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Influence of TiN coating on the biocompatibility of medical NiTi alloy
Colloids and Surfaces B: Biointerfaces 101 (2013) 343–349
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Influence of TiN coating on the biocompatibility of medical NiTi alloy Shi Jin a , Yang Zhang a,∗ , Qiang Wang a , Dan Zhang a , Song Zhang b a b
School of Stomatology, China Medical University, Shenyang 110002, China School of Materials Science and Engineering, Shenyang University of Technology, Shenyang 110023, China
a r t i c l e
i n f o
Article history: Received 15 March 2012 Received in revised form 10 June 2012 Accepted 22 June 2012 Available online 3 July 2012 Keywords: NiTi alloy TiN coating Biocompatibility Proliferation
a b s t r a c t The biocompatibility of TiN coated nickel–titanium shape memory alloy (NiTi-SMA) was evaluated to compare with that of the uncoated NiTi-SMA. Based on the orthodontic clinical application, the surface properties and biocompatibility were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), wettability test, mechanical test and in vitro tests including MTT, cell apoptosis and cell adhesion tests. It was observed that the bonding between the substrate and TiN coating is excellent. The roughness and wettability increased as for the TiN coating compared with the uncoated NiTi-SMA. MTT test showed no significant difference between the coated and uncoated NiTi-SMA, however the percentage of early cell apoptosis was significantly higher as for the uncoated NiTi alloy. SEM results showed that TiN coating could enhance the cell attachment, spreading and proliferation on NiTi-SMA. The results indicated that TiN coating bonded with the substrate well and could lead to a better biocompatibility. © 2012 Elsevier B.V. All rights reserved.
1. Introduction NiTi-SMA, with nearly equiatomic ratio of nickel and titanium elements, is well known as its shape memory effect and superelasticity effect for several years [1–7]. Of good clinical and mechanical properties, NiTi-SMA is one of the most common orthodontic archwires in conventional orthodontic treatment [8–10]. The shape memory and superelasticity effect are due to the transformation between martensite and austenite, which can be activated by thermal or mechanical loads [11]. When the austenite reaches the martensite start temperature (Ms ), its mechanically properties are unstable and the martensitic reaction begins. It is suitable for orthopaedic implants, orthodontic archwires, orthopaedics, artificial joints, coronary stents and interventional therapy [12]. In previous studies, Ni2+ , which will release after corrosion, was proven to cause toxicity, carcinogen and immune–sensitizing effects. Therefore, it is necessary to modify the surfaces of NiTiSMA to improve its corrosion resistance and biocompatibility. The inhibitory film of TiN, TiO2 and HA was made on the NiTi-SMA substrate which might effectively reduce the release of Ni2+ and improve the biocompatibility [11,13–18]. Among all the coatings, TiN is allowed to be used in producing coatings and cutting tools for its hardness and its remarkable resistance to wear and corrosion.
∗ Corresponding author at: School of Stomatology, China Medical University, 117# Nanjing North Street, Shenyang 110002, China. Tel.: +86 24 22891418. E-mail address: [email protected] (Y. Zhang). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.06.029
It has been used with a TiN coating for the heads of hip prostheses to improve their wear and fatigue resistance [12]. Zhao et al. [23] explored the underlying biological mechanisms of biocompatibility differences between the changes of bare and TiN-coated NiTi alloys, and the TiN-coated alloy was tend to be used as cardiovascular biomaterials. Cell–material interactions are strongly influenced by surface topography, surface roughness, chemical and physicochemical property including surface wettability and surface modification [19]. The behavior of cell adhesion is closely related to biocompatibility. Hallab et al. [20] found that surface energy may be a more important determinant of cell adhesion and proliferation than surface roughness for directing cell adhesion and cell colonization onto engineered tissue scaffoldings. Cell adhesion was also affected by the physico–chemical linkages between cells, van der Waals and electrostatic forces between materials. Manso et al. [21] found that sputtered TiN coatings deposited on Ti-Al-V alloy surfaces showed enhanced biological responses. Chien et al. [22] found that cell viability, proliferation and adherence increased significantly with nitride film coatings in comparison to the uncoated Ni-based alloy surfaces. We hypothesize that the biocompatibility will increase because of the TiN coating and TiN coated NiTi-SMA can be used as orthodontic archwires. To investigate this hypothesis, a study was designed to find out the difference between coated and uncoated NiTi-SMA surfaces. We estimate the physical and mechanical properties by SEM, XRD, wettability assay and mechanical test, to find out the surface characterization, and to see the load and
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displacement of the TiN-coating. Then the cell viability, proliferation, adhesion and morphology were tested to study the biocompatibility of the samples. Thus, this paper focuses on evaluating the effect of TiN coating, to as certain whether it can be used as orthodontic archwires. 2. Materials and methods
between the centers of the two supports was 30 mm. A crosshead at the surface center of the sample was engaged to move downward at a rate of 0.05 mm/min, causing samples to bend to a final displacement of 2 mm under the applied load. Load and displacement were recorded and analyzed by a test analyzing software. The surface morphology was assessed by SEM to detect whether cracks can be discovered.
2.1. Sample preparation
2.3. Biocompatibility studies
A commercially available NiTi-SMA plate (nominal composition: 50.7 at% Ni, made by General Research Institute for Nonferrous Metals, Beijing, China) for medical applications was used in this study. TiN coating was prepared using a filtered arcing ion plate technique, and its thickness was about 2.86 m. The coated and uncoated NiTi-SMA samples were cut into the sizes of 37 mm × 20 mm × 4 mm and ˚10 mm × 1 mm. Lead samples with the dimension of 20 mm × 20 mm × 1 mm were used as positive control. All the samples were mechanically polished sequentially with SiC papers of grit 200, 400, 800 and 1200. Then ultrasonically cleaned with acetone and deionized water, dried in air and then autoclaving for 30 min. Polished samples were ultrasonically cleaned with ethanol and rinsed with deionised water before leaving to dry overnight in air at room temperature.
2.3.1. Cell culture L-929 murine fibroblast cell line was obtained from Central Laboratory (School of Stomatology, China Medical University, China). Cells were cultured in RMPI 1640 medium with 10% FBS and maintained in an incubator at 37 ◦ C, 5% CO2 . Cells were subcultured by 0.25% trypsinization (Sigma Chemical Co., St. Louis, MO) when the monolayer reached subconfluence. In the following tests, L-929 cells were used in order to test the biocompatibility of TiN coatings to compare with the uncoated surface of the NiTi-SMA.
