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Hierarchical structures on nickel-titanium fabricated by ultrasonic nanocrystal surface modification
Materials Science & Engineering C 93 (2018) 12–20
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Hierarchical structures on nickel-titanium fabricated by ultrasonic nanocrystal surface modification
T
Xiaoning Houa, Steven Mankocib, Nicholas Waltersc, Hongyu Gaoc, Ruixia Zhanga, Shengxi Lid, Haifeng Qine, Zhencheng Rena, Gary L. Dolle, Hongbo Congd, Ashlie Martinic, ⁎ ⁎⁎ Vijay K. Vasudevanf, Xianfeng Zhoua,b,g, , Nita Sahaib,h,i, Yalin Donga, Chang Yea, a
Department of Mechanical Engineering, University of Akron, Akron, OH 44325, USA Department of Polymer Science, University of Akron, Akron, OH 44325, USA c Department of Mechanical Engineering, University of California - Merced, Merced, CA 95343, USA d Department of Chemical and Biomolecular Engineering, University of Akron, Akron, OH, USA e Timken Engineered Surfaces Laboratories, University of Akron, Akron, OH 44325, USA f Department of Mechanical and Materials Engineering, University of Cincinnati, Cincinnati, OH 45221, USA g School of Polymer Science and Engineering, Qingdao University of Science and Engineering, Qingdao 266042, China h Department of Geosciences, University of Akron, Akron, OH 44325, USA i Integrated Bioscience Program, University of Akron, Akron, OH 44325, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hierarchical structures Ultrasonic nanocrystal surface modification Biomedical implant Severe plastic deformation Biocompatibility Corrosion
Hierarchical structures on metallic implants can enhance the interaction between cells and implants and thus increase their biocompatibility. However, it is difficult to directly fabricate hierarchical structures on metallic implants. In this study, we used a simple one-step method, ultrasonic nanocrystal surface modification (UNSM), to fabricate hierarchical surface structures on a nickel-titanium (NiTi) alloy. During UNSM, a tungsten carbide ball hits metal surfaces at ultrasonic frequency. The overlapping of the ultrasonic strikes generates hierarchical structures with microscale grooves and embedded nanoscale wrinkles. Cell culture experiments showed that cells adhere better and grow more prolifically on the UNSM-treated samples. Compared with the untreated samples, the UNSM-treated samples have higher corrosion resistance. In addition, the surface hardness increased from 243 Hv to 296 Hv and the scratch hardness increased by 22%. Overall, the improved biocompatibility, higher corrosion resistance, and enhanced mechanical properties demonstrate that UNSM is a simple and effective method to process metallic implant materials.
1. Introduction A good implant material needs hierarchical surface structures that can act as scaffold with multiscale dimensions for cell adhesion and growth [1,2]. Mammal bones, for example, consist of different dimensions of collagen fibrils and mineral apatite particles [3]. The collagen fibrils, assembled collagen molecules in the form of higher-order polymers, normally have micro- or submicroscale lamellar structures that function as scaffold for cell growth [4,5]. The nanoscale apatite particles nucleate and grow in the gap zone of collagen fibrils [6]. These hierarchical structures provide a matrix for cell attachment, promote cell proliferation and differentiation, as well as meet the mechanical requirements for bone tissue. Inspired by natural hierarchical structures [7], many researchers ⁎
used artificial hierarchical structures to enhance cell-matrix interactions in tissue engineering. It has been reported that materials with hierarchical structures can improve biocompatibility of biomaterials [8,9]. However, fabricating hierarchical structures on biomaterials, especially metallic implants, is challenging. Most methods used for the fabrication of hierarchical structures involve building a macroscale template followed by the creation of nanoscale structures [8,10]. During these procedures, more than one fabrication techniques such as photolithography [11–13], sol–gel method [14,15], acid treatment [13,16], anodization [17,18], etc., are usually needed. For example, hierarchically ordered structures with multiple length scales were fabricated by combining micro-molding, polystyrene sphere templating, and cooperative assembly of inorganic sol-gel species with amphiphilic triblock copolymers [19]. These multi-step processes make fabrication
Corresponding author. Correspondence to: C. Ye, Department of Mechanical Engineering, University of Akron, Akron, OH 44325, USA. E-mail addresses: [email protected] (X. Zhou), [email protected] (C. Ye).
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https://doi.org/10.1016/j.msec.2018.07.032 Received 11 October 2017; Received in revised form 7 June 2018; Accepted 12 July 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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also used to characterize surface morphology. 2.3. Microstructure Cross-sections of the UNSM samples were ground up to 1200 grit with silicon carbide grinding paper before etching. After etching (with nitric acid and hydrofluoric acid at volume ratio of 3:5) for 1 to 2 s at room temperature, samples were immediately rinsed with flowing water. The microstructures were characterized with a digital optical microscope (MU130, AmScope). 2.4. In vitro cell viability Scheme 1. Schematic of the UNSM process.
To evaluate the effect of UNSM on cell survivability, cytotoxicity study was carried out using a LIVE/DEAD cell viability assay. At first, three samples from the control group and the UNSM group were ultrasonically cleaned in dichloromethane, acetone, ethanol, deionized water, and ethanol again for 5 min each, using a 3510 Branson ultrasonic cleaner with an ice-water mixture bath, followed by sterilization by ultraviolet radiation in a biosafety cabinet for 12 h. Adipose-derived stem cells (ADSCs, from Lonza®, Switzerland) at passage 5 were cultured in a regular growth medium (α-MEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin) at 37 °C in a 5% CO2 atmosphere for 72 h. After that, cells were harvested, seeded, and incubated on the cleaned samples that were already transferred into 24well cell culture plates. Cells within the regular growth medium were seeded at 5000 cells/cm2. After incubation for 24 h, 1 ml combined LIVE/DEAD cell staining solution (2 μM calcein AM and 4 μM EthD-1 in phosphate-buffered saline (PBS)) was added to each well and incubated for 5–10 min at room temperature. Images were recorded using an IX51 Epifluorescence microscope (Olympus Co., Japan) equipped with a fluorescent light source and filters. The viability was calculated by manually counting the number of live cells and dead cells. Five randomly selected images from each group were used for this calculation. This part of cell culture experiment was repeated twice. To examine cell adhesion and spread, cell patterns were examined using Millipore's Actin Cytoskeleton and Focal Adhesion Staining Kit (FAK100). At first, after the aforementioned cleaning and sterilizing steps, disk samples were transferred into 24-well plates, followed by seeding ADSCs on the 24-well plates at 5000 cells/cm2 and culturing for 48 h. Then, the cells were washed with PBS and fixed in 5% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. After the blocking solution was aspirated, the cells were washed with PBS twice, followed by treatment with anti-vinculin monoclonal antibody (diluted 1:500) for 1 h at room temperature for vinculin staining. Next, TRITCconjugated phalloidin (diluted 1:1000), in combination with a FITCtagged secondary antibody (diluted 1:1000), were used for 30 min at room temperature for F-actin cytoskeleton staining. Finally, nuclei were stained by DAPI (diluted 1:1000) for 5 min at room temperature. Cells were washed three times with PBS after each staining. Finally, the stained cells were imaged with the same IX51 Epifluorescence microscope. The area per cell and the ratio of cell length to width from the two groups were obtained after randomly counting ~30 cells from three images of each group with the ImageJ software. This part of cell culture experiment was repeated three times.
