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Selectable surface nitridation of titanium using focused pulsed Nd:YAG laser irradiation with nitrogen gas blow
Surface & Coatings Technology 246 (2014) 52–56
Contents lists available at ScienceDirect
Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
Selectable surface nitridation of titanium using focused pulsed Nd:YAG laser irradiation with nitrogen gas blow Naofumi Ohtsu ⁎, Wataru Saito, Misao Yamane Instrumental Analysis Center, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan
a r t i c l e
i n f o
Article history: Received 27 December 2013 Accepted in revised form 28 February 2014 Available online 12 March 2014 Keywords: Selectable nitridation Titanium Focused laser irradiation Surface hardness Cell attachment
a b s t r a c t In the present study, we demonstrated that a surface nitride layer with high surface hardness and good cell attachment performance could be produced on an arbitrary area of a titanium (Ti) substrate by using focused pulsed Nd:YAG laser irradiation accompanied by nitrogen gas blow. A focused laser beam induced nitrogen plasma in the vicinity of the Ti substrate surface. This plasma made it possible to form a nitride layer with a sufficient thickness, even when the laser power was low. The produced surface layer was approximately 1100 nm in thickness and was composed of TiN and Ti; the concentration of TiN eventually decreased with increasing depth. The hardness of the laser-treated surface was approximately 7.5 GPa, which was approximately five times that of the untreated Ti surface. Furthermore, the surface wettability of the Ti substrate was drastically improved by treating it with focused laser, which enhanced the cell attachment performance of the substrate. We concluded that the surface nitriding process using focused pulsed Nd:YAG laser is an excellent surface modification technique for Ti materials that can simultaneously improve their wear resistance and cell attachment performance. © 2014 Published by Elsevier B.V.
1. Introduction Titanium (Ti) implants have become essential medical products in the fields of surgery and dentistry as substitutes for hard tissues. Their excellent biocompatibility, high mechanical strength, and resistance to corrosion make them suitable for use as medical implants [1–6]. However, low wear resistance is a considerable disadvantage of Ti materials, which often restricts their application [7,8]. For instance, Ti materials are considered to be inappropriate for use as an artificial femoral head material because powders are generated owing to friction and cause an inflammation reaction in the surrounding tissues [9]. Ti nitride is known to have excellent hardness. Hence, coating Ti with a nitride layer is a significant possibility for overcoming the low wear resistance of Ti materials. In industries, nitride layers are synthesized directly on substrates by using surface nitridation, a process that involves the diffusion of nitrogen. Heretofore, nitrogen diffusion has been achieved by heating substrates in an ammonia atmosphere [10–12] and exposing them to nitrogen plasmas [13–15]. These processes can lead to the formation of homogeneous nitride layers over an entire surface but cannot be applied to a restricted area. Biomedical materials are small pieces with complex shapes in general; therefore, surface treatments that form nitride layers only on selected small areas are considered innovative techniques. Laser irradiated on a metallic surface induces several different phenomena such as the ablation of the naturally formed oxide, melting of ⁎ Corresponding author. Tel./fax: +81 157 26 9563. E-mail address: [email protected] (N. Ohtsu).
http://dx.doi.org/10.1016/j.surfcoat.2014.02.068 0257-8972/© 2014 Published by Elsevier B.V.
the metal, and reaction of the metal with atmospheric gases. These phenomena are likely to form a new surface layer, and laser processing is thus expected to accomplish the desired selectable surface treatment. Ettaqui et al. demonstrated the formation of a surface layer consisting of cubic Ti nitride (TiN) and hexagonal alpha-Ti nitride (TN0.3) by radiating a pulsed Nd:YAG laser having a power of 35 J pulse−1 and accompanied by nitrogen gas flow [16]. Abboud et al. also demonstrated the formation of a TiN layer on Ti–6Al–4V alloy by using a high-power CO2 laser and that the resulting surface had high surface hardness, exceeding 1000 HV [17]. Several researchers have heretofore reported the formation of a nitride layer on Ti materials through laser irradiation [18–20]. However, extensive energy applied to a substrate in a short period of time can change its microstructure, thereby deteriorating its mechanical properties. Furthermore, high-power lasers are expensive and require large instruments, in general. It is well known that plasma consisting of atmospheric gases and solid constituents is induced on a solid surface if a pulsed laser beam is focused on its surface, owing to a high laser energy density. This phenomenon has been recently utilized for developing a rapid analysis technique called laser-induced breakdown spectroscopy [21]. We considered that radical nitrogen species within plasma facilitate the diffusion of nitrogen into the surface of a substrate [22], and hence, a nitride surface may be synthesized by using a compact pulsed laser if a laser beam is focused on the surface. Motivated by this consideration, we have made an effort to develop a laser nitridation process utilizing a focused beam [23–26]. To date, we have attempted to determine the optimal settings for fabricating a nitride layer on a Ti substrate. For this purpose, laser irradiation was performed on a Ti substrate placed in an atmosphere-controlled chamber