2.2. Surface characterization 2.2.1. Surface morphology and phase composition The surface morphology and composition of the coated and uncoated NiTi-SMA were assessed by scanning electron microscope (SEM, SSX-550, SHIMADZU Inc., Japan) and analyzed by X-ray diffractometer (XRD, D/MAX-RB, RIGAKU Inc., Japan). 2.2.2. Surface roughness analysis Alpha-step IQ® surface profiler (KLA Tencor Co., USA) was used to evaluate the surface roughness. 2.2.3. Wettability Contact angles were used to access the hydrophobicity or hydrophilicity of the sample surfaces. Those of the tested surfaces with and without TiN coating were measured by using JC2000A optical contact angle system (POWEREACH Inc., China). Five test liquids were used to test the contact angle: distilled water, RMPI 1640 medium (Gibico, Rockville, MD) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, St. Louis, MO) (pH 7.0–7.2), 1-bromonaphthalene (Sinopharm Chemical Reagent Co. Ltd), Hank’s solution [24] and artificial saliva (NaCl 0.4 g/l, KCl 0.4 g/l, CaCl2 0.6004 g/l, NaH2 PO4 ·2H2 O 0.78 g/l, KSCN 0.300 g/l, Na2 S·9H2 O 0.005 g/l, urea 1.000 g/l) [25]. A microsyringe was used to administer the droplets on to samples’ surface (0.6 l/droplet). The samples were cleaned with ethanol in an ultrasonic bath for 10 min and then air dried between measurements. The images were captured and transferred to the computer screen, and the contact angle was measured and determined from the images. The surface energy was calculated according to the following p
p 1/2
p
equation: 1 (1 + cos ) = 2 [(l s ) + (ld s )1/2. This equation can be rearranged as by Owens and Wendt to yield: l (1 + 1/2
p 1/2
p 1/2
1/2
2.3.2. Cytotoxicity test The MTT [27] [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] tests were carried out in order to confirm the basic biocompatibility of these surfaces. Extracts were prepared using RMPI 1640 medium supplemented with 10% FBS as the extraction medium with the surface area of extraction medium ratio 1.25 ml/cm2 in an incubator at 37 ◦ C, 5% CO2 for 1, 3, 7, 14, 28 days respectively. The positive control group was lead extraction medium which was prepared using RMPI 1640 medium supplemented with 10% FBS as the extraction medium with the surface area of extraction medium ratio 3 ml/cm2 in a humidified atmosphere with 37 ◦ C, 5% CO2 for 72 h. The extracts were refrigerated at 4 ◦ C before the cytotoxicity test. Cells were seeded in 96-well plates (Corning, NY) at 4 × 103 cells/200 l medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 200 l of extracts. After incubating the cells in an incubator for 24 h, 48 h, 72 h, respectively, 20 l MTT was added to each well. Samples were incubated for 4 h at 37 ◦ C then 150 l dimethyl sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) was added in each well. Ten wells of each group were prepared and measured. The absorbance of each well was read by using Multimode Plate Readers (Infinite M200 Pro, Tecan, Switzerland) at a wavelength of 490 nm. 2.3.3. Cell apoptosis test Extracts were prepared using RMPI 1640 medium supplemented with 10% FBS as the extraction medium with the surface area of extraction medium ratio 1.25 ml/cm2 in a humidified atmosphere with 5% CO2 , 37 ◦ C for 72 h. Cells were seeded in 6-well plates (Corning, NY) at 3 × 104 cells/3 ml medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 3 ml of extracts, and then incubated for 48 h. Detected the cells by Flow Cytometry (FACS, FACSCalibur, Becton Dickinson, USA) following the instruction of Annexin V-FITC/PI Apoptosis Detection Kit (Biosea Biotechnology Co., Ltd., China) at a provocation wavelength of 488 nm and analyzed the results by Modfit LT3.0 Software.
1/2
cos )/(ld ) = (s ) [(l ) /(ld ) ] + (sd ) , where is the contact angle, l is liquid surface tension and s is the solid surface tension. The total free surface energy is merely the sum of its two component forces. [26] 2.2.4. Mechanical test Three-point bending test was performed on an AG-I 500 kN (SHIMADZU Inc., Japan) mechanical testing machine. The distance
2.3.4. Morphologic examination L-929 cells were seeded onto the coated and uncoated samples in 6-well plates at a density of 3 × 104 /3 ml for direct cell adhesion observation. 3 samples were prepared for each group. After 24 h, 48 h and 72 h incubation respectively, the culture media was removed and specimens were fixed with 2.5% glutaraldehyde solution for 4 h at 4 ◦ C and rinsed 3 times with phosphate
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Fig. 1. SEM images show the surface morphologies of (a) surface and (b) vertical section of TiN coated NiTi-SMA.
buffer solution (PBS, pH = 7.4), followed by dehydration in a gradient ethanol/distilled water mixture (30%, 50%, 75%, 95%, 100%) for 10 min each and dried in air. SEM was used to observe the morphologic characteristics of the cells cultured onto the test surfaces. 2.4. Statistical analysis Data of these measurements were analyzed with software SPSS 13.0. The quantitative results from wettability tests and surface roughness test were analyzed using the two-sample t-test. Statistical analysis of results of MTT tests and cell apoptosis test was accomplished by one-way analysis of variance (ANOVA). P values <0.05 were considered to be statistically significant. 3. Result 3.1. Surface characterization 3.1.1. Surface morphology and phase composition The surface morphologies of TiN coated NiTi-SMA are shown in Fig. 1. Compared to the ground NiTi alloy sample, many uniformly distributed bright spots can be seen on the surface of coated sample. XRD profile proves the formation of TiN coating on the samples’ surfaces. Fig. 2 presents the X-ray diffraction patterns of coated and uncoated NiTi-SMA. The sharp NiTi, Ni4 Ti3 , TiN (2 0 0) peaks
can be found, while two apparent TiN (1 1 1 and 2 2 0) peaks appear for the TiN coated samples, NiTi (2 0 0) is also observed. 3.1.2. Surface roughness analysis Roughness measurement proves the increased surface roughness after surface modification as the Alpha-step IQ® surface profiler results show. The Ra and Rz value of coated samples are 18.952 ± 1.553 nm and 148.590 ± 16.860 nm. While the Ra and Rz values of uncoated samples are 7.306 ± 0.480 nm and 37.562 ± 3.449 nm. The surface conditions of NiTi-SMA are rather smooth than TiN coating. The mean roughness values of two groups are significantly different (P < 0.05). 3.1.3. Wettability In this study, wettability results in Table 1 show that the contact angle was significantly larger for NiTi-SMA with all five test liquids, indicating that TiN coated sample was more hydrophilic (P < 0.05). Table 1 also shows the surface energy of coated group is larger than uncoated group. 3.1.4. Mechanical test Fig. 3 shows the schematic diagram of three-point bending test and the SEM image of the bending area of TiN coating. When the sample bent to a displacement of 2 mm, the load was 4099 ± 3 N. The angle is 7.595 ± 0.023◦ . As shown in Fig. 3, no crack can be seen on the sample’s bending area after bending test. 3.2. Biocompatibility studies 3.2.1. Cytotoxicity test Table 2 shows the optical density of L-929 cells in extraction medium of the test samples measured by MTT test. There was no significant difference between five extraction times in both coated and uncoated group (P > 0.05). So the average values of optical density at different culture time points were obtained and the relative growth rates were calculated. There was no significant difference Table 1 Contact angles of two samples with respect to five different solutions and the surface energy of coated and uncoated NiTi-SMA, n = 6. Uncoated*
Coated* Deionized water (◦ ) 1-Bromonaphthalene (◦ ) RMPI 1640 (◦ ) Hank’s solution (◦ ) Artificial saliva (◦ ) Surface energy (mN/m) Fig. 2. XRD profiles of coated and uncoated NiTi-SMA surfaces.