of hierarchical structures complex, costly, and time consuming. Additionally, for biomaterials, both biomedical and mechanical requirements should be considered. Therefore, it is desirable to fabricate hierarchical structures with a simple method, which at the same time can improve the mechanical properties of metallic implants. In this study, we report a simple one-step method, ultrasonic nanocrystal surface modification (UNSM), to fabricate hierarchical surface structures on Nickle-Titanium (NiTi). During the UNSM process (Scheme 1), a tungsten carbide ball attached to an ultrasonic device scans over the sample surface while striking it at high frequency (20 kHz). UNSM can be used to fabricate hierarchical surface pattern by manipulating the process parameters including tip diameter, strike intensity, and strike density, all of which can be precisely controlled. In addition to hierarchical surface structures, the grain refinement induced by UNSM is believed to promote cellular activity [20–23] and to improve mechanical properties [24–30]. A previous study showed that three-pass UNSM can generate an amorphous surface layer on NiTi [31]. In this study, by fabricating hierarchical surface structure on NiTi using UNSM with carefully selected process parameters, not only was the biocompatibility of the NiTi alloy enhanced, but also the corrosion performance and the mechanical properties were significantly improved. 2. Experimental section 2.1. Material preparation NiTi (50.4 at.% Ni, thickness 1.5 mm, active Af 54.6 °C) plates from Special Metals (WV, USA) were cut into disks with diameters of 15 mm. Disk samples ground up to 1200 grit with silicon carbide grinding paper (Buehler, Microcut®, 8 in.) were used as control. For the UNSM group, the disk samples were ground up to 1200 grit followed by UNSM treatment. For the cell culture study, the control samples were ground up to 800 grit to reduce the difference in surface roughness between the control and the UNSM groups. In this study, the UNSM experiment was carried out with the following conditions: a static load of 20 N, a vibration amplitude of 12 μm, a scanning speed (Scheme 1, V1) of 1000 mm/min and an interval (distance between neighboring scans) of 10 μm. The details of the process paths are shown in Scheme 1. In this study, the diameter of the UNSM-treated area was set as 15 mm, leaving an annulus with ~0.2 mm width on the edge not treated.
2.5. Corrosion test The corrosion behavior of NiTi samples was investigated using electrochemical testing, including open circuit potential (OCP), potentiodynamic polarization (PDP), and electrochemical impedance spectroscopy (EIS). All electrochemical measurements were carried out using a VersaSTAT4 electrochemical workstation (Princeton Applied Research) at room temperature. A standard three-electrode flat cell was used. The reference, counter, and working electrodes were a saturated calomel electrode (SCE, Hg/Hg2Cl2/KCl), a platinum mesh, and a test
2.2. Surface morphology The surface morphology of samples before and after UNSM was characterized using a Zygo NewView 7300 surface profiler. The scanning area was 0.18 mm × 0.13 mm for 400× magnification. Each scanning area generated a roughness average, Ra, of the surface, and five measurements were carried out to obtain the mean Ra. A scanning electron microscope (SEM) (LYRA3, TESCAN) operating at 20 kV was 13
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Table 1 Chemical composition of 1000 ml SBF. NaCl
NaHCO3
KCl
K2HPO4·3H2O
MgCl2·6H2O
HCl (1.0 M)
CaCl2
Na2SO4
Tris
HCl (1.0 M)
8.035 g
0.355 g
0.225 g
0.231 g
0.311 g
39 ml
0.292 g
0.072 g
6.118 g
0–5 ml
Fig. 1. (a) 2-D surface morphology of NiTi before and after UNSM (scanning area: 0.18 mm × 0.13 mm). (b) Height distribution (identified by the black dashed arrow) of the UNSM sample surface along the black dashed line in (a). The surface profile was split into waviness (blue curve) and nanoscale roughness (red curve). Groove shape was highlighted by the black dotted line. (c) SEM image of the nanoscale wrinkles within grooves (left) and height distribution of the wrinkles (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
sample, respectively. The exposed area of the sample surface was 1 cm2. Simulated body fluid (SBF) was used as the testing medium (pH value of 7.40 ± 0.05). The chemical composition of SBF was listed in Table 1 [32]. Each type of experiment was repeated more than three times for both the control and the UNSM groups to ensure reliability of the data. All samples were exposed to the SBF solution for 1800 s to obtain the OCP curves, followed by PDP testing (scan rate 0.5 mV/s and scan range from −0.3 to 1.5 V vs OCP) to obtain the corrosion potential, corrosion current density, and breakdown/pitting potential. EIS spectra were obtained with a sinusoidal AC potential wave of 20 mV in amplitude applied to the working electrode around OCP (obtained for a duration of 3600 s) at a frequency from 100 kHz to 10 mHz. The ZView software was employed to fit the EIS data using Randles equivalent circuit to obtain the impedance Rct.
friction from a sharp object was evaluated according to the ASTM G171-03(2009) Standard Test Method for Scratch Hardness. To minimize the effect of the difference of surface roughness on scratch hardness from the control and the UNSM samples, all samples were gently polished with 3-μm diamond polycrystalline diamond suspension before scratch testing. A linear reciprocating tribometer (Rtec Instruments Multi-Function Tribometer) with a rounded conical diamond tip with a radius of 200 μm and conical apex of 120° was used. A scratching speed of 0.2 mm/s, a stroke length of 1.5 mm, and a normal force of 100 N were applied in this study. Scratch hardness was calculated according to the following equation:
HSp = kP / w 2,
(1)
where, HSp is the scratch hardness (GPa), k is a geometrical constant (24.98), P is the applied normal force (gf), and w is the scratch width (μm). The average scratch hardness was calculated from an average of three scratches per specimen and three width measurement locations per scratch. The 3-D profiles of the scratches were measured using a white light interferometer mounted on the tribometer.
2.6. Hardness and scratch tests Vickers microhardness was measured on sample cross sections using a Wilson Tukon 1202 system. A load of 50 g and a holding time of 10 s were used. Five measurements were made to calculate the average surface microhardness for both the control and the UNSM groups. The resistance of the UNSM-treated samples to fracture due to 14
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Fig. 2. Microstructures of NiTi before and after UNSM. The optical images were obtained after etching the cross-section of samples for 2 s (left) and for 1 s (right), respectively. The dashed red lines represent the top surfaces of samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
manipulating the UNSM process parameters (scanning speed, interval, amplitude, and static load) [33]. Thus, we have demonstrated that UNSM can generate hierarchical structures with microscale grooves embedded with nanoscale wrinkles. Note that even though only NiTi was reported here, UNSM can also be used to generated hierarchical structures on other metals. Fig. 2 compares the cross-sectional microstructures of samples before and after UNSM. After etching the cross-section for 2 s (left column in Fig. 2), grain boundaries can be observed. Slightly refined grains (identified by white arrows in Fig. 2) can be observed in the top surface layer of the UNSM-treated sample. One interesting feature is that upon reducing the etching time to 1 s, only the grains in the near surface region were etched (right column in Fig. 2). Specifically, the UNSM group had a larger depth of etching compared with the control group. This might be caused by the high defect density in the UNSM-treated samples.