N. Ohtsu et al. / Surface & Coatings Technology 246 (2014) 52–56
to strictly control the pressure and composition of a reaction gas. Further, the effects of irradiation settings such as laser power and nitrogen gas pressure on the characteristics of the surface layer have been investigated [23–26]. The obtained results revealed that the nitrogen gas pressure is the most important factor for controlling the surface layer characteristics and that a nitrogen gas pressure beyond the atmospheric pressure is required to form a nitride layer with a sufficient thickness for improving the wear resistance of Ti materials [26]. Similar results were reported by other research group. For instance, Höche et al. demonstrated that a TiN layer was formed by radiating a pulsed Nd:YAG laser onto Ti substrate placed in a chamber filled with pure nitrogen at a pressure of 3 × 105 Pa, notwithstanding a comparatively low laser power of about 40 mJ [27,28]. The most important conclusion of the previous studies implied that an atmosphere-controlled chamber is no longer required for the nitridation of Ti materials [26]. This is because a high-pressure nitride gas atmosphere on a restricted surface area can also be realized by blowing nitrogen gas. This was a welcome finding because a laser nitridation instrument becomes very simple if it is not required to use an atmosphere-controlled chamber. In the present study, therefore, a Ti substrate was treated using focused Nd:YAG laser irradiation accompanied with the blowing of nitrogen gas. The surface structure and hardness of the laser-treated surface were then evaluated. Furthermore, the biocompatibility of the laser-irradiated surface was evaluated by determining the cell attachment behavior of MC3T3-E1 cells on the surface. 2. Experimental procedure 2.1. Surface treatment using focused pulsed Nd:YAG laser A Ti plate (10 mm × 10 mm × 1 mm) with a purity of 99.5% (Furuuchi Chem. Co., Japan) was used as a substrate. The substrate was chemically polished using a colloidal silica suspension with an average particle size of 40 nm and was then ultrasonically washed in ethanol. The substrate was placed on a stepping-motor-driven XY-axis stage (ALD-4011-G1M-R, Chuo Precision Industrial, Japan), and the stage was scanned at a speed of 1 mm s− 1. A Nd:YAG Q-switched laser (Minilite II, Continuum Inc, USA) emitting at 532 nm (SHG mode) with a 4 ± 1 ns pulse width was used for nitridation. The laser energy per pulse, determined using a laser power meter, was 25 mJ. The laser was repeatedly and automatically triggered at a rate of 8 Hz. The laser beam was focused with a convex lens and was then radiated on the Ti substrate, and this irradiation was accompanied by the blowing of nitrogen gas. The theoretical spot size of focused laser was about 30 μm in diameter; accordingly, the calculated energy density was about 30 MJ m−2. A gas injection port was placed at a tilt angle of 60° against the substrate surface. The gas injection port was 12.7 mm in diameter, and its distance from the substrate surface was approximately 3 cm. The speed of nitrogen gas blow, monitored using a mass flow meter, was set to 7 × 104 cm3 min−1. Laser treatment of the substrate was finished within about 300 s. 2.2. Characterization of laser-treated Ti surfaces Image of the laser-treated Ti surfaces were obtained using a digital microscope (KH-7700, Hirox, Japan). Changes in the chemical states and compositions of the surfaces in the depth direction were analyzed by X-ray photoelectron spectroscopy (XPS; PHI 5000 VersaProbe, UlvacPhi, Japan), using monochromatic Al Kα radiation (hν = 1486.6 eV). The photoelectron take-off angle was set to 45° and the pass energy was set to 58.7 eV. An Ar ion gun with an acceleration voltage of 4 kV was used to obtain elemental depth profiles. The etching rate, estimated from a SiO2 layer, was approximately 12.5 nm min−1. The structure of the laser-treated surfaces was observed using conventional transmission electron microscopy (TEM; H9000NAR, Hitachi, Japan). TEM specimens