*
P > 0.05.
60.85 14.35 49.75 58.25 59.75 53.992
± ± ± ± ± ±
2.219 0.877 1.250 1.403 2.243 1.083
65.65 23.35 67.50 68.20 69.20 50.089
± ± ± ± ± ±
1.607 2.111 2.598 1.052 2.618 0.695
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Table 2 The optical density of L-929 cells in extraction solution of coated and uncoated NiTi-SMA as measured by the MTT test, n = 10; P > 0.05. Extracting time of extraction solution
Detection period (h)
1d
3d
7d
14 d
28 d
Average
Coated
24 48 72
0.321 ± 0.005 0.467 ± 0.022 0.779 ± 0.016
0.318 ± 0.002 0.463 ± 0.013 0.776 ± 0.029
0.327 ± 0.002 0.481 ± 0.007 0.771 ± 0.028
0.329 ± 0.009 0.481 ± 0.023 0.804 ± 0.021
0.319 ± 0.000 0.482 ± 0.007 0.762 ± 0.021
0.323 ± 0.006 0.475 ± 0.017 0.778 ± 0.026
Uncoated
24 48 72
0.325 ± 0.006 0.478 ± 0.010 0.786 ± 0.016
0.319 ± 0.004 0.479 ± 0.004 0.794 ± 0.023
0.317 ± 0.005 0.472 ± 0.008 0.735 ± 0.030
0.330 ± 0.003 0.467 ± 0.022 0.764 ± 0.016
0.321 ± 0.012 0.468 ± 0.038 0.742 ± 0.005
0.323 ± 0.008 0.473 ± 0.019 0.764 ± 0.030
Table 3 Relative growth rate (RGR) and cytotoxicity level at different detection period, n = 10. Samples
Negative control Coated group* Uncoated group* Positive control# * #
24 h
48 h
72 h
RGR%
CTG
RGR%
CTG
RGR%
CTG
100 94.25 ± 1.257 94.17 ± 1.380 84.45 ± 3.098
0 1 1 1
100 99.03 ± 1.646 98.70 ± 1.043 49.97 ± 1.158
0 1 1 2
100 97.43 ± 1.756 95.68 ± 2.893 26.39 ± 1.970
0 1 1 4
P > 0.05. P < 0.01.
between every two groups of coated, uncoated NiTi-SMA and negative group (P > 0.05), while there was significant difference between positive control group and the other three groups (P < 0.01). In Table 3, the relative growth rate (RGR) of the coated, uncoated and positive groups was assessed. RGR was calculated as following: RGR = ODe /ODc × 100%, where ODe is the average OD value of the experimental and positive control groups, and ODc represents to the negative control group. The cell toxicity grade (CTG) was obtained by the relationship between RGR and CTG, according to the standard United States Pharmacopeia. The results show that CTG of the coated and uncoated groups is in grade 1 which represents no toxicity. After 48 h and 72 h of incubation, the positive
control group is in grade 2 and grade 4 respectively which displays slightly and obvious toxicity. Fig. 4 shows the mean values of early apoptosis rates and the scatter plots detected by FCM. The early apoptosis rate of the negative, coated group and uncoated group is 2.38%, 3.21%, 5.63%, respectively. That of the negative and coated group were significantly higher than uncoated group (P < 0.05), while there was no significant difference between negative group and coated group (P > 0.05). 3.2.2. Morphologic examination Fig. 5 shows the morphologies of L-929 cells cultured on the coated and uncoated NiTi-SMA for 24 h, 48 h and 72 h. It indicates that L-929 cells attached well after 24 h culture, and proliferated after 48 h and 72 h culture on all the sample surfaces. As shown in Fig. 4, the morphologic features of L-929 adherent to NiTi-SMA are completely different. The cells on coated group had a large quantity and were more spreading than those on uncoated group at 24 h culture. After 48 h and 72 h culture, there were more cells on coated compared to uncoated group. 4. Discussion
Fig. 3. (a) Schematic diagram of three-point bending test and (b) the SEM image of the bending area of TiN-coating; n = 3.
NiTi-SMAs have been investigated to be used as biomedical instrument for several years [2,4,5]. The thin film of TiN, TiO2 , and HA has been made at alloy surface. In previous studies, TiN was focused on as diffusion barriers in orthopedic prostheses and cardiac valves in order to enhance surface characteristics and hemocompatibility [23,28,29]. In this paper the surfaces of coated and uncoated NiTi-SMA were characterized using SEM, XRD, surface profiler and mechanical test. After loading a force of 4099 N, no crack appeared on the TiN coating. It can be indicated that the TiN coating can bear the orthodontic force caused by the deformation of orthodontic archwires. The higher roughness of TiN coating may be caused by the bright spots (Fig. 1), which constituted mainly by TiN. Surface roughness and surface energy results also confirmed that the coated group was rougher and had a lower surface energy which relatively means more hydrophilic than the uncoated group. Wenzel et al. [30] figured out that there was a relationship between contact angles and surface roughness. The results are in agreement with much of the work previously published that the biocompatibility of TiN coated NiTi is good [23]. This study
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Fig. 4. Flow cytometric analysis of early apoptosis rates detected by Annexin V-FITC/PI apoptosis detection method. Scatter plots are shown in (a) control group, (b) coated group, (c) uncoated group respectively and (d) shows the early apoptosis rates of L-929 cells cultured in different medium. Error bars represent means ± SD, n = 5; *P > 0.05, **P < 0.05.