2.7. Phase identification To identify the phases of the samples, an X-ray diffraction (XRD) system (Rigaku Ultima IV) with a Cu-Kα radiation source (λ = 1.5418 Å), operated at a voltage of 40 kV and a current of 35 mA, was used to characterize the samples before and after UNSM treatment. XRD patterns were recorded at a scanning speed of 1°/min and a step size of 0.04°. The measured 2θ angle was from 30° to 80°. 3. Results and discussion 3.1. Surface morphology and microstructures Fig. 1 shows the surface morphology of NiTi before and after UNSM. The average Ra increased from 17.4 ± 1.1 nm (mean value ± standard deviation, the same below) for the control to 32.0 ± 2.8 nm after UNSM treatment. The control sample has a smooth surface while the UNSM-treated sample exhibits a groove-patterned surface morphology (Fig. 1a). The groove patterns were also confirmed by the height distribution curves in Fig. 1b. The surface height distribution from the UNSM group can be divided into two parts: waviness and nanoscale roughness (Fig. 1b). The waviness is due to the grooves generated by UNSM scanning in the direction paralleled with V1 in Scheme 1. In addition to the grooves, nanoscale wrinkles are observed within the grooves. The wrinkles and their nanoscale dimensions can be confirmed from the SEM image and the height distribution curve in Fig. 1c, which shows that the maximum peak-to-valley height of the wrinkles is only a few tens of nanometers. These nanoscale wrinkles were generated by the overlap of neighbor ultrasonic strikes. The feature size of the nanoscale wrinkles is controlled by the scanning speed and the ultrasonic frequency. Groove structures are generated by the ploughing effect during UNSM scanning, while the repeated, high frequency strikes can generate nanoscale wrinkles within the grooves. The feature size of these grooves and wrinkles can be controlled by
3.2. Cell viability evaluation in vitro To study the biocompatibility of NiTi after UNSM, ADSCs were used for cell culture study. Biocompatibility was evaluated based on the cell viability in vitro. Representative cell viability images of the two groups are shown in Fig. 3a and b. Compared with that on the control samples, fewer dead cells (red dots) were observed on the UNSM-treated samples. In addition, most cells on the control sample exhibited a round shape while cells on the UNSM-treated sample appeared to be spindleshaped. This indicates that the cells adhered better on the UNSMtreated surfaces than on the unprocessed surfaces [34,35] The average survival rate increased from 90.7 ± 3.2% to 98.6 ± 1.6% after UNSM. Focal adhesions (FAs) are specialized adhesive contacts between cells and extracellular matrix (ECM) [36]. FAs not only transmit mechanical forces that anchor the intracellular cytoskeleton to bound integrins and the ECM, but also function as signaling centers to regulate 15
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Fig. 3. Cell viability visualized by LIVE/DEAD cell staining (cells were cultured for 24 h) on control (a) and on UNSM (b) samples. The live cells were stained fluorescent green, and the dead cells were stained red. Scale bar: 100 μm. (c–j) Fluorescence microscopy of focal adhesions and actin cytoskeleton in ADSCs after culturing for 48 h on NiTi samples. Scale bar: 20 μm. (c, d) Nuclear counterstaining (blue) revealed with DAPI; (e, f) F-actin cytoskeleton (red) detected using TRITC-conjugated phalloidin; (g, h) focal contacts (green) revealed using anti-vinculin monoclonal antibody and a FITC-conjugated secondary antibody; (i, j) monochrome images of DAPI (blue), TRITC-conjugated phalloidin (red), and anti-vinculin (green) were overlaid and displayed in pseudo-colors. Left column: control; right column: UNSM. Cell area of each group was highlighted on (i, j) in the form of mean ± standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
numerous biochemical signal pathways between the interacting cells and ECM [36–38]. Therefore, FAs play a crucial role in regulating cell behaviors such as cell spreading, mobility, and growth [39–42]. Stronger cell-substrate adhesion usually leads to better cell morphology and more focal adhesion sites [43–45], leading to improved biocompatibility. Herein, better cell morphology was viewed as larger cell area and smaller cell length/width ratio. To evaluate the biocompatibility of UNSM-treated NiTi, ADSCs were cultured on the control and UNSM samples for 48 h. Then FAs were evaluated, and cell morphology was compared by analyzing the cell area and the cell length/width ratio. Fig. 3c–j shows the fluorescence microscopy images of cell morphology on the control and UNSM samples. The cell FAs (bright green dots) cultured on the control and the UNSM groups can be seen in Fig. 3g and h, respectively. Cells grow and spread better on the UNSM-treated samples than that on the untreated NiTi samples. After UNSM, the average area per cell on samples increased from 394.9 ± 146.4 μm2 to 556.2 ± 309.3 μm2, corresponding to an increase of 41%. However, this data is not statistically significant (p ≥ 0.05, single factor ANOVA) due to the high variation of the UNSM group. The average ratio of cell length to width decreased from 2.8 ± 1.0 to 2.1 ± 0.7 (p < 0.05, single factor ANOVA). The reduced length/width ratio of the cells on the UNSM-treated samples means that cells spread well without showing obvious preferred alignment, which can be directly confirmed from the more extended cells on the UNSM group (Fig. 3j) in comparison with the slender cells on the control group (Fig. 3i). Higher cell viability and better cell adhesion indicate that UNSM increased the biocompatibility of NiTi. Biocompatibility of surfaces is closely related to the interaction of cells with surfaces. It is recognized that several ECM proteins play a key role in the interaction of cells and surfaces. These proteins have a specific amino acid sequence, such as the Arginine-Glycine-Aspartic (RGD) loop, which provide binding sites for integrins that are embedded in cell membranes [46,47]. When a cell adheres or moves, thin actin-rich plasma-membrane protrusions, named filopodia, play a role in probing the environment [48]. Once filopodia find suitable binding sites, more integrins will accumulate in filopodia in that region to probe matrix, promoting cell adhesion and migration [49], and thus good biocompatibility of the matrix is shown. For NiTi alloys, the release of toxic Ni irons is considered to be the main cause of decreasing biocompatibility, but the formation of protective TiO2 layer on NiTi is beneficial of improving corrosion resistance and biocompatibility [50]. The enhanced adhesion of cells herein may benefit from the improved oxide passivation layer on NiTi arising from the UNSM treatment. This will be further discussed later in the corrosion section. Cell behaviors can be guided by surface properties of materials such as roughness [51], nanostructured surfaces [45,52,53], and feature types (e.g., micro- and nanoscale ridges and grooves) [54,55]. Different cells respond differently to nanoscale surface features [56]. Ponsonnet and co-workers [51] investigated fibroblast adhesion on NiTi with different roughness. Their results revealed that human fibroblast prefer to adhere and spread on the smoothest NiTi surface with Rz value (peak16
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Fig. 4. (a) The OCP profiles of the control and UNSM-processed NiTi in SBF solution. (b) Representative polarization Tafel curves of NiTi from the two groups. (c) Electrochemical parameters, icorr and Ecorr, obtained from the polarization curves of each group. (d) Nyquist plots of the control and UNSM-processed NiTi after EIS test in SBF.
