Ti3C2 exhibits good biocompatibility and affects osteogenic differentiation [25,26]. Ke et al., reported that the thin layer of Ti3C2 showed better osteoinductivity than that of graphene oxide layer. Based on these properties, Ke et al. fabricated Ti3C2 and a poly-lactic acid (PLA) composite membrane for guided bone regeneration applications [14]. The osteoinductivity of the PLA/MXene composite membrane were enhanced compared to those For use as biomedical implants, materials should exhibit both biocompatibility and biofunctionality. Biocompatibility was investigated using a simple cell attachment, as shown in Figure 3a,b. The hMSCs attached to the bare Ti (one of the most biocompatible metallic implant materials) exhibited a widely stretched morphology. This stretched morphology of cells generally means that the cells are actively attached to the substrate [21]. The number and morphology of hMSCs attached to the Ti 3 C 2 -coated Ti surface were similar to those of the hMSCs attached to the bare Ti. The biocompatibility of the Ti 3 C 2 -coated layer was also evaluated in terms of cell proliferation. The MTS results, shown in Figure 3c, revealed that the cell viability varied only slightly between the two groups. In other words, the biocompatibility of the Ti 3 C 2 coating layer is equivalent to that of the bare Ti. Although there was no statistical significance, the mean value of cell viability decreased after Ti 3 C 2 coating. In some cases, under bioactive circumstance, stem cells showed more differentiating properties rather than proliferating [22,23].  [27]. This is very notable because carboxides are also observed on Ti3C2 MXene [28]. The behaviors of hMSCs would also be affected by nanostructures of the Ti3C2-coated Ti surface. Nanostructured surfaces do not always demonstrate an increased cell proliferation level. Sometimes, it even suppressed cell proliferation [29,30]. Many studies, however, have exhibited that osteogenic differentiation of stem cells was promoted on a nanostructured surface [31,32]. The resulting hydrophilicity of Ti3C2-coated Ti ( Figure S2) would be another reason of increased cellular activity [33]. One of the limits of this study was that effects of the MXene on the hMSCs were not clear because both surface chemistry and morphologies were changed simultaneously by the EPD process. A comparative study using either a single layer or a few layers of MXeneshould be performed to minimize the morphology change to confirm the effect of the Ti3C2 layer itself. In summary, the EPD technique was quite useful for creating a Ti3C2 coating layer, which was effective in promoting the osteogenic differentiation of hMSCs. This EPD coating method can yield coatings of Ti3C2 as well as coatings with a mixture of Ti3C2 and the relevant drug, which can be released from the biomedical implant surface after implantation.

Conclusions
This study proposed a simple strategy for improving the osteoinductivity of Ti by depositing a Ti3C2 coating on the material surface. A uniform Ti3C2 layer was successfully created on the surface using a simple EPD coating technique. This layer was characterized by particulate morphology and a higher roughness than that of the bare Ti surface, as  The biofuctionality of Ti was investigated by considering the osteogenic differentiation of hMSCs. The ALP activity of hMSCs after 14 days of culturing the bare and Ti 3 C 2 -coated Ti substrate is shown in Figure 3d. As shown in the figure, the osteogenic differentiation of the coated Ti group is significantly higher than that of the bare Ti sample group. Moreover, the effect of the Ti 3 C 2 coating on the osteogenic differentiation of hMSCs is also studied using an ARS staining method. The level of hMSC absorbance after staining is shown in Figure 3e. The ARS staining method can be used to quantitatively evaluate the amount of calcium deposited by cells. The optical absorbance of hMSCs gathered from the Ti 3 C 2coated Ti layer was considerably higher than that of the hMSCs collected from the bare Ti substrate. Cellular behaviors are affected by various surface properties, such as surface chemistry, morphology, and wettability [24]. Several studies have reported that Ti 3 C 2 exhibits good biocompatibility and affects osteogenic differentiation [25,26]. Ke et al., reported that the thin layer of Ti 3 C 2 showed better osteoinductivity than that of graphene oxide layer. Based on these properties, Ke et al. fabricated Ti 3 C 2 and a poly-lactic acid (PLA) composite membrane for guided bone regeneration applications [14]. The osteoinductivity of the PLA/MXene composite membrane were enhanced compared to those of the pure PLA membrane. Ke et al. also demonstrated that the TiC x O y layer, a partially oxidized layer of MAX phases (Ti 3 AlC 2 and Ti 3 SiC 2 ), had high affinity to Ca 2+ ions [27]. This is very notable because carboxides are also observed on Ti 3 C 2 MXene [28]. The behaviors of hMSCs would also be affected by nanostructures of the Ti 3 C 2 -coated Ti surface. Nanostructured surfaces do not always demonstrate an increased cell proliferation level. Sometimes, it even suppressed cell proliferation [29,30]. Many studies, however, have exhibited that osteogenic differentiation of stem cells was promoted on a nanostructured surface [31,32]. The resulting hydrophilicity of Ti 3 C 2 -coated Ti ( Figure S2) would be another reason of increased cellular activity [33]. One of the limits of this study was that effects of the MXene on the hMSCs were not clear because both surface chemistry and morphologies were changed simultaneously by the EPD process. A comparative study using either a single layer or a few layers of MXeneshould be performed to minimize the morphology change to confirm the effect of the Ti 3 C 2 layer itself. In summary, the EPD technique was quite useful for creating a Ti 3 C 2 coating layer, which was effective in promoting the osteogenic differentiation of hMSCs. This EPD coating method can yield coatings of Ti 3 C 2 as well as coatings with a mixture of Ti 3 C 2 and the relevant drug, which can be released from the biomedical implant surface after implantation.

Conclusions
This study proposed a simple strategy for improving the osteoinductivity of Ti by depositing a Ti 3 C 2 coating on the material surface. A uniform Ti 3 C 2 layer was successfully created on the surface using a simple EPD coating technique. This layer was characterized by particulate morphology and a higher roughness than that of the bare Ti surface, as revealed by SEM and AFM analyses. Furthermore, XRD and Raman spectroscopy confirmed that the coated layer was composed of a Ti 3 C 2 phase. The b cellular tests using hMSCs demonstrated that the biocompatibility of the Ti 3 C 2 coating layer was comparable to that of the bare Ti. Moreover, ALP activity and ARS staining tests demonstrated that the level of hMSC osteogenic differentiation was enhanced due to the effect of the coating layer. The results suggest that EPD coating of Ti 3 C 2 onto the Ti surface can improve the potential of the surface for use in biomedical implants.