appeared that the roughness of TiN coated NiTi-SMA was higher, while the surface energy has a similar trend. Generally, the contact angles were larger for the low-energy, hydrophobic polymers than for the higher energy, more hydrophilic materials. The contact angle of coated surface was determined to be 49.75◦ with RMPI 1640 medium (Table 1), which obviously lower than that of the uncoated surface. The surface energies, calculated to be 54 mN/m, were higher than those of uncoated surfaces (Table 1). Den Barber et al. [31] evaluated those physicochemical parameters such as wettability and surface free energy influence cell growth. The TiN coating may affects the biocompatibility, so more tests are considered to further validate this conclusion. In vitro cytotoxicity assays are most commonly used for testing the biocompatibility of biological materials [11,32–39]. As there was no significant difference between different extraction times in coated and uncoated group (P > 0.05) in MTT test, it may indicate that the extraction time has almost no effect on cell viability and proliferation. After 24 h, 48 h and 72 h of incubation, the CTGs of the coated and uncoated groups all represent no toxicity. As NiTi-SMA is used as biomedical instrument for several years, it is considered that the NiTi-SMAs with TiN coating are no toxicity which corresponds with the basic requirements of medical materials. Flow cytometry is now a widely used method for analyzing expression of cell surface and intracellular molecules [40–43]. It is selected to detect the apoptosis of L-929 cells in different leaching solutions. The results of early apoptosis rate shows: negative group < coated group < uncoated group. The early apoptosis rate of uncoated group rise significantly (P < 0.05). Compared with the uncoated NiTi-SMA, the TiN-coating may protect the cells from apoptosis. Zhao et al. [23] found that compared with the bare NiTi
alloys, TiN coating significantly reduced the release of Ni ions from the alloys. The result of FCM test indicated that the TiN film may reduce the release of Ni2+ and improve the biocompatibility. To observe the morphology and quantity of L-929 cells adhering to the substrate directly, SEM was applicable. Three time points, considering about the stages of cell viability test, were chosen to assess the attachment, adhesion and spreading of L-929 cells on the coated and uncoated NiTi-SMA in this study. The 24 h detection period represents early attachment, the 48 h and 72 h detection period show the adhesion and spreading processes of fibroblasts on the tested surfaces. Cell adhesion was related to various natural phenomena such as embryogenesis, maintenance of tissue structure, wound healing, immune response, metastasis, and tissue integration of biomaterials [44]. The changes of cellular morphology are revealed in Fig. 5. The results of the cell morphologic examination were consistent with the conclusion reported by Lampin et al. [45]. They found that a slight roughness raised the migration area to an upper extent no matter which cell type and enhancement of the cell adhesion potential was related to the degree of roughness and the hydrophobicity. Groessner-Schreiber et al. [46] found that fibroblasts intimately adherent to the surface of TiN compared to the oxidized titanium surfaces. Fibroblast cells cultured on coated and uncoated NiTi-SMA were observed to exhibit polygonal (24 h), spindle–shaped (48 h) patterns and spreading stacked (72 h) with a well-developed cytoskeleton. Morphologic examination indicated that the number of cells on the TiN coating was larger than those on NiTi-SMA. These features show that the TiN layer on the NiTi-SMA has the ability to induce the fibroblasts attachment, spreading, proliferation and growth on their surfaces from RMPI 1640 medium.
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Fig. 5. SEM images show the surface morphologies of L-929 cells cultured on coated and uncoated NiTi-SMA at different detection period.
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5. Conclusion It seems that TiN coating presents greater roughness and good wettability which increases surface affinity, expected in cell adherence. TiN-coating has a good bonding strength which can bear a load of 4099 N after bending to a displacement of 2 mm. In MTT test, OD values represent that cell viability and proliferation were similar among the coated, uncoated and negative control group. MTT test results show no significant difference between five extraction times in both coated and uncoated group. However, the early apoptosis rates of TiN coated group were significantly lower than uncoated group. Cell attachment, spreading and proliferation were apparently enhanced on TiN coating, TiN coating modify the surface contents of NiTi-SMA and increase the roughness and wettability, which enhance the proliferation and fibroblast adherence. In addition, TiN coating could lead to a better biocompatibility on NiTiSMA, which provided valuable insights into the potential use of TiN coating on NiTi orthodontics archwires. Acknowledgements The authors are grateful for the supports from National Basic Research Program of China (973 Program, No. 2012CB619101) and Liaoning Province of China (20082088). We also appreciated the kind help from Central Laboratory, School of Stomatology, China Medical University, during cell culture. References [1] A. Falvo, F.M. Furgiuele, C. Maletta, Laser welding of a NiTi alloy: mechanical and shape memory behaviour, Mater. Sci. Eng. A 412 (2005) 235. [2] B.P. Khasenov, A.A. Kadnikov, D.I. Rabkin, Use in technology and medicine of TiNi alloys revealing the shape memory effect, Met. Sci. Heat Treat. 30 (1988) 300. [3] T.W. Duerig, A.R. Pelton, D. Stockel, The use of superelasticity in medicine, Metallurgy 50 (1996) 569. [4] T.W. Duerig, Present and future applications of shape memory and superelastic materials, Mater. Res. Soc. Symp. Proc. 360 (1995) 497. [5] L.S. Castleman, S.M. Motzkin, F.P. Alicandri, V.L. Bonawit, Biocompatibility of nitinol alloy as an implant material, J. Biomed. Mater. Res. 10 (1976) 695. [6] J. Ryhanen, E. Niemi, W. Serlo, E. Niemela, P. Sandvik, H. Pernu, T. Salo, Biocompatibility of nickel–titanium shape memory metal and its corrosion behavior in human cell cultures, J. Biomed. Mater. Res. 35 (1997) 451. [7] D.J. Wever, A.G. Veldhuizen, M.M. Sanders, J.M. Schakenraad, J.R. van Horn, Cytotoxic, allergic and genotoxic activity of a nickel–titanium alloy, Biomaterials 18 (1997) 1115. [8] W.V. Moorleghem, M. Chandrasekaran, D. Reynaerts, J. Peirs, H.V. Brussel, Shape memory and superelastic alloys: the new medical materials with growing demand, Biomed. Mater. Eng. 8 (1998) 55. [9] W.A. Brantley, T. Eliades, Orthodontic Materials: Scientific and Clinical Aspects, Thieme, 2001. [10] L. Torrisi, The NiTi superelastic alloy application to the dentistry field, Biomed. Mater. Eng. 9 (1999) 39. [11] R.P. Kusy, J.Q. Whitley, Thermal and mechanical characteristics of stainless steel, titanium–molybdenum, and nickel–titanium archwires, Am. J. Orthod. Dentofacial Orthop. 131 (2007) 229. [12] S. Piscanec, L.C. Ciacchi, E. Vesselli, G. Comelli, O. Sbaizero, S. Meriani, A. De Vita, Bioactivity of TiN-coated titanium implants, Acta Mater. 52 (2004) 1237. [13] M. Es-Souni, H. Fischer-Brandies, Assessing the biocompatibility of NiTi shape memory alloys used for medical applications, Anal. Bioanal. Chem. 381 (2005) 557. [14] D. Budziak, E. Martendal, E. Carasek, Preparation and characterization of new solid–phase microextraction fibers obtained by sol–gel technology and zirconium oxide electrodeposited on NiTi alloy, J. Chromatogr. A 1187 (2008) 34. [15] R. Matos de Souza, L. Macedo de Menezes, Nickel, chromium and iron levels in the saliva of patients with simulated fixed orthodontic appliances, Angle Orthod. 78 (2008) 345. [16] R. Fors, M. Persson, Nickel in dental plaque and saliva in patients with and without orthodontic appliances, Eur. J. Orthod. 28 (2006) 292.