to-valley roughness) of approximately 0.5 μm among all the mechanically ground samples. Different orientations of cells were observed on NiTi ground to Rz > 5 μm [51]. This observation is consistent with previously reported results by Clark and co-authors [57], who cultured cells on grooved substrates with different depths and pitches. Three types of cells (BHK cells, MDCK cells, and chick embryo cerebral neurons) showed oriented growth on surfaces with deeper grooves (2 μm). Both groove pitch and depth affected cell adhesion, although the depth of grooves affected cell adhesion more significantly [57]. Additionally, many studies have shown that hierarchical surfaces with both microscale and nanoscale structures can provide outstanding biocompatibility [18,52]. For instance, hierarchical micro-/nano-textured titanium surface topographies with titania nanotubes enhanced multiple osteoblast behaviors including cell adhesion and osteogenesisrelated gene expressions [18]. Attachment, spreading, adhesion, proliferation, and differentiation of osteoblasts were enhanced on titanium surfaces with TiO2 nanonodules and titanium micropit architectures [9]. Furthermore, even inherent antibacterial ability as well as osteogenic inducing activity were achieved on titanium surfaces with micro-/submciro-/nanoscale structures [58]. Compared with surfaces with microscale structures, anti-aging ability was improved on nanomicro-hierarchical titanium surfaces due to their ability to retain hydrophilicity and bioactivity [59]. In fact, not only surface morphology and exposed surface area, but hydrophilicity [13] and surface free energy [60] can change due to hierarchical structures. As a result, hierarchical structures with both micro-/submicro-structural features and nanoscale features enable the modulation of cell-surface interactions. In this study, UNSM not only generated a microscale groove pattern, but also generated wrinkles with nanoscale within each groove by tip strikes. The local average Ra of UNSM-treated NiTi is only ~32 nm (scanning area: 0.18 mm × 0.13 mm) with hierarchical structures. These features positively affected the final cell adhesion and spread in vitro. Specifically, improved biocompatibility was obtained in the
UNSM-treated group as evidenced by the higher cell viability and better cell morphology. The increased surface area due to hierarchical structures, for example, can present more binding sites to cell membrane receptors and thus enhance cellular activities [61,62]. Besides the improved biocompatibility, it is reported that a remarkable decrease in the adhesion of gram-positive bacteria (S. aureus and S. epidermidis) was realized on 316L stainless steel surface with nanoscale surface roughness generated by shot peening in comparison with that on the nontreated samples [21]. Therefore, the hierarchical surface of NiTi with nanoscale Ra (32.0 ± 2.8 nm) after UNSM may also provide the potential of antibacterial features. This would be investigated in our future work. 3.3. Corrosion resistance The corrosion performance of NiTi was studied using electrochemical tests. OCP, also known as open circuit voltage (OCV), is the electrical potential difference between the working electrode and the reference electrode when there is no current, potential, or external load in the standard cell. Normally, the metal with low OCP will dissolve faster in the electrolyte than the metal with high OCP. Fig. 4a illustrates the representative OCP curves for NiTi from the control and the UNSM groups obtained in SBF solution. It shows that the UNSM-processed NiTi has a higher OCP than the control sample, indicating that the UNSMprocessed NiTi is more corrosion resistant than the control sample. The representative electrochemical Tafel curves of the two groups (Fig. 4b) show that the corrosion resistance was improved after UNSM based on the enhanced corrosion potential (Ecorr) and the decreased corrosion current density (icorr). Specifically, as shown in Fig. 4c, Ecorr changed from −452 ± 29 mV/SCE to −270 ± 46 mV/SCE; icorr decreased from 2145 ± 643 nA/cm2 to 352 ± 225 nA/cm2, corresponding to an 84% reduction in corrosion current density. The increase of Ecorr and the decrease of icorr indicate that the UNSM-processed NiTi samples 17
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Fig. 5. (a) Hardness depth profile of NiTi. (b) Interferometer images of surfaces after scratch hardness testing on NiTi (left) and average scratch hardness measurements (right). (c) XRD patterns of NiTi before and after UNSM treatment.
plays a dominant role in the protection of NiTi stents against corrosion. Another study showed that NiTi has better corrosion resistance in both Hanks' solution and MEM solution than Ti and Ti-6Al-4V alloys because of the formation of a passive TiO2 film [64]. The freshly formed oxide layer was also evidenced by XPS measurements on NiTi after sandblasting [65]. Furthermore, a higher corrosion resistance was achieved on Ti–6Al–4V with refined grains (grain size ~ 50 nm) after surface mechanical attrition treatment (SMAT) due to the formation of a passivation layer [66]. Studies also found that compared with coarsegrained structures, Ti with ultra-fine grains had higher corrosion resistance due to the rapid formation of a passivation layer [67,68]. Thus, Ti-based alloys provide high corrosion resistance, mainly due to the passivation layer formed on sample surface. UNSM-induced grain refinement leads to an increase of grain boundaries that is beneficial to the formation of a thicker and more homogeneous passivation layers [68,69]. Thus, it can be inferred that the UNSM-treated samples, with hierarchical surfaces and refined grains, show higher corrosion resistance than the untreated samples probably because of the denser and more stable passive oxide film.
have higher corrosion resistance than the unprocessed samples. Furthermore, the breakdown potential (Eb) of NiTi, identified by the rapid increase of the anodic current density (circle area in Fig. 4b), also increased from 212.5 ± 41.9 mV/SCE to 293.3 ± 58.6 mV/SCE after UNSM. This means that pitting initiation on the UNSM-treated samples became more difficult compared with that in the untreated samples. The corrosion resistance of NiTi was also investigated using the EIS method. Fig. 4d shows representative Nyquist diagrams of the control and UNSM groups. It is apparent that the radius of the semicircle for the UNSM group is larger than that for the control sample, revealing enhanced corrosion resistance of NiTi after UNSM treatment. To further evaluate the corrosion resistance of NiTi with and without UNSM treatment, the Nyquist plots were fitted using Randles circuit (Rs(CdlRct)). In this model, Rs is the electrolyte resistance, Cdl is the capacitor properties of the interface between the electrode and its surrounding electrolyte, and Rct is the polarization/charge transfer resistance of the surface layer. The fitting result shows that the Rct of the UNSM-processed NiTi (1.18 × 105 ± 0.40 × 105 Ω) is almost twice as high as that of the unprocessed NiTi (6.80 × 104 ± 0.75 × 104 Ω). Generally, higher Rct indicates that the surface layer of the sample has higher resistance to its dissolution. Hence, the larger Rct from the UNSM group corresponds to higher corrosion resistance of the UNSM-treated NiTi compared with the untreated group. Trepanier and co-workers [63] demonstrated that the oxide layer
3.4. Hardness and phases As shown in Fig. 5a, the surface microhardness of NiTi increased from 243 ± 8 Hv to 296 ± 7 Hv after UNSM, corresponding to 22% 18
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References
improvement. During the UNSM process, plastic strain was generated on sample surface, resulting in the hierarchical structures and grain refinement. UNSM also leaded to a higher dislocation density beneath the hierarchical surface and thus higher hardness. The strengthening effect of UNSM weakened as it goes deeper into the material. This agrees well with a previous study on amorphous NiTi produced by three-pass UNSM [31]. Scratch hardness tests were carried out to evaluate the scratch resistance of the samples. Representative scratches on the control and the UNSM groups are shown in Fig. 5b (left). A higher average scratch hardness was observed for the UNSM group (Fig. 5b), indicating that the UNSM treatment increases the surface scratch resistance of NiTi. This is attributed to the grain refinement and higher dislocation density after UNSM. Fig. 5c shows the XRD patterns of the control and UNSM samples. Before UNSM, the main phases of NiTi used in this study were martensite with orthorhombic B19 and B19΄ structures and austenite with the cubic B2 structure. After UNSM, the broadening of multiple peaks from 38° to 45° makes it impossible to identify any peaks. This is a result of lattice distortion and decreased crystallinity [65]. Peaks with 2θ larger than 45°, indicating the phases of B19΄ and B2, disappeared after UNSM.
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4. Conclusions In this study, hierarchical structures with microscale grooves embedded with nanoscale wrinkles were fabricated on NiTi using UNSM. The changes of surface morphology and microstructures near the top surface after UNSM resulted in improved biocompatibility, corrosion resistance, and mechanical properties. The following conclusions can be drawn: 1) UNSM provides a simple one-step method to fabricate hierarchical surface structures on NiTi. 2) The biocompatibility of NiTi was improved after UNSM, which was mainly attributed to the hierarchical surface structures. 3) The corrosion resistance of the UNSM-processed NiTi was significantly improved, as confirmed by the higher corrosion potential, lower corrosion current density, and higher impedance in the electrochemical tests. 4) The hardness of NiTi was enhanced by UNSM, confirmed by both the in-depth hardness and the scratch hardness data. Thus, we have demonstrated that UNSM can fabricate hierarchical surface structures and refine grains that can enhance the biocompatibility, suppress the corrosion rate, and improve the mechanical properties of a biomedical NiTi alloy. By changing UNSM parameters such as applied load, tip diameter, distance between neighboring scans (interval), scanning speed, etc., hierarchical surfaces with different micro-/ submicroscale groove widths and depths and nanoscale wrinkles can be fabricated. Note that the capability to generated hierarchical microstructure using UNSM is not limited to NiTi alloys. UNSM can be used to generate hierarchical microstructure on other metals and alloys.