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were prepared using an ion slicer (EM-09100IS, JEOL, Japan). X-ray diffraction (XRD; New D8 ADVANCE, Bruker AXS, Germany) patterns of the specimens were obtained by utilizing grazing-incident-angle geometry using Cu Kα radiation. The incident angle of X-ray was set to 2°, and the effective measurement depth of this setting was a few tens of micrometers. Nanoindentation tests were performed using a nanomechanical test instrument (TS-75 TriboScope, Hysitron, USA) mounted on a scanning probe microscope (SPM; SPM-9700, Shimadz, Japan). Load– displacement curves of the treated surfaces were measured using a Berkovich diamond indenter tip. The indentation load was varied from 0 to 1000 μN to achieve different penetration depths. The hardness of the surfaces was calculated from load–displacement curves by using the Oliver and Pharr method [29]. The contact angle of distilled water was measured using a contact angle meter (DM-CE1, Kyowa Interface Science, Japan). 2.3. Cell attachment behavior of MC3T3-E1 cells on treated Ti surfaces MC3T3-E1 cells (RIKEN BioResource Center, Japan), an osteoblastlike cell line, were cultured in an α-modified minimum essential medium (α-MEM; GIBCO BRL, USA) containing 10% fetal bovine serum (FBS; JR Scientific, USA) and 1% antibiotic-antimycotic (100 U mL− 1 penicillin, 100 μg mL−1 streptomycin, and 0.25 μg mL−1 amphotericin B; GIBCO BRL, USA) at 37 °C under 5% CO2 in a humidified atmosphere. Laser-treated and untreated Ti disks (ϕ 15 mm × 1 mm) were sterilized by autoclaving for 30 min at 121 °C, and were then placed in an individual well of a 24-well cell culture polystyrene plate. MC3T3-E1 cells were seeded at the rate of 1 × 104 cells mL−1 on intact polystyrene, untreated Ti, or treated Ti disks in the well. The plates were incubated for 24 h, after which the attached cells were measured using a WST-8 assay (Cell Counting Kit-8, Dogin, Japan). Experiments were conducted on three disks (n = 3) for each specimen. The number of cells was represented as mean ± S.D., and statistical analyses were performed using Student's t-test to identify the levels of significance (p b 0.05) between two groups. 3. Results and discussion 3.1. Microstructure, crystallinity, and chemical composition of treated Ti surfaces Fig. 1 shows the surface of a laser-treated Ti substrate. The surface color was yellow. A regular surface including grooves, generated by melting and solidification caused by laser irradiation, was observed. The width and depth of the grooves were approximately 500 μm and 35 μm, respectively. Fig. 2 shows a cross-sectional surface image of a treated Ti substrate at the bottom of a groove. This image shows that the treated Ti substrate is covered with a 1100-nm-thick layer (Fig. 2(a)). Furthermore, another thin layer of about 30 nm in thickness is also observed (Arrowed part in Fig. 2(b)) at the topmost surface. In the selected area diffraction (SAD) pattern corresponding to the main part of the surface layer (Fig. 2(c)), diffraction spots assigned to Ti with a hcp structure were detected, but the presence of other phases could not be ascertained. Thus, detailed analyses of the constituent phases by XRD and XPS were conducted and are described below. The XRD pattern of a treated Ti surface is shown in Fig. 3. The peaks in the pattern were assigned to δ-TiN with a cubic structure and α-Ti with an hcp structure. It is plausible that the peaks for TiN originated from the surface layer. Meanwhile, in the corresponding SAD pattern (Fig. 2(c)), no spot due to TiN is observed, but only those due to Ti can be seen. We conjectured that the concentration and/or crystallinity of TiN were relatively low, and thus, no spot corresponding to TiN appeared in the SAD pattern. XPS survey spectra obtained from the topmost surface indicated the presence of Ti, N, and O. The in-depth elemental profiles of Ti, N, and O for a laser-treated Ti surface, as measured using XPS, are shown in Fig. 4.
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N. Ohtsu et al. / Surface & Coatings Technology 246 (2014) 52–56
Fig. 1. Surface of a laser-treated Ti substrate: (a) digital camera image and (b) digital microscope image.
Fig. 2. (a) Cross-sectional bright field (BF) TEM image of a laser-treated Ti surface. (b) Higher-magnification image of the topmost surface area. (c) Selected area diffraction (SAD) pattern taken from the surface layer.
Atomic concentrations were calculated from the spectral intensities of Ti 2p, N 1s, and O 1s, and the background of spectra was subtracted by the Shirley method. The obtained in-depth profiles can be roughly divided into two regions. A thin layer, at the topmost surface, of about 30 nm in thickness contained Ti and O. This layer is likely to be an oxide layer like TiO2, and is also observed in the TEM image (Fig. 2(b)).
The main part of the surface layer consisted of Ti and N, and the concentration of N gradually decreased as the depth increased. Changes in the N 1s spectra as a function of the etching time, corresponding to the depth, are shown in Fig. 5. The N 1s spectral shapes were similar regardless of the etching time, and the binding energy of N 1s was 397.3 eV, corresponding to TiN [30]. These results indicate that the
δ-TiN α-Ti
θ Fig. 3. XRD pattern of a laser-treated Ti surface measured using grazing incident-angle geometry with an incident angle of 2°.
Fig. 4. In-depth elemental profiles of the surface layer obtained from XPS equipped with an Ar etching system.
N. Ohtsu et al. / Surface & Coatings Technology 246 (2014) 52–56
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μ
N 1s
Fig. 6. Load–displacement curves for untreated and laser-treated Ti substrates.