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Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Influence of TiN coating on the biocompatibility of medical NiTi alloy Shi Jin a , Yang Zhang a,∗ , Qiang Wang a , Dan Zhang a , Song Zhang b a b
School of Stomatology, China Medical University, Shenyang 110002, China School of Materials Science and Engineering, Shenyang University of Technology, Shenyang 110023, China
a r t i c l e
i n f o
Article history: Received 15 March 2012 Received in revised form 10 June 2012 Accepted 22 June 2012 Available online 3 July 2012 Keywords: NiTi alloy TiN coating Biocompatibility Proliferation
a b s t r a c t The biocompatibility of TiN coated nickel–titanium shape memory alloy (NiTi-SMA) was evaluated to compare with that of the uncoated NiTi-SMA. Based on the orthodontic clinical application, the surface properties and biocompatibility were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), wettability test, mechanical test and in vitro tests including MTT, cell apoptosis and cell adhesion tests. It was observed that the bonding between the substrate and TiN coating is excellent. The roughness and wettability increased as for the TiN coating compared with the uncoated NiTi-SMA. MTT test showed no significant difference between the coated and uncoated NiTi-SMA, however the percentage of early cell apoptosis was significantly higher as for the uncoated NiTi alloy. SEM results showed that TiN coating could enhance the cell attachment, spreading and proliferation on NiTi-SMA. The results indicated that TiN coating bonded with the substrate well and could lead to a better biocompatibility. © 2012 Elsevier B.V. All rights reserved.
1. Introduction NiTi-SMA, with nearly equiatomic ratio of nickel and titanium elements, is well known as its shape memory effect and superelasticity effect for several years [1–7]. Of good clinical and mechanical properties, NiTi-SMA is one of the most common orthodontic archwires in conventional orthodontic treatment [8–10]. The shape memory and superelasticity effect are due to the transformation between martensite and austenite, which can be activated by thermal or mechanical loads [11]. When the austenite reaches the martensite start temperature (Ms ), its mechanically properties are unstable and the martensitic reaction begins. It is suitable for orthopaedic implants, orthodontic archwires, orthopaedics, artificial joints, coronary stents and interventional therapy [12]. In previous studies, Ni2+ , which will release after corrosion, was proven to cause toxicity, carcinogen and immune–sensitizing effects. Therefore, it is necessary to modify the surfaces of NiTiSMA to improve its corrosion resistance and biocompatibility. The inhibitory film of TiN, TiO2 and HA was made on the NiTi-SMA substrate which might effectively reduce the release of Ni2+ and improve the biocompatibility [11,13–18]. Among all the coatings, TiN is allowed to be used in producing coatings and cutting tools for its hardness and its remarkable resistance to wear and corrosion.
∗ Corresponding author at: School of Stomatology, China Medical University, 117# Nanjing North Street, Shenyang 110002, China. Tel.: +86 24 22891418. E-mail address: [email protected] (Y. Zhang). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.06.029
It has been used with a TiN coating for the heads of hip prostheses to improve their wear and fatigue resistance [12]. Zhao et al. [23] explored the underlying biological mechanisms of biocompatibility differences between the changes of bare and TiN-coated NiTi alloys, and the TiN-coated alloy was tend to be used as cardiovascular biomaterials. Cell–material interactions are strongly influenced by surface topography, surface roughness, chemical and physicochemical property including surface wettability and surface modification [19]. The behavior of cell adhesion is closely related to biocompatibility. Hallab et al. [20] found that surface energy may be a more important determinant of cell adhesion and proliferation than surface roughness for directing cell adhesion and cell colonization onto engineered tissue scaffoldings. Cell adhesion was also affected by the physico–chemical linkages between cells, van der Waals and electrostatic forces between materials. Manso et al. [21] found that sputtered TiN coatings deposited on Ti-Al-V alloy surfaces showed enhanced biological responses. Chien et al. [22] found that cell viability, proliferation and adherence increased significantly with nitride film coatings in comparison to the uncoated Ni-based alloy surfaces. We hypothesize that the biocompatibility will increase because of the TiN coating and TiN coated NiTi-SMA can be used as orthodontic archwires. To investigate this hypothesis, a study was designed to find out the difference between coated and uncoated NiTi-SMA surfaces. We estimate the physical and mechanical properties by SEM, XRD, wettability assay and mechanical test, to find out the surface characterization, and to see the load and
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displacement of the TiN-coating. Then the cell viability, proliferation, adhesion and morphology were tested to study the biocompatibility of the samples. Thus, this paper focuses on evaluating the effect of TiN coating, to as certain whether it can be used as orthodontic archwires. 2. Materials and methods
between the centers of the two supports was 30 mm. A crosshead at the surface center of the sample was engaged to move downward at a rate of 0.05 mm/min, causing samples to bend to a final displacement of 2 mm under the applied load. Load and displacement were recorded and analyzed by a test analyzing software. The surface morphology was assessed by SEM to detect whether cracks can be discovered.
2.1. Sample preparation
2.3. Biocompatibility studies
A commercially available NiTi-SMA plate (nominal composition: 50.7 at% Ni, made by General Research Institute for Nonferrous Metals, Beijing, China) for medical applications was used in this study. TiN coating was prepared using a filtered arcing ion plate technique, and its thickness was about 2.86 m. The coated and uncoated NiTi-SMA samples were cut into the sizes of 37 mm × 20 mm × 4 mm and ˚10 mm × 1 mm. Lead samples with the dimension of 20 mm × 20 mm × 1 mm were used as positive control. All the samples were mechanically polished sequentially with SiC papers of grit 200, 400, 800 and 1200. Then ultrasonically cleaned with acetone and deionized water, dried in air and then autoclaving for 30 min. Polished samples were ultrasonically cleaned with ethanol and rinsed with deionised water before leaving to dry overnight in air at room temperature.
2.3.1. Cell culture L-929 murine fibroblast cell line was obtained from Central Laboratory (School of Stomatology, China Medical University, China). Cells were cultured in RMPI 1640 medium with 10% FBS and maintained in an incubator at 37 ◦ C, 5% CO2 . Cells were subcultured by 0.25% trypsinization (Sigma Chemical Co., St. Louis, MO) when the monolayer reached subconfluence. In the following tests, L-929 cells were used in order to test the biocompatibility of TiN coatings to compare with the uncoated surface of the NiTi-SMA.