Acknowledgements The authors (X. Hou, R. Zhang, X. Zhou, Z. Ren, N. Sahai, Y. Dong and C. Ye) are grateful for the financial support of this research by the start-up funds provided by the College of Engineering and the College of Polymer Science and Engineering at The University of Akron. Scratch hardness experiments were performed with equipment purchased using funding from the US Army Research Office under contract/grant number W911NF1610549.
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Hierarchical structures on nickel-titanium fabricated by ultrasonic nanocrystal surface modification
T
Xiaoning Houa, Steven Mankocib, Nicholas Waltersc, Hongyu Gaoc, Ruixia Zhanga, Shengxi Lid, Haifeng Qine, Zhencheng Rena, Gary L. Dolle, Hongbo Congd, Ashlie Martinic, ⁎ ⁎⁎ Vijay K. Vasudevanf, Xianfeng Zhoua,b,g, , Nita Sahaib,h,i, Yalin Donga, Chang Yea, a
Department of Mechanical Engineering, University of Akron, Akron, OH 44325, USA Department of Polymer Science, University of Akron, Akron, OH 44325, USA c Department of Mechanical Engineering, University of California - Merced, Merced, CA 95343, USA d Department of Chemical and Biomolecular Engineering, University of Akron, Akron, OH, USA e Timken Engineered Surfaces Laboratories, University of Akron, Akron, OH 44325, USA f Department of Mechanical and Materials Engineering, University of Cincinnati, Cincinnati, OH 45221, USA g School of Polymer Science and Engineering, Qingdao University of Science and Engineering, Qingdao 266042, China h Department of Geosciences, University of Akron, Akron, OH 44325, USA i Integrated Bioscience Program, University of Akron, Akron, OH 44325, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hierarchical structures Ultrasonic nanocrystal surface modification Biomedical implant Severe plastic deformation Biocompatibility Corrosion
Hierarchical structures on metallic implants can enhance the interaction between cells and implants and thus increase their biocompatibility. However, it is difficult to directly fabricate hierarchical structures on metallic implants. In this study, we used a simple one-step method, ultrasonic nanocrystal surface modification (UNSM), to fabricate hierarchical surface structures on a nickel-titanium (NiTi) alloy. During UNSM, a tungsten carbide ball hits metal surfaces at ultrasonic frequency. The overlapping of the ultrasonic strikes generates hierarchical structures with microscale grooves and embedded nanoscale wrinkles. Cell culture experiments showed that cells adhere better and grow more prolifically on the UNSM-treated samples. Compared with the untreated samples, the UNSM-treated samples have higher corrosion resistance. In addition, the surface hardness increased from 243 Hv to 296 Hv and the scratch hardness increased by 22%. Overall, the improved biocompatibility, higher corrosion resistance, and enhanced mechanical properties demonstrate that UNSM is a simple and effective method to process metallic implant materials.
1. Introduction A good implant material needs hierarchical surface structures that can act as scaffold with multiscale dimensions for cell adhesion and growth [1,2]. Mammal bones, for example, consist of different dimensions of collagen fibrils and mineral apatite particles [3]. The collagen fibrils, assembled collagen molecules in the form of higher-order polymers, normally have micro- or submicroscale lamellar structures that function as scaffold for cell growth [4,5]. The nanoscale apatite particles nucleate and grow in the gap zone of collagen fibrils [6]. These hierarchical structures provide a matrix for cell attachment, promote cell proliferation and differentiation, as well as meet the mechanical requirements for bone tissue. Inspired by natural hierarchical structures [7], many researchers ⁎
used artificial hierarchical structures to enhance cell-matrix interactions in tissue engineering. It has been reported that materials with hierarchical structures can improve biocompatibility of biomaterials [8,9]. However, fabricating hierarchical structures on biomaterials, especially metallic implants, is challenging. Most methods used for the fabrication of hierarchical structures involve building a macroscale template followed by the creation of nanoscale structures [8,10]. During these procedures, more than one fabrication techniques such as photolithography [11–13], sol–gel method [14,15], acid treatment [13,16], anodization [17,18], etc., are usually needed. For example, hierarchically ordered structures with multiple length scales were fabricated by combining micro-molding, polystyrene sphere templating, and cooperative assembly of inorganic sol-gel species with amphiphilic triblock copolymers [19]. These multi-step processes make fabrication
Corresponding author. Correspondence to: C. Ye, Department of Mechanical Engineering, University of Akron, Akron, OH 44325, USA. E-mail addresses: [email protected] (X. Zhou), [email protected] (C. Ye).
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https://doi.org/10.1016/j.msec.2018.07.032 Received 11 October 2017; Received in revised form 7 June 2018; Accepted 12 July 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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also used to characterize surface morphology. 2.3. Microstructure Cross-sections of the UNSM samples were ground up to 1200 grit with silicon carbide grinding paper before etching. After etching (with nitric acid and hydrofluoric acid at volume ratio of 3:5) for 1 to 2 s at room temperature, samples were immediately rinsed with flowing water. The microstructures were characterized with a digital optical microscope (MU130, AmScope). 2.4. In vitro cell viability Scheme 1. Schematic of the UNSM process.
To evaluate the effect of UNSM on cell survivability, cytotoxicity study was carried out using a LIVE/DEAD cell viability assay. At first, three samples from the control group and the UNSM group were ultrasonically cleaned in dichloromethane, acetone, ethanol, deionized water, and ethanol again for 5 min each, using a 3510 Branson ultrasonic cleaner with an ice-water mixture bath, followed by sterilization by ultraviolet radiation in a biosafety cabinet for 12 h. Adipose-derived stem cells (ADSCs, from Lonza®, Switzerland) at passage 5 were cultured in a regular growth medium (α-MEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin) at 37 °C in a 5% CO2 atmosphere for 72 h. After that, cells were harvested, seeded, and incubated on the cleaned samples that were already transferred into 24well cell culture plates. Cells within the regular growth medium were seeded at 5000 cells/cm2. After incubation for 24 h, 1 ml combined LIVE/DEAD cell staining solution (2 μM calcein AM and 4 μM EthD-1 in phosphate-buffered saline (PBS)) was added to each well and incubated for 5–10 min at room temperature. Images were recorded using an IX51 Epifluorescence microscope (Olympus Co., Japan) equipped with a fluorescent light source and filters. The viability was calculated by manually counting the number of live cells and dead cells. Five randomly selected images from each group were used for this calculation. This part of cell culture experiment was repeated twice. To examine cell adhesion and spread, cell patterns were examined using Millipore's Actin Cytoskeleton and Focal Adhesion Staining Kit (FAK100). At first, after the aforementioned cleaning and sterilizing steps, disk samples were transferred into 24-well plates, followed by seeding ADSCs on the 24-well plates at 5000 cells/cm2 and culturing for 48 h. Then, the cells were washed with PBS and fixed in 5% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. After the blocking solution was aspirated, the cells were washed with PBS twice, followed by treatment with anti-vinculin monoclonal antibody (diluted 1:500) for 1 h at room temperature for vinculin staining. Next, TRITCconjugated phalloidin (diluted 1:1000), in combination with a FITCtagged secondary antibody (diluted 1:1000), were used for 30 min at room temperature for F-actin cytoskeleton staining. Finally, nuclei were stained by DAPI (diluted 1:1000) for 5 min at room temperature. Cells were washed three times with PBS after each staining. Finally, the stained cells were imaged with the same IX51 Epifluorescence microscope. The area per cell and the ratio of cell length to width from the two groups were obtained after randomly counting ~30 cells from three images of each group with the ImageJ software. This part of cell culture experiment was repeated three times.