Etching Time
decreased with increasing the scanning velocity, due to the decrease of the layer thickness [28]. Accordingly, we assumed that the scanning speed is the key factors for forming a hard surface. 3.3. Cellular attachment of laser-treated Ti surfaces
Fig. 5. N 1s XPS spectral changes in the surface layer with increasing etching time.
main part of the surface layer comprised TiN and Ti, and that the concentration of TiN eventually decreased with increasing depth. At the topmost surface, a thin oxide layer was formed after the laser treatment. In our process, the reaction gas must include a small amount of oxygen because the nitrogen blow included oxygen gas present in the air. We thus considered that the oxide layer was probably formed via a reaction with oxygen during the lowering of the surface temperature after irradiation. On the other hand, in our previous study [26], a reaction gas was prepared in an atmosphere-controlled chamber, and thus, oxygen gas was hardly included. The nitride layer formed in the chamber did not show the presence of a thin oxide layer, and this result supports our consideration. 3.2. Hardness of laser-irradiated Ti surface The hardness of a laser-irradiated surface was measured using a nanoindentation test. The load–displacement curves obtained for untreated and laser-treated Ti substrates are shown in Fig. 6. It is clear that tip penetration depth for the laser-irradiated Ti surfaces is shallower than that of the untreated Ti surfaces. The hardness values of the surfaces, calculated from their load–displacement curves, are also shown in the figure. The hardness of a treated Ti surface was 7.5 GPa, approximately five times that of an untreated Ti surface, which almost agreed with the value reported by Höche et al. [28]. This result indicates that a Ti surface can be hardened by focused pulsed laser irradiation accompanied by a nitrogen gas blow. However, the hardness obtained after this treatment was much lower than that obtained in our previous study [26]. In this study, the substrate was placed on a stepping-motor-driven XY-axis stage and the stage was scanned at a speed of 1 mm s− 1. On the other hand, the laser shots were repeated 50 times on one spot without moving the substrate in our previous study. As a result, the number of laser shots radiated on a specific area was reduced as compared with the previous case. Höche et al. demonstrated that the hardness of a treated surface was
The quantitative results for cellular number, normalized to the initial number of seeded cells, on laser-treated and untreated Ti surfaces after cultivation for 24 h are shown in Fig. 7. The result for an intact polystyrene (PS) surface of the culture plate is also shown in the figure as a control result. A significant statistical difference was found between the cellular number for the treated and untreated surfaces. The cellular number on a laser-treated Ti surface was approximately three times that on an untreated Ti surface. This result indicates that the surface treatment using focused laser irradiation can significantly enhance the cell attachment performance of Ti surfaces. To explore the reasons for this enhancement, we measured the contact angle of distilled water on the surfaces after sterilization. The photograph of distilled water dropped on the surfaces is shown in Fig. 8. No water drop was formed on the treated surface, while an apparent water drop was observed on the untreated Ti surface. This result demonstrates that the Ti surface was converted to a hydrophilic surface by the laser nitridation treatment. Hao et al. reported that the wettability of Ti–6Al–4V alloy was improved by treating its surface with a high-power diode laser with oxygen gas assist, and that osteoblast cells grew better on a treated surface [31]. They concluded that the wettability characteristic dominates the osteoblast response rather than the surface roughness. In our treatment, laser irradiation was assisted by nitrogen gas blow. However, the surface was covered with a thin oxide layer, as shown in Fig. 4, which implies that the surface chemical state was similar to that reported by
**
Fig. 7. Cell numbers on laser-treated and untreated Ti surfaces after cultivation for 24 h, normalized to the initial number of seeded cells. Cell numbers on the intact polystyrene (PS) surface of the culture plate are shown as a control result. All error bars are shown as mean ± S.D. (n = 3). Data were statistically analyzed using Student's t-test (* p b 0.05).
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Fig. 8. Photograph of distilled water dropped on: (a) a laser-treated Ti surface and (b) untreated Ti surface.
Hao et al. Upon considering these observations, it was concluded that the enhancement in the cell attachment performance was due to the improvement in surface wettability by the laser nitridation treatment.