2.2. Surface characterization 2.2.1. Surface morphology and phase composition The surface morphology and composition of the coated and uncoated NiTi-SMA were assessed by scanning electron microscope (SEM, SSX-550, SHIMADZU Inc., Japan) and analyzed by X-ray diffractometer (XRD, D/MAX-RB, RIGAKU Inc., Japan). 2.2.2. Surface roughness analysis Alpha-step IQ® surface profiler (KLA Tencor Co., USA) was used to evaluate the surface roughness. 2.2.3. Wettability Contact angles were used to access the hydrophobicity or hydrophilicity of the sample surfaces. Those of the tested surfaces with and without TiN coating were measured by using JC2000A optical contact angle system (POWEREACH Inc., China). Five test liquids were used to test the contact angle: distilled water, RMPI 1640 medium (Gibico, Rockville, MD) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, St. Louis, MO) (pH 7.0–7.2), 1-bromonaphthalene (Sinopharm Chemical Reagent Co. Ltd), Hank’s solution [24] and artificial saliva (NaCl 0.4 g/l, KCl 0.4 g/l, CaCl2 0.6004 g/l, NaH2 PO4 ·2H2 O 0.78 g/l, KSCN 0.300 g/l, Na2 S·9H2 O 0.005 g/l, urea 1.000 g/l) [25]. A microsyringe was used to administer the droplets on to samples’ surface (0.6 l/droplet). The samples were cleaned with ethanol in an ultrasonic bath for 10 min and then air dried between measurements. The images were captured and transferred to the computer screen, and the contact angle was measured and determined from the images. The surface energy was calculated according to the following p
p 1/2
p
equation: 1 (1 + cos ) = 2 [(l s ) + (ld s )1/2. This equation can be rearranged as by Owens and Wendt to yield: l (1 + 1/2
p 1/2
p 1/2
1/2
2.3.2. Cytotoxicity test The MTT [27] [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] tests were carried out in order to confirm the basic biocompatibility of these surfaces. Extracts were prepared using RMPI 1640 medium supplemented with 10% FBS as the extraction medium with the surface area of extraction medium ratio 1.25 ml/cm2 in an incubator at 37 ◦ C, 5% CO2 for 1, 3, 7, 14, 28 days respectively. The positive control group was lead extraction medium which was prepared using RMPI 1640 medium supplemented with 10% FBS as the extraction medium with the surface area of extraction medium ratio 3 ml/cm2 in a humidified atmosphere with 37 ◦ C, 5% CO2 for 72 h. The extracts were refrigerated at 4 ◦ C before the cytotoxicity test. Cells were seeded in 96-well plates (Corning, NY) at 4 × 103 cells/200 l medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 200 l of extracts. After incubating the cells in an incubator for 24 h, 48 h, 72 h, respectively, 20 l MTT was added to each well. Samples were incubated for 4 h at 37 ◦ C then 150 l dimethyl sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) was added in each well. Ten wells of each group were prepared and measured. The absorbance of each well was read by using Multimode Plate Readers (Infinite M200 Pro, Tecan, Switzerland) at a wavelength of 490 nm. 2.3.3. Cell apoptosis test Extracts were prepared using RMPI 1640 medium supplemented with 10% FBS as the extraction medium with the surface area of extraction medium ratio 1.25 ml/cm2 in a humidified atmosphere with 5% CO2 , 37 ◦ C for 72 h. Cells were seeded in 6-well plates (Corning, NY) at 3 × 104 cells/3 ml medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 3 ml of extracts, and then incubated for 48 h. Detected the cells by Flow Cytometry (FACS, FACSCalibur, Becton Dickinson, USA) following the instruction of Annexin V-FITC/PI Apoptosis Detection Kit (Biosea Biotechnology Co., Ltd., China) at a provocation wavelength of 488 nm and analyzed the results by Modfit LT3.0 Software.
1/2
cos )/(ld ) = (s ) [(l ) /(ld ) ] + (sd ) , where is the contact angle, l is liquid surface tension and s is the solid surface tension. The total free surface energy is merely the sum of its two component forces. [26] 2.2.4. Mechanical test Three-point bending test was performed on an AG-I 500 kN (SHIMADZU Inc., Japan) mechanical testing machine. The distance
2.3.4. Morphologic examination L-929 cells were seeded onto the coated and uncoated samples in 6-well plates at a density of 3 × 104 /3 ml for direct cell adhesion observation. 3 samples were prepared for each group. After 24 h, 48 h and 72 h incubation respectively, the culture media was removed and specimens were fixed with 2.5% glutaraldehyde solution for 4 h at 4 ◦ C and rinsed 3 times with phosphate
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Fig. 1. SEM images show the surface morphologies of (a) surface and (b) vertical section of TiN coated NiTi-SMA.
buffer solution (PBS, pH = 7.4), followed by dehydration in a gradient ethanol/distilled water mixture (30%, 50%, 75%, 95%, 100%) for 10 min each and dried in air. SEM was used to observe the morphologic characteristics of the cells cultured onto the test surfaces. 2.4. Statistical analysis Data of these measurements were analyzed with software SPSS 13.0. The quantitative results from wettability tests and surface roughness test were analyzed using the two-sample t-test. Statistical analysis of results of MTT tests and cell apoptosis test was accomplished by one-way analysis of variance (ANOVA). P values <0.05 were considered to be statistically significant. 3. Result 3.1. Surface characterization 3.1.1. Surface morphology and phase composition The surface morphologies of TiN coated NiTi-SMA are shown in Fig. 1. Compared to the ground NiTi alloy sample, many uniformly distributed bright spots can be seen on the surface of coated sample. XRD profile proves the formation of TiN coating on the samples’ surfaces. Fig. 2 presents the X-ray diffraction patterns of coated and uncoated NiTi-SMA. The sharp NiTi, Ni4 Ti3 , TiN (2 0 0) peaks
can be found, while two apparent TiN (1 1 1 and 2 2 0) peaks appear for the TiN coated samples, NiTi (2 0 0) is also observed. 3.1.2. Surface roughness analysis Roughness measurement proves the increased surface roughness after surface modification as the Alpha-step IQ® surface profiler results show. The Ra and Rz value of coated samples are 18.952 ± 1.553 nm and 148.590 ± 16.860 nm. While the Ra and Rz values of uncoated samples are 7.306 ± 0.480 nm and 37.562 ± 3.449 nm. The surface conditions of NiTi-SMA are rather smooth than TiN coating. The mean roughness values of two groups are significantly different (P < 0.05). 3.1.3. Wettability In this study, wettability results in Table 1 show that the contact angle was significantly larger for NiTi-SMA with all five test liquids, indicating that TiN coated sample was more hydrophilic (P < 0.05). Table 1 also shows the surface energy of coated group is larger than uncoated group. 3.1.4. Mechanical test Fig. 3 shows the schematic diagram of three-point bending test and the SEM image of the bending area of TiN coating. When the sample bent to a displacement of 2 mm, the load was 4099 ± 3 N. The angle is 7.595 ± 0.023◦ . As shown in Fig. 3, no crack can be seen on the sample’s bending area after bending test. 3.2. Biocompatibility studies 3.2.1. Cytotoxicity test Table 2 shows the optical density of L-929 cells in extraction medium of the test samples measured by MTT test. There was no significant difference between five extraction times in both coated and uncoated group (P > 0.05). So the average values of optical density at different culture time points were obtained and the relative growth rates were calculated. There was no significant difference Table 1 Contact angles of two samples with respect to five different solutions and the surface energy of coated and uncoated NiTi-SMA, n = 6. Uncoated*
Coated* Deionized water (◦ ) 1-Bromonaphthalene (◦ ) RMPI 1640 (◦ ) Hank’s solution (◦ ) Artificial saliva (◦ ) Surface energy (mN/m) Fig. 2. XRD profiles of coated and uncoated NiTi-SMA surfaces.