of hierarchical structures complex, costly, and time consuming. Additionally, for biomaterials, both biomedical and mechanical requirements should be considered. Therefore, it is desirable to fabricate hierarchical structures with a simple method, which at the same time can improve the mechanical properties of metallic implants. In this study, we report a simple one-step method, ultrasonic nanocrystal surface modification (UNSM), to fabricate hierarchical surface structures on Nickle-Titanium (NiTi). During the UNSM process (Scheme 1), a tungsten carbide ball attached to an ultrasonic device scans over the sample surface while striking it at high frequency (20 kHz). UNSM can be used to fabricate hierarchical surface pattern by manipulating the process parameters including tip diameter, strike intensity, and strike density, all of which can be precisely controlled. In addition to hierarchical surface structures, the grain refinement induced by UNSM is believed to promote cellular activity [20–23] and to improve mechanical properties [24–30]. A previous study showed that three-pass UNSM can generate an amorphous surface layer on NiTi [31]. In this study, by fabricating hierarchical surface structure on NiTi using UNSM with carefully selected process parameters, not only was the biocompatibility of the NiTi alloy enhanced, but also the corrosion performance and the mechanical properties were significantly improved. 2. Experimental section 2.1. Material preparation NiTi (50.4 at.% Ni, thickness 1.5 mm, active Af 54.6 °C) plates from Special Metals (WV, USA) were cut into disks with diameters of 15 mm. Disk samples ground up to 1200 grit with silicon carbide grinding paper (Buehler, Microcut®, 8 in.) were used as control. For the UNSM group, the disk samples were ground up to 1200 grit followed by UNSM treatment. For the cell culture study, the control samples were ground up to 800 grit to reduce the difference in surface roughness between the control and the UNSM groups. In this study, the UNSM experiment was carried out with the following conditions: a static load of 20 N, a vibration amplitude of 12 μm, a scanning speed (Scheme 1, V1) of 1000 mm/min and an interval (distance between neighboring scans) of 10 μm. The details of the process paths are shown in Scheme 1. In this study, the diameter of the UNSM-treated area was set as 15 mm, leaving an annulus with ~0.2 mm width on the edge not treated.
2.5. Corrosion test The corrosion behavior of NiTi samples was investigated using electrochemical testing, including open circuit potential (OCP), potentiodynamic polarization (PDP), and electrochemical impedance spectroscopy (EIS). All electrochemical measurements were carried out using a VersaSTAT4 electrochemical workstation (Princeton Applied Research) at room temperature. A standard three-electrode flat cell was used. The reference, counter, and working electrodes were a saturated calomel electrode (SCE, Hg/Hg2Cl2/KCl), a platinum mesh, and a test
2.2. Surface morphology The surface morphology of samples before and after UNSM was characterized using a Zygo NewView 7300 surface profiler. The scanning area was 0.18 mm × 0.13 mm for 400× magnification. Each scanning area generated a roughness average, Ra, of the surface, and five measurements were carried out to obtain the mean Ra. A scanning electron microscope (SEM) (LYRA3, TESCAN) operating at 20 kV was 13
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Table 1 Chemical composition of 1000 ml SBF. NaCl
NaHCO3
KCl
K2HPO4·3H2O
MgCl2·6H2O
HCl (1.0 M)
CaCl2
Na2SO4
Tris
HCl (1.0 M)
8.035 g
0.355 g
0.225 g
0.231 g
0.311 g
39 ml
0.292 g
0.072 g
6.118 g
0–5 ml
Fig. 1. (a) 2-D surface morphology of NiTi before and after UNSM (scanning area: 0.18 mm × 0.13 mm). (b) Height distribution (identified by the black dashed arrow) of the UNSM sample surface along the black dashed line in (a). The surface profile was split into waviness (blue curve) and nanoscale roughness (red curve). Groove shape was highlighted by the black dotted line. (c) SEM image of the nanoscale wrinkles within grooves (left) and height distribution of the wrinkles (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
sample, respectively. The exposed area of the sample surface was 1 cm2. Simulated body fluid (SBF) was used as the testing medium (pH value of 7.40 ± 0.05). The chemical composition of SBF was listed in Table 1 [32]. Each type of experiment was repeated more than three times for both the control and the UNSM groups to ensure reliability of the data. All samples were exposed to the SBF solution for 1800 s to obtain the OCP curves, followed by PDP testing (scan rate 0.5 mV/s and scan range from −0.3 to 1.5 V vs OCP) to obtain the corrosion potential, corrosion current density, and breakdown/pitting potential. EIS spectra were obtained with a sinusoidal AC potential wave of 20 mV in amplitude applied to the working electrode around OCP (obtained for a duration of 3600 s) at a frequency from 100 kHz to 10 mHz. The ZView software was employed to fit the EIS data using Randles equivalent circuit to obtain the impedance Rct.
friction from a sharp object was evaluated according to the ASTM G171-03(2009) Standard Test Method for Scratch Hardness. To minimize the effect of the difference of surface roughness on scratch hardness from the control and the UNSM samples, all samples were gently polished with 3-μm diamond polycrystalline diamond suspension before scratch testing. A linear reciprocating tribometer (Rtec Instruments Multi-Function Tribometer) with a rounded conical diamond tip with a radius of 200 μm and conical apex of 120° was used. A scratching speed of 0.2 mm/s, a stroke length of 1.5 mm, and a normal force of 100 N were applied in this study. Scratch hardness was calculated according to the following equation:
HSp = kP / w 2,
(1)
where, HSp is the scratch hardness (GPa), k is a geometrical constant (24.98), P is the applied normal force (gf), and w is the scratch width (μm). The average scratch hardness was calculated from an average of three scratches per specimen and three width measurement locations per scratch. The 3-D profiles of the scratches were measured using a white light interferometer mounted on the tribometer.
2.6. Hardness and scratch tests Vickers microhardness was measured on sample cross sections using a Wilson Tukon 1202 system. A load of 50 g and a holding time of 10 s were used. Five measurements were made to calculate the average surface microhardness for both the control and the UNSM groups. The resistance of the UNSM-treated samples to fracture due to 14
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Fig. 2. Microstructures of NiTi before and after UNSM. The optical images were obtained after etching the cross-section of samples for 2 s (left) and for 1 s (right), respectively. The dashed red lines represent the top surfaces of samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
manipulating the UNSM process parameters (scanning speed, interval, amplitude, and static load) [33]. Thus, we have demonstrated that UNSM can generate hierarchical structures with microscale grooves embedded with nanoscale wrinkles. Note that even though only NiTi was reported here, UNSM can also be used to generated hierarchical structures on other metals. Fig. 2 compares the cross-sectional microstructures of samples before and after UNSM. After etching the cross-section for 2 s (left column in Fig. 2), grain boundaries can be observed. Slightly refined grains (identified by white arrows in Fig. 2) can be observed in the top surface layer of the UNSM-treated sample. One interesting feature is that upon reducing the etching time to 1 s, only the grains in the near surface region were etched (right column in Fig. 2). Specifically, the UNSM group had a larger depth of etching compared with the control group. This might be caused by the high defect density in the UNSM-treated samples.