by a Grant-in-aid for Scientific Research (C) (No. 24560841) from the Ministry of Education, Science, Sports, and Culture (MEXT) of Japan. References
4. Conclusions In the present study, we prepared a nitride layer on a titanium (Ti) substrate by using focused pulsed Nd:YAG laser irradiation accompanied by nitrogen gas blow. The main part of the produced surface layer was composed of TiN and Ti, and the concentration of TiN decreased with increasing depth. The thickness of the surface layer was approximately 1100 nm. Furthermore, at the topmost surface, a thin oxide layer 30 nm in thickness was also formed. The hardness of the laser-treated Ti surface was 7.5 GPa, and this value was approximately five times that for an untreated Ti surface. Furthermore, the surface wettability of the Ti substrate was drastically improved by treating it with focused laser irradiation, which enhanced the cell attachment performance of the substrate. In conclusion, surface treatment using focused pulsed Nd:YAG laser irradiation accompanied by nitrogen gas blow is an excellent surface modification technique that can simultaneously improve the wear resistance and cell attachment performance of Ti materials. This new process is thus considered to be an innovative technique for use on biomedical materials. Conflict of interest None. Acknowledgment
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28]
The authors gratefully acknowledge Mr. Kozuka, an undergraduate student in our laboratory for his support in the cell attachment test. The authors also acknowledge Mr. Tokuda from the Kitami Institute of Technology for his help in TEM analysis. Part of this work was supported
[29] [30] [31]
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Contents lists available at ScienceDirect
Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
Selectable surface nitridation of titanium using focused pulsed Nd:YAG laser irradiation with nitrogen gas blow Naofumi Ohtsu ⁎, Wataru Saito, Misao Yamane Instrumental Analysis Center, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan
a r t i c l e
i n f o
Article history: Received 27 December 2013 Accepted in revised form 28 February 2014 Available online 12 March 2014 Keywords: Selectable nitridation Titanium Focused laser irradiation Surface hardness Cell attachment
a b s t r a c t In the present study, we demonstrated that a surface nitride layer with high surface hardness and good cell attachment performance could be produced on an arbitrary area of a titanium (Ti) substrate by using focused pulsed Nd:YAG laser irradiation accompanied by nitrogen gas blow. A focused laser beam induced nitrogen plasma in the vicinity of the Ti substrate surface. This plasma made it possible to form a nitride layer with a sufficient thickness, even when the laser power was low. The produced surface layer was approximately 1100 nm in thickness and was composed of TiN and Ti; the concentration of TiN eventually decreased with increasing depth. The hardness of the laser-treated surface was approximately 7.5 GPa, which was approximately five times that of the untreated Ti surface. Furthermore, the surface wettability of the Ti substrate was drastically improved by treating it with focused laser, which enhanced the cell attachment performance of the substrate. We concluded that the surface nitriding process using focused pulsed Nd:YAG laser is an excellent surface modification technique for Ti materials that can simultaneously improve their wear resistance and cell attachment performance. © 2014 Published by Elsevier B.V.
1. Introduction Titanium (Ti) implants have become essential medical products in the fields of surgery and dentistry as substitutes for hard tissues. Their excellent biocompatibility, high mechanical strength, and resistance to corrosion make them suitable for use as medical implants [1–6]. However, low wear resistance is a considerable disadvantage of Ti materials, which often restricts their application [7,8]. For instance, Ti materials are considered to be inappropriate for use as an artificial femoral head material because powders are generated owing to friction and cause an inflammation reaction in the surrounding tissues [9]. Ti nitride is known to have excellent hardness. Hence, coating Ti with a nitride layer is a significant possibility for overcoming the low wear resistance of Ti materials. In industries, nitride layers are synthesized directly on substrates by using surface nitridation, a process that involves the diffusion of nitrogen. Heretofore, nitrogen diffusion has been achieved by heating substrates in an ammonia atmosphere [10–12] and exposing them to nitrogen plasmas [13–15]. These processes can lead to the formation of homogeneous nitride layers over an entire surface but cannot be applied to a restricted area. Biomedical materials are small pieces with complex shapes in general; therefore, surface treatments that form nitride layers only on selected small areas are considered innovative techniques. Laser irradiated on a metallic surface induces several different phenomena such as the ablation of the naturally formed oxide, melting of ⁎ Corresponding author. Tel./fax: +81 157 26 9563. E-mail address: [email protected] (N. Ohtsu).
http://dx.doi.org/10.1016/j.surfcoat.2014.02.068 0257-8972/© 2014 Published by Elsevier B.V.