*
P > 0.05.
60.85 14.35 49.75 58.25 59.75 53.992
± ± ± ± ± ±
2.219 0.877 1.250 1.403 2.243 1.083
65.65 23.35 67.50 68.20 69.20 50.089
± ± ± ± ± ±
1.607 2.111 2.598 1.052 2.618 0.695
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Table 2 The optical density of L-929 cells in extraction solution of coated and uncoated NiTi-SMA as measured by the MTT test, n = 10; P > 0.05. Extracting time of extraction solution
Detection period (h)
1d
3d
7d
14 d
28 d
Average
Coated
24 48 72
0.321 ± 0.005 0.467 ± 0.022 0.779 ± 0.016
0.318 ± 0.002 0.463 ± 0.013 0.776 ± 0.029
0.327 ± 0.002 0.481 ± 0.007 0.771 ± 0.028
0.329 ± 0.009 0.481 ± 0.023 0.804 ± 0.021
0.319 ± 0.000 0.482 ± 0.007 0.762 ± 0.021
0.323 ± 0.006 0.475 ± 0.017 0.778 ± 0.026
Uncoated
24 48 72
0.325 ± 0.006 0.478 ± 0.010 0.786 ± 0.016
0.319 ± 0.004 0.479 ± 0.004 0.794 ± 0.023
0.317 ± 0.005 0.472 ± 0.008 0.735 ± 0.030
0.330 ± 0.003 0.467 ± 0.022 0.764 ± 0.016
0.321 ± 0.012 0.468 ± 0.038 0.742 ± 0.005
0.323 ± 0.008 0.473 ± 0.019 0.764 ± 0.030
Table 3 Relative growth rate (RGR) and cytotoxicity level at different detection period, n = 10. Samples
Negative control Coated group* Uncoated group* Positive control# * #
24 h
48 h
72 h
RGR%
CTG
RGR%
CTG
RGR%
CTG
100 94.25 ± 1.257 94.17 ± 1.380 84.45 ± 3.098
0 1 1 1
100 99.03 ± 1.646 98.70 ± 1.043 49.97 ± 1.158
0 1 1 2
100 97.43 ± 1.756 95.68 ± 2.893 26.39 ± 1.970
0 1 1 4
P > 0.05. P < 0.01.
between every two groups of coated, uncoated NiTi-SMA and negative group (P > 0.05), while there was significant difference between positive control group and the other three groups (P < 0.01). In Table 3, the relative growth rate (RGR) of the coated, uncoated and positive groups was assessed. RGR was calculated as following: RGR = ODe /ODc × 100%, where ODe is the average OD value of the experimental and positive control groups, and ODc represents to the negative control group. The cell toxicity grade (CTG) was obtained by the relationship between RGR and CTG, according to the standard United States Pharmacopeia. The results show that CTG of the coated and uncoated groups is in grade 1 which represents no toxicity. After 48 h and 72 h of incubation, the positive
control group is in grade 2 and grade 4 respectively which displays slightly and obvious toxicity. Fig. 4 shows the mean values of early apoptosis rates and the scatter plots detected by FCM. The early apoptosis rate of the negative, coated group and uncoated group is 2.38%, 3.21%, 5.63%, respectively. That of the negative and coated group were significantly higher than uncoated group (P < 0.05), while there was no significant difference between negative group and coated group (P > 0.05). 3.2.2. Morphologic examination Fig. 5 shows the morphologies of L-929 cells cultured on the coated and uncoated NiTi-SMA for 24 h, 48 h and 72 h. It indicates that L-929 cells attached well after 24 h culture, and proliferated after 48 h and 72 h culture on all the sample surfaces. As shown in Fig. 4, the morphologic features of L-929 adherent to NiTi-SMA are completely different. The cells on coated group had a large quantity and were more spreading than those on uncoated group at 24 h culture. After 48 h and 72 h culture, there were more cells on coated compared to uncoated group. 4. Discussion
Fig. 3. (a) Schematic diagram of three-point bending test and (b) the SEM image of the bending area of TiN-coating; n = 3.
NiTi-SMAs have been investigated to be used as biomedical instrument for several years [2,4,5]. The thin film of TiN, TiO2 , and HA has been made at alloy surface. In previous studies, TiN was focused on as diffusion barriers in orthopedic prostheses and cardiac valves in order to enhance surface characteristics and hemocompatibility [23,28,29]. In this paper the surfaces of coated and uncoated NiTi-SMA were characterized using SEM, XRD, surface profiler and mechanical test. After loading a force of 4099 N, no crack appeared on the TiN coating. It can be indicated that the TiN coating can bear the orthodontic force caused by the deformation of orthodontic archwires. The higher roughness of TiN coating may be caused by the bright spots (Fig. 1), which constituted mainly by TiN. Surface roughness and surface energy results also confirmed that the coated group was rougher and had a lower surface energy which relatively means more hydrophilic than the uncoated group. Wenzel et al. [30] figured out that there was a relationship between contact angles and surface roughness. The results are in agreement with much of the work previously published that the biocompatibility of TiN coated NiTi is good [23]. This study
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Fig. 4. Flow cytometric analysis of early apoptosis rates detected by Annexin V-FITC/PI apoptosis detection method. Scatter plots are shown in (a) control group, (b) coated group, (c) uncoated group respectively and (d) shows the early apoptosis rates of L-929 cells cultured in different medium. Error bars represent means ± SD, n = 5; *P > 0.05, **P < 0.05.
appeared that the roughness of TiN coated NiTi-SMA was higher, while the surface energy has a similar trend. Generally, the contact angles were larger for the low-energy, hydrophobic polymers than for the higher energy, more hydrophilic materials. The contact angle of coated surface was determined to be 49.75◦ with RMPI 1640 medium (Table 1), which obviously lower than that of the uncoated surface. The surface energies, calculated to be 54 mN/m, were higher than those of uncoated surfaces (Table 1). Den Barber et al. [31] evaluated those physicochemical parameters such as wettability and surface free energy influence cell growth. The TiN coating may affects the biocompatibility, so more tests are considered to further validate this conclusion. In vitro cytotoxicity assays are most commonly used for testing the biocompatibility of biological materials [11,32–39]. As there was no significant difference between different extraction times in coated and uncoated group (P > 0.05) in MTT test, it may indicate that the extraction time has almost no effect on cell viability and proliferation. After 24 h, 48 h and 72 h of incubation, the CTGs of the coated and uncoated groups all represent no toxicity. As NiTi-SMA is used as biomedical instrument for several years, it is considered that the NiTi-SMAs with TiN coating are no toxicity which corresponds with the basic requirements of medical materials. Flow cytometry is now a widely used method for analyzing expression of cell surface and intracellular molecules [40–43]. It is selected to detect the apoptosis of L-929 cells in different leaching solutions. The results of early apoptosis rate shows: negative group < coated group < uncoated group. The early apoptosis rate of uncoated group rise significantly (P < 0.05). Compared with the uncoated NiTi-SMA, the TiN-coating may protect the cells from apoptosis. Zhao et al. [23] found that compared with the bare NiTi
alloys, TiN coating significantly reduced the release of Ni ions from the alloys. The result of FCM test indicated that the TiN film may reduce the release of Ni2+ and improve the biocompatibility. To observe the morphology and quantity of L-929 cells adhering to the substrate directly, SEM was applicable. Three time points, considering about the stages of cell viability test, were chosen to assess the attachment, adhesion and spreading of L-929 cells on the coated and uncoated NiTi-SMA in this study. The 24 h detection period represents early attachment, the 48 h and 72 h detection period show the adhesion and spreading processes of fibroblasts on the tested surfaces. Cell adhesion was related to various natural phenomena such as embryogenesis, maintenance of tissue structure, wound healing, immune response, metastasis, and tissue integration of biomaterials [44]. The changes of cellular morphology are revealed in Fig. 5. The results of the cell morphologic examination were consistent with the conclusion reported by Lampin et al. [45]. They found that a slight roughness raised the migration area to an upper extent no matter which cell type and enhancement of the cell adhesion potential was related to the degree of roughness and the hydrophobicity. Groessner-Schreiber et al. [46] found that fibroblasts intimately adherent to the surface of TiN compared to the oxidized titanium surfaces. Fibroblast cells cultured on coated and uncoated NiTi-SMA were observed to exhibit polygonal (24 h), spindle–shaped (48 h) patterns and spreading stacked (72 h) with a well-developed cytoskeleton. Morphologic examination indicated that the number of cells on the TiN coating was larger than those on NiTi-SMA. These features show that the TiN layer on the NiTi-SMA has the ability to induce the fibroblasts attachment, spreading, proliferation and growth on their surfaces from RMPI 1640 medium.
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Fig. 5. SEM images show the surface morphologies of L-929 cells cultured on coated and uncoated NiTi-SMA at different detection period.
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5. Conclusion It seems that TiN coating presents greater roughness and good wettability which increases surface affinity, expected in cell adherence. TiN-coating has a good bonding strength which can bear a load of 4099 N after bending to a displacement of 2 mm. In MTT test, OD values represent that cell viability and proliferation were similar among the coated, uncoated and negative control group. MTT test results show no significant difference between five extraction times in both coated and uncoated group. However, the early apoptosis rates of TiN coated group were significantly lower than uncoated group. Cell attachment, spreading and proliferation were apparently enhanced on TiN coating, TiN coating modify the surface contents of NiTi-SMA and increase the roughness and wettability, which enhance the proliferation and fibroblast adherence. In addition, TiN coating could lead to a better biocompatibility on NiTiSMA, which provided valuable insights into the potential use of TiN coating on NiTi orthodontics archwires. Acknowledgements The authors are grateful for the supports from National Basic Research Program of China (973 Program, No. 2012CB619101) and Liaoning Province of China (20082088). We also appreciated the kind help from Central Laboratory, School of Stomatology, China Medical University, during cell culture. References [1] A. Falvo, F.M. Furgiuele, C. Maletta, Laser welding of a NiTi alloy: mechanical and shape memory behaviour, Mater. Sci. Eng. A 412 (2005) 235. [2] B.P. Khasenov, A.A. Kadnikov, D.I. Rabkin, Use in technology and medicine of TiNi alloys revealing the shape memory effect, Met. Sci. Heat Treat. 30 (1988) 300. [3] T.W. Duerig, A.R. Pelton, D. Stockel, The use of superelasticity in medicine, Metallurgy 50 (1996) 569. [4] T.W. Duerig, Present and future applications of shape memory and superelastic materials, Mater. Res. Soc. Symp. Proc. 360 (1995) 497. [5] L.S. Castleman, S.M. Motzkin, F.P. Alicandri, V.L. Bonawit, Biocompatibility of nitinol alloy as an implant material, J. Biomed. Mater. Res. 10 (1976) 695. [6] J. Ryhanen, E. Niemi, W. Serlo, E. Niemela, P. Sandvik, H. Pernu, T. Salo, Biocompatibility of nickel–titanium shape memory metal and its corrosion behavior in human cell cultures, J. Biomed. Mater. Res. 35 (1997) 451. [7] D.J. Wever, A.G. Veldhuizen, M.M. Sanders, J.M. Schakenraad, J.R. van Horn, Cytotoxic, allergic and genotoxic activity of a nickel–titanium alloy, Biomaterials 18 (1997) 1115. [8] W.V. Moorleghem, M. Chandrasekaran, D. Reynaerts, J. Peirs, H.V. Brussel, Shape memory and superelastic alloys: the new medical materials with growing demand, Biomed. Mater. Eng. 8 (1998) 55. [9] W.A. Brantley, T. Eliades, Orthodontic Materials: Scientific and Clinical Aspects, Thieme, 2001. [10] L. Torrisi, The NiTi superelastic alloy application to the dentistry field, Biomed. Mater. Eng. 9 (1999) 39. [11] R.P. Kusy, J.Q. Whitley, Thermal and mechanical characteristics of stainless steel, titanium–molybdenum, and nickel–titanium archwires, Am. J. Orthod. Dentofacial Orthop. 131 (2007) 229. [12] S. Piscanec, L.C. Ciacchi, E. Vesselli, G. Comelli, O. Sbaizero, S. Meriani, A. De Vita, Bioactivity of TiN-coated titanium implants, Acta Mater. 52 (2004) 1237. [13] M. Es-Souni, H. Fischer-Brandies, Assessing the biocompatibility of NiTi shape memory alloys used for medical applications, Anal. Bioanal. Chem. 381 (2005) 557. [14] D. Budziak, E. Martendal, E. Carasek, Preparation and characterization of new solid–phase microextraction fibers obtained by sol–gel technology and zirconium oxide electrodeposited on NiTi alloy, J. Chromatogr. A 1187 (2008) 34. [15] R. Matos de Souza, L. Macedo de Menezes, Nickel, chromium and iron levels in the saliva of patients with simulated fixed orthodontic appliances, Angle Orthod. 78 (2008) 345. [16] R. Fors, M. Persson, Nickel in dental plaque and saliva in patients with and without orthodontic appliances, Eur. J. Orthod. 28 (2006) 292.
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