2.7. Phase identification To identify the phases of the samples, an X-ray diffraction (XRD) system (Rigaku Ultima IV) with a Cu-Kα radiation source (λ = 1.5418 Å), operated at a voltage of 40 kV and a current of 35 mA, was used to characterize the samples before and after UNSM treatment. XRD patterns were recorded at a scanning speed of 1°/min and a step size of 0.04°. The measured 2θ angle was from 30° to 80°. 3. Results and discussion 3.1. Surface morphology and microstructures Fig. 1 shows the surface morphology of NiTi before and after UNSM. The average Ra increased from 17.4 ± 1.1 nm (mean value ± standard deviation, the same below) for the control to 32.0 ± 2.8 nm after UNSM treatment. The control sample has a smooth surface while the UNSM-treated sample exhibits a groove-patterned surface morphology (Fig. 1a). The groove patterns were also confirmed by the height distribution curves in Fig. 1b. The surface height distribution from the UNSM group can be divided into two parts: waviness and nanoscale roughness (Fig. 1b). The waviness is due to the grooves generated by UNSM scanning in the direction paralleled with V1 in Scheme 1. In addition to the grooves, nanoscale wrinkles are observed within the grooves. The wrinkles and their nanoscale dimensions can be confirmed from the SEM image and the height distribution curve in Fig. 1c, which shows that the maximum peak-to-valley height of the wrinkles is only a few tens of nanometers. These nanoscale wrinkles were generated by the overlap of neighbor ultrasonic strikes. The feature size of the nanoscale wrinkles is controlled by the scanning speed and the ultrasonic frequency. Groove structures are generated by the ploughing effect during UNSM scanning, while the repeated, high frequency strikes can generate nanoscale wrinkles within the grooves. The feature size of these grooves and wrinkles can be controlled by
3.2. Cell viability evaluation in vitro To study the biocompatibility of NiTi after UNSM, ADSCs were used for cell culture study. Biocompatibility was evaluated based on the cell viability in vitro. Representative cell viability images of the two groups are shown in Fig. 3a and b. Compared with that on the control samples, fewer dead cells (red dots) were observed on the UNSM-treated samples. In addition, most cells on the control sample exhibited a round shape while cells on the UNSM-treated sample appeared to be spindleshaped. This indicates that the cells adhered better on the UNSMtreated surfaces than on the unprocessed surfaces [34,35] The average survival rate increased from 90.7 ± 3.2% to 98.6 ± 1.6% after UNSM. Focal adhesions (FAs) are specialized adhesive contacts between cells and extracellular matrix (ECM) [36]. FAs not only transmit mechanical forces that anchor the intracellular cytoskeleton to bound integrins and the ECM, but also function as signaling centers to regulate 15
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Fig. 3. Cell viability visualized by LIVE/DEAD cell staining (cells were cultured for 24 h) on control (a) and on UNSM (b) samples. The live cells were stained fluorescent green, and the dead cells were stained red. Scale bar: 100 μm. (c–j) Fluorescence microscopy of focal adhesions and actin cytoskeleton in ADSCs after culturing for 48 h on NiTi samples. Scale bar: 20 μm. (c, d) Nuclear counterstaining (blue) revealed with DAPI; (e, f) F-actin cytoskeleton (red) detected using TRITC-conjugated phalloidin; (g, h) focal contacts (green) revealed using anti-vinculin monoclonal antibody and a FITC-conjugated secondary antibody; (i, j) monochrome images of DAPI (blue), TRITC-conjugated phalloidin (red), and anti-vinculin (green) were overlaid and displayed in pseudo-colors. Left column: control; right column: UNSM. Cell area of each group was highlighted on (i, j) in the form of mean ± standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
numerous biochemical signal pathways between the interacting cells and ECM [36–38]. Therefore, FAs play a crucial role in regulating cell behaviors such as cell spreading, mobility, and growth [39–42]. Stronger cell-substrate adhesion usually leads to better cell morphology and more focal adhesion sites [43–45], leading to improved biocompatibility. Herein, better cell morphology was viewed as larger cell area and smaller cell length/width ratio. To evaluate the biocompatibility of UNSM-treated NiTi, ADSCs were cultured on the control and UNSM samples for 48 h. Then FAs were evaluated, and cell morphology was compared by analyzing the cell area and the cell length/width ratio. Fig. 3c–j shows the fluorescence microscopy images of cell morphology on the control and UNSM samples. The cell FAs (bright green dots) cultured on the control and the UNSM groups can be seen in Fig. 3g and h, respectively. Cells grow and spread better on the UNSM-treated samples than that on the untreated NiTi samples. After UNSM, the average area per cell on samples increased from 394.9 ± 146.4 μm2 to 556.2 ± 309.3 μm2, corresponding to an increase of 41%. However, this data is not statistically significant (p ≥ 0.05, single factor ANOVA) due to the high variation of the UNSM group. The average ratio of cell length to width decreased from 2.8 ± 1.0 to 2.1 ± 0.7 (p < 0.05, single factor ANOVA). The reduced length/width ratio of the cells on the UNSM-treated samples means that cells spread well without showing obvious preferred alignment, which can be directly confirmed from the more extended cells on the UNSM group (Fig. 3j) in comparison with the slender cells on the control group (Fig. 3i). Higher cell viability and better cell adhesion indicate that UNSM increased the biocompatibility of NiTi. Biocompatibility of surfaces is closely related to the interaction of cells with surfaces. It is recognized that several ECM proteins play a key role in the interaction of cells and surfaces. These proteins have a specific amino acid sequence, such as the Arginine-Glycine-Aspartic (RGD) loop, which provide binding sites for integrins that are embedded in cell membranes [46,47]. When a cell adheres or moves, thin actin-rich plasma-membrane protrusions, named filopodia, play a role in probing the environment [48]. Once filopodia find suitable binding sites, more integrins will accumulate in filopodia in that region to probe matrix, promoting cell adhesion and migration [49], and thus good biocompatibility of the matrix is shown. For NiTi alloys, the release of toxic Ni irons is considered to be the main cause of decreasing biocompatibility, but the formation of protective TiO2 layer on NiTi is beneficial of improving corrosion resistance and biocompatibility [50]. The enhanced adhesion of cells herein may benefit from the improved oxide passivation layer on NiTi arising from the UNSM treatment. This will be further discussed later in the corrosion section. Cell behaviors can be guided by surface properties of materials such as roughness [51], nanostructured surfaces [45,52,53], and feature types (e.g., micro- and nanoscale ridges and grooves) [54,55]. Different cells respond differently to nanoscale surface features [56]. Ponsonnet and co-workers [51] investigated fibroblast adhesion on NiTi with different roughness. Their results revealed that human fibroblast prefer to adhere and spread on the smoothest NiTi surface with Rz value (peak16
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Fig. 4. (a) The OCP profiles of the control and UNSM-processed NiTi in SBF solution. (b) Representative polarization Tafel curves of NiTi from the two groups. (c) Electrochemical parameters, icorr and Ecorr, obtained from the polarization curves of each group. (d) Nyquist plots of the control and UNSM-processed NiTi after EIS test in SBF.