the metal, and reaction of the metal with atmospheric gases. These phenomena are likely to form a new surface layer, and laser processing is thus expected to accomplish the desired selectable surface treatment. Ettaqui et al. demonstrated the formation of a surface layer consisting of cubic Ti nitride (TiN) and hexagonal alpha-Ti nitride (TN0.3) by radiating a pulsed Nd:YAG laser having a power of 35 J pulse−1 and accompanied by nitrogen gas flow [16]. Abboud et al. also demonstrated the formation of a TiN layer on Ti–6Al–4V alloy by using a high-power CO2 laser and that the resulting surface had high surface hardness, exceeding 1000 HV [17]. Several researchers have heretofore reported the formation of a nitride layer on Ti materials through laser irradiation [18–20]. However, extensive energy applied to a substrate in a short period of time can change its microstructure, thereby deteriorating its mechanical properties. Furthermore, high-power lasers are expensive and require large instruments, in general. It is well known that plasma consisting of atmospheric gases and solid constituents is induced on a solid surface if a pulsed laser beam is focused on its surface, owing to a high laser energy density. This phenomenon has been recently utilized for developing a rapid analysis technique called laser-induced breakdown spectroscopy [21]. We considered that radical nitrogen species within plasma facilitate the diffusion of nitrogen into the surface of a substrate [22], and hence, a nitride surface may be synthesized by using a compact pulsed laser if a laser beam is focused on the surface. Motivated by this consideration, we have made an effort to develop a laser nitridation process utilizing a focused beam [23–26]. To date, we have attempted to determine the optimal settings for fabricating a nitride layer on a Ti substrate. For this purpose, laser irradiation was performed on a Ti substrate placed in an atmosphere-controlled chamber
N. Ohtsu et al. / Surface & Coatings Technology 246 (2014) 52–56
to strictly control the pressure and composition of a reaction gas. Further, the effects of irradiation settings such as laser power and nitrogen gas pressure on the characteristics of the surface layer have been investigated [23–26]. The obtained results revealed that the nitrogen gas pressure is the most important factor for controlling the surface layer characteristics and that a nitrogen gas pressure beyond the atmospheric pressure is required to form a nitride layer with a sufficient thickness for improving the wear resistance of Ti materials [26]. Similar results were reported by other research group. For instance, Höche et al. demonstrated that a TiN layer was formed by radiating a pulsed Nd:YAG laser onto Ti substrate placed in a chamber filled with pure nitrogen at a pressure of 3 × 105 Pa, notwithstanding a comparatively low laser power of about 40 mJ [27,28]. The most important conclusion of the previous studies implied that an atmosphere-controlled chamber is no longer required for the nitridation of Ti materials [26]. This is because a high-pressure nitride gas atmosphere on a restricted surface area can also be realized by blowing nitrogen gas. This was a welcome finding because a laser nitridation instrument becomes very simple if it is not required to use an atmosphere-controlled chamber. In the present study, therefore, a Ti substrate was treated using focused Nd:YAG laser irradiation accompanied with the blowing of nitrogen gas. The surface structure and hardness of the laser-treated surface were then evaluated. Furthermore, the biocompatibility of the laser-irradiated surface was evaluated by determining the cell attachment behavior of MC3T3-E1 cells on the surface. 2. Experimental procedure 2.1. Surface treatment using focused pulsed Nd:YAG laser A Ti plate (10 mm × 10 mm × 1 mm) with a purity of 99.5% (Furuuchi Chem. Co., Japan) was used as a substrate. The substrate was chemically polished using a colloidal silica suspension with an average particle size of 40 nm and was then ultrasonically washed in ethanol. The substrate was placed on a stepping-motor-driven XY-axis stage (ALD-4011-G1M-R, Chuo Precision Industrial, Japan), and the stage was scanned at a speed of 1 mm s− 1. A Nd:YAG Q-switched laser (Minilite II, Continuum Inc, USA) emitting at 532 nm (SHG mode) with a 4 ± 1 ns pulse width was used for nitridation. The laser energy per pulse, determined using a laser power meter, was 25 mJ. The laser was repeatedly and automatically triggered at a rate of 8 Hz. The laser beam was focused with a convex lens and was then radiated on the Ti substrate, and this irradiation was accompanied by the blowing of nitrogen gas. The theoretical spot size of focused laser was about 30 μm in diameter; accordingly, the calculated energy density was about 30 MJ m−2. A gas injection port was placed at a tilt angle of 60° against the substrate surface. The gas injection port was 12.7 mm in diameter, and its distance from the substrate surface was approximately 3 cm. The speed of nitrogen gas blow, monitored using a mass flow meter, was set to 7 × 104 cm3 min−1. Laser treatment of the substrate was finished within about 300 s. 2.2. Characterization of laser-treated Ti surfaces Image of the laser-treated Ti surfaces were obtained using a digital microscope (KH-7700, Hirox, Japan). Changes in the chemical states and compositions of the surfaces in the depth direction were analyzed by X-ray photoelectron spectroscopy (XPS; PHI 5000 VersaProbe, UlvacPhi, Japan), using monochromatic Al Kα radiation (hν = 1486.6 eV). The photoelectron take-off angle was set to 45° and the pass energy was set to 58.7 eV. An Ar ion gun with an acceleration voltage of 4 kV was used to obtain elemental depth profiles. The etching rate, estimated from a SiO2 layer, was approximately 12.5 nm min−1. The structure of the laser-treated surfaces was observed using conventional transmission electron microscopy (TEM; H9000NAR, Hitachi, Japan). TEM specimens
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were prepared using an ion slicer (EM-09100IS, JEOL, Japan). X-ray diffraction (XRD; New D8 ADVANCE, Bruker AXS, Germany) patterns of the specimens were obtained by utilizing grazing-incident-angle geometry using Cu Kα radiation. The incident angle of X-ray was set to 2°, and the effective measurement depth of this setting was a few tens of micrometers. Nanoindentation tests were performed using a nanomechanical test instrument (TS-75 TriboScope, Hysitron, USA) mounted on a scanning probe microscope (SPM; SPM-9700, Shimadz, Japan). Load– displacement curves of the treated surfaces were measured using a Berkovich diamond indenter tip. The indentation load was varied from 0 to 1000 μN to achieve different penetration depths. The hardness of the surfaces was calculated from load–displacement curves by using the Oliver and Pharr method [29]. The contact angle of distilled water was measured using a contact angle meter (DM-CE1, Kyowa Interface Science, Japan). 2.3. Cell attachment behavior of MC3T3-E1 cells on treated Ti surfaces MC3T3-E1 cells (RIKEN BioResource Center, Japan), an osteoblastlike cell line, were cultured in an α-modified minimum essential medium (α-MEM; GIBCO BRL, USA) containing 10% fetal bovine serum (FBS; JR Scientific, USA) and 1% antibiotic-antimycotic (100 U mL− 1 penicillin, 100 μg mL−1 streptomycin, and 0.25 μg mL−1 amphotericin B; GIBCO BRL, USA) at 37 °C under 5% CO2 in a humidified atmosphere. Laser-treated and untreated Ti disks (ϕ 15 mm × 1 mm) were sterilized by autoclaving for 30 min at 121 °C, and were then placed in an individual well of a 24-well cell culture polystyrene plate. MC3T3-E1 cells were seeded at the rate of 1 × 104 cells mL−1 on intact polystyrene, untreated Ti, or treated Ti disks in the well. The plates were incubated for 24 h, after which the attached cells were measured using a WST-8 assay (Cell Counting Kit-8, Dogin, Japan). Experiments were conducted on three disks (n = 3) for each specimen. The number of cells was represented as mean ± S.D., and statistical analyses were performed using Student's t-test to identify the levels of significance (p b 0.05) between two groups. 3. Results and discussion 3.1. Microstructure, crystallinity, and chemical composition of treated Ti surfaces Fig. 1 shows the surface of a laser-treated Ti substrate. The surface color was yellow. A regular surface including grooves, generated by melting and solidification caused by laser irradiation, was observed. The width and depth of the grooves were approximately 500 μm and 35 μm, respectively. Fig. 2 shows a cross-sectional surface image of a treated Ti substrate at the bottom of a groove. This image shows that the treated Ti substrate is covered with a 1100-nm-thick layer (Fig. 2(a)). Furthermore, another thin layer of about 30 nm in thickness is also observed (Arrowed part in Fig. 2(b)) at the topmost surface. In the selected area diffraction (SAD) pattern corresponding to the main part of the surface layer (Fig. 2(c)), diffraction spots assigned to Ti with a hcp structure were detected, but the presence of other phases could not be ascertained. Thus, detailed analyses of the constituent phases by XRD and XPS were conducted and are described below. The XRD pattern of a treated Ti surface is shown in Fig. 3. The peaks in the pattern were assigned to δ-TiN with a cubic structure and α-Ti with an hcp structure. It is plausible that the peaks for TiN originated from the surface layer. Meanwhile, in the corresponding SAD pattern (Fig. 2(c)), no spot due to TiN is observed, but only those due to Ti can be seen. We conjectured that the concentration and/or crystallinity of TiN were relatively low, and thus, no spot corresponding to TiN appeared in the SAD pattern. XPS survey spectra obtained from the topmost surface indicated the presence of Ti, N, and O. The in-depth elemental profiles of Ti, N, and O for a laser-treated Ti surface, as measured using XPS, are shown in Fig. 4.
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Fig. 1. Surface of a laser-treated Ti substrate: (a) digital camera image and (b) digital microscope image.
Fig. 2. (a) Cross-sectional bright field (BF) TEM image of a laser-treated Ti surface. (b) Higher-magnification image of the topmost surface area. (c) Selected area diffraction (SAD) pattern taken from the surface layer.
Atomic concentrations were calculated from the spectral intensities of Ti 2p, N 1s, and O 1s, and the background of spectra was subtracted by the Shirley method. The obtained in-depth profiles can be roughly divided into two regions. A thin layer, at the topmost surface, of about 30 nm in thickness contained Ti and O. This layer is likely to be an oxide layer like TiO2, and is also observed in the TEM image (Fig. 2(b)).
The main part of the surface layer consisted of Ti and N, and the concentration of N gradually decreased as the depth increased. Changes in the N 1s spectra as a function of the etching time, corresponding to the depth, are shown in Fig. 5. The N 1s spectral shapes were similar regardless of the etching time, and the binding energy of N 1s was 397.3 eV, corresponding to TiN [30]. These results indicate that the
δ-TiN α-Ti
θ Fig. 3. XRD pattern of a laser-treated Ti surface measured using grazing incident-angle geometry with an incident angle of 2°.
Fig. 4. In-depth elemental profiles of the surface layer obtained from XPS equipped with an Ar etching system.
N. Ohtsu et al. / Surface & Coatings Technology 246 (2014) 52–56
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μ
N 1s
Fig. 6. Load–displacement curves for untreated and laser-treated Ti substrates.
Etching Time
decreased with increasing the scanning velocity, due to the decrease of the layer thickness [28]. Accordingly, we assumed that the scanning speed is the key factors for forming a hard surface. 3.3. Cellular attachment of laser-treated Ti surfaces
Fig. 5. N 1s XPS spectral changes in the surface layer with increasing etching time.
main part of the surface layer comprised TiN and Ti, and that the concentration of TiN eventually decreased with increasing depth. At the topmost surface, a thin oxide layer was formed after the laser treatment. In our process, the reaction gas must include a small amount of oxygen because the nitrogen blow included oxygen gas present in the air. We thus considered that the oxide layer was probably formed via a reaction with oxygen during the lowering of the surface temperature after irradiation. On the other hand, in our previous study [26], a reaction gas was prepared in an atmosphere-controlled chamber, and thus, oxygen gas was hardly included. The nitride layer formed in the chamber did not show the presence of a thin oxide layer, and this result supports our consideration. 3.2. Hardness of laser-irradiated Ti surface The hardness of a laser-irradiated surface was measured using a nanoindentation test. The load–displacement curves obtained for untreated and laser-treated Ti substrates are shown in Fig. 6. It is clear that tip penetration depth for the laser-irradiated Ti surfaces is shallower than that of the untreated Ti surfaces. The hardness values of the surfaces, calculated from their load–displacement curves, are also shown in the figure. The hardness of a treated Ti surface was 7.5 GPa, approximately five times that of an untreated Ti surface, which almost agreed with the value reported by Höche et al. [28]. This result indicates that a Ti surface can be hardened by focused pulsed laser irradiation accompanied by a nitrogen gas blow. However, the hardness obtained after this treatment was much lower than that obtained in our previous study [26]. In this study, the substrate was placed on a stepping-motor-driven XY-axis stage and the stage was scanned at a speed of 1 mm s− 1. On the other hand, the laser shots were repeated 50 times on one spot without moving the substrate in our previous study. As a result, the number of laser shots radiated on a specific area was reduced as compared with the previous case. Höche et al. demonstrated that the hardness of a treated surface was
The quantitative results for cellular number, normalized to the initial number of seeded cells, on laser-treated and untreated Ti surfaces after cultivation for 24 h are shown in Fig. 7. The result for an intact polystyrene (PS) surface of the culture plate is also shown in the figure as a control result. A significant statistical difference was found between the cellular number for the treated and untreated surfaces. The cellular number on a laser-treated Ti surface was approximately three times that on an untreated Ti surface. This result indicates that the surface treatment using focused laser irradiation can significantly enhance the cell attachment performance of Ti surfaces. To explore the reasons for this enhancement, we measured the contact angle of distilled water on the surfaces after sterilization. The photograph of distilled water dropped on the surfaces is shown in Fig. 8. No water drop was formed on the treated surface, while an apparent water drop was observed on the untreated Ti surface. This result demonstrates that the Ti surface was converted to a hydrophilic surface by the laser nitridation treatment. Hao et al. reported that the wettability of Ti–6Al–4V alloy was improved by treating its surface with a high-power diode laser with oxygen gas assist, and that osteoblast cells grew better on a treated surface [31]. They concluded that the wettability characteristic dominates the osteoblast response rather than the surface roughness. In our treatment, laser irradiation was assisted by nitrogen gas blow. However, the surface was covered with a thin oxide layer, as shown in Fig. 4, which implies that the surface chemical state was similar to that reported by
**
Fig. 7. Cell numbers on laser-treated and untreated Ti surfaces after cultivation for 24 h, normalized to the initial number of seeded cells. Cell numbers on the intact polystyrene (PS) surface of the culture plate are shown as a control result. All error bars are shown as mean ± S.D. (n = 3). Data were statistically analyzed using Student's t-test (* p b 0.05).
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Fig. 8. Photograph of distilled water dropped on: (a) a laser-treated Ti surface and (b) untreated Ti surface.
Hao et al. Upon considering these observations, it was concluded that the enhancement in the cell attachment performance was due to the improvement in surface wettability by the laser nitridation treatment.
by a Grant-in-aid for Scientific Research (C) (No. 24560841) from the Ministry of Education, Science, Sports, and Culture (MEXT) of Japan. References
4. Conclusions In the present study, we prepared a nitride layer on a titanium (Ti) substrate by using focused pulsed Nd:YAG laser irradiation accompanied by nitrogen gas blow. The main part of the produced surface layer was composed of TiN and Ti, and the concentration of TiN decreased with increasing depth. The thickness of the surface layer was approximately 1100 nm. Furthermore, at the topmost surface, a thin oxide layer 30 nm in thickness was also formed. The hardness of the laser-treated Ti surface was 7.5 GPa, and this value was approximately five times that for an untreated Ti surface. Furthermore, the surface wettability of the Ti substrate was drastically improved by treating it with focused laser irradiation, which enhanced the cell attachment performance of the substrate. In conclusion, surface treatment using focused pulsed Nd:YAG laser irradiation accompanied by nitrogen gas blow is an excellent surface modification technique that can simultaneously improve the wear resistance and cell attachment performance of Ti materials. This new process is thus considered to be an innovative technique for use on biomedical materials. Conflict of interest None. Acknowledgment
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28]
The authors gratefully acknowledge Mr. Kozuka, an undergraduate student in our laboratory for his support in the cell attachment test. The authors also acknowledge Mr. Tokuda from the Kitami Institute of Technology for his help in TEM analysis. Part of this work was supported
[29] [30] [31]
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