to-valley roughness) of approximately 0.5 μm among all the mechanically ground samples. Different orientations of cells were observed on NiTi ground to Rz > 5 μm [51]. This observation is consistent with previously reported results by Clark and co-authors [57], who cultured cells on grooved substrates with different depths and pitches. Three types of cells (BHK cells, MDCK cells, and chick embryo cerebral neurons) showed oriented growth on surfaces with deeper grooves (2 μm). Both groove pitch and depth affected cell adhesion, although the depth of grooves affected cell adhesion more significantly [57]. Additionally, many studies have shown that hierarchical surfaces with both microscale and nanoscale structures can provide outstanding biocompatibility [18,52]. For instance, hierarchical micro-/nano-textured titanium surface topographies with titania nanotubes enhanced multiple osteoblast behaviors including cell adhesion and osteogenesisrelated gene expressions [18]. Attachment, spreading, adhesion, proliferation, and differentiation of osteoblasts were enhanced on titanium surfaces with TiO2 nanonodules and titanium micropit architectures [9]. Furthermore, even inherent antibacterial ability as well as osteogenic inducing activity were achieved on titanium surfaces with micro-/submciro-/nanoscale structures [58]. Compared with surfaces with microscale structures, anti-aging ability was improved on nanomicro-hierarchical titanium surfaces due to their ability to retain hydrophilicity and bioactivity [59]. In fact, not only surface morphology and exposed surface area, but hydrophilicity [13] and surface free energy [60] can change due to hierarchical structures. As a result, hierarchical structures with both micro-/submicro-structural features and nanoscale features enable the modulation of cell-surface interactions. In this study, UNSM not only generated a microscale groove pattern, but also generated wrinkles with nanoscale within each groove by tip strikes. The local average Ra of UNSM-treated NiTi is only ~32 nm (scanning area: 0.18 mm × 0.13 mm) with hierarchical structures. These features positively affected the final cell adhesion and spread in vitro. Specifically, improved biocompatibility was obtained in the
UNSM-treated group as evidenced by the higher cell viability and better cell morphology. The increased surface area due to hierarchical structures, for example, can present more binding sites to cell membrane receptors and thus enhance cellular activities [61,62]. Besides the improved biocompatibility, it is reported that a remarkable decrease in the adhesion of gram-positive bacteria (S. aureus and S. epidermidis) was realized on 316L stainless steel surface with nanoscale surface roughness generated by shot peening in comparison with that on the nontreated samples [21]. Therefore, the hierarchical surface of NiTi with nanoscale Ra (32.0 ± 2.8 nm) after UNSM may also provide the potential of antibacterial features. This would be investigated in our future work. 3.3. Corrosion resistance The corrosion performance of NiTi was studied using electrochemical tests. OCP, also known as open circuit voltage (OCV), is the electrical potential difference between the working electrode and the reference electrode when there is no current, potential, or external load in the standard cell. Normally, the metal with low OCP will dissolve faster in the electrolyte than the metal with high OCP. Fig. 4a illustrates the representative OCP curves for NiTi from the control and the UNSM groups obtained in SBF solution. It shows that the UNSM-processed NiTi has a higher OCP than the control sample, indicating that the UNSMprocessed NiTi is more corrosion resistant than the control sample. The representative electrochemical Tafel curves of the two groups (Fig. 4b) show that the corrosion resistance was improved after UNSM based on the enhanced corrosion potential (Ecorr) and the decreased corrosion current density (icorr). Specifically, as shown in Fig. 4c, Ecorr changed from −452 ± 29 mV/SCE to −270 ± 46 mV/SCE; icorr decreased from 2145 ± 643 nA/cm2 to 352 ± 225 nA/cm2, corresponding to an 84% reduction in corrosion current density. The increase of Ecorr and the decrease of icorr indicate that the UNSM-processed NiTi samples 17
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Fig. 5. (a) Hardness depth profile of NiTi. (b) Interferometer images of surfaces after scratch hardness testing on NiTi (left) and average scratch hardness measurements (right). (c) XRD patterns of NiTi before and after UNSM treatment.
plays a dominant role in the protection of NiTi stents against corrosion. Another study showed that NiTi has better corrosion resistance in both Hanks' solution and MEM solution than Ti and Ti-6Al-4V alloys because of the formation of a passive TiO2 film [64]. The freshly formed oxide layer was also evidenced by XPS measurements on NiTi after sandblasting [65]. Furthermore, a higher corrosion resistance was achieved on Ti–6Al–4V with refined grains (grain size ~ 50 nm) after surface mechanical attrition treatment (SMAT) due to the formation of a passivation layer [66]. Studies also found that compared with coarsegrained structures, Ti with ultra-fine grains had higher corrosion resistance due to the rapid formation of a passivation layer [67,68]. Thus, Ti-based alloys provide high corrosion resistance, mainly due to the passivation layer formed on sample surface. UNSM-induced grain refinement leads to an increase of grain boundaries that is beneficial to the formation of a thicker and more homogeneous passivation layers [68,69]. Thus, it can be inferred that the UNSM-treated samples, with hierarchical surfaces and refined grains, show higher corrosion resistance than the untreated samples probably because of the denser and more stable passive oxide film.
have higher corrosion resistance than the unprocessed samples. Furthermore, the breakdown potential (Eb) of NiTi, identified by the rapid increase of the anodic current density (circle area in Fig. 4b), also increased from 212.5 ± 41.9 mV/SCE to 293.3 ± 58.6 mV/SCE after UNSM. This means that pitting initiation on the UNSM-treated samples became more difficult compared with that in the untreated samples. The corrosion resistance of NiTi was also investigated using the EIS method. Fig. 4d shows representative Nyquist diagrams of the control and UNSM groups. It is apparent that the radius of the semicircle for the UNSM group is larger than that for the control sample, revealing enhanced corrosion resistance of NiTi after UNSM treatment. To further evaluate the corrosion resistance of NiTi with and without UNSM treatment, the Nyquist plots were fitted using Randles circuit (Rs(CdlRct)). In this model, Rs is the electrolyte resistance, Cdl is the capacitor properties of the interface between the electrode and its surrounding electrolyte, and Rct is the polarization/charge transfer resistance of the surface layer. The fitting result shows that the Rct of the UNSM-processed NiTi (1.18 × 105 ± 0.40 × 105 Ω) is almost twice as high as that of the unprocessed NiTi (6.80 × 104 ± 0.75 × 104 Ω). Generally, higher Rct indicates that the surface layer of the sample has higher resistance to its dissolution. Hence, the larger Rct from the UNSM group corresponds to higher corrosion resistance of the UNSM-treated NiTi compared with the untreated group. Trepanier and co-workers [63] demonstrated that the oxide layer
3.4. Hardness and phases As shown in Fig. 5a, the surface microhardness of NiTi increased from 243 ± 8 Hv to 296 ± 7 Hv after UNSM, corresponding to 22% 18
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References
improvement. During the UNSM process, plastic strain was generated on sample surface, resulting in the hierarchical structures and grain refinement. UNSM also leaded to a higher dislocation density beneath the hierarchical surface and thus higher hardness. The strengthening effect of UNSM weakened as it goes deeper into the material. This agrees well with a previous study on amorphous NiTi produced by three-pass UNSM [31]. Scratch hardness tests were carried out to evaluate the scratch resistance of the samples. Representative scratches on the control and the UNSM groups are shown in Fig. 5b (left). A higher average scratch hardness was observed for the UNSM group (Fig. 5b), indicating that the UNSM treatment increases the surface scratch resistance of NiTi. This is attributed to the grain refinement and higher dislocation density after UNSM. Fig. 5c shows the XRD patterns of the control and UNSM samples. Before UNSM, the main phases of NiTi used in this study were martensite with orthorhombic B19 and B19΄ structures and austenite with the cubic B2 structure. After UNSM, the broadening of multiple peaks from 38° to 45° makes it impossible to identify any peaks. This is a result of lattice distortion and decreased crystallinity [65]. Peaks with 2θ larger than 45°, indicating the phases of B19΄ and B2, disappeared after UNSM.
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4. Conclusions In this study, hierarchical structures with microscale grooves embedded with nanoscale wrinkles were fabricated on NiTi using UNSM. The changes of surface morphology and microstructures near the top surface after UNSM resulted in improved biocompatibility, corrosion resistance, and mechanical properties. The following conclusions can be drawn: 1) UNSM provides a simple one-step method to fabricate hierarchical surface structures on NiTi. 2) The biocompatibility of NiTi was improved after UNSM, which was mainly attributed to the hierarchical surface structures. 3) The corrosion resistance of the UNSM-processed NiTi was significantly improved, as confirmed by the higher corrosion potential, lower corrosion current density, and higher impedance in the electrochemical tests. 4) The hardness of NiTi was enhanced by UNSM, confirmed by both the in-depth hardness and the scratch hardness data. Thus, we have demonstrated that UNSM can fabricate hierarchical surface structures and refine grains that can enhance the biocompatibility, suppress the corrosion rate, and improve the mechanical properties of a biomedical NiTi alloy. By changing UNSM parameters such as applied load, tip diameter, distance between neighboring scans (interval), scanning speed, etc., hierarchical surfaces with different micro-/ submicroscale groove widths and depths and nanoscale wrinkles can be fabricated. Note that the capability to generated hierarchical microstructure using UNSM is not limited to NiTi alloys. UNSM can be used to generate hierarchical microstructure on other metals and alloys.
Acknowledgements The authors (X. Hou, R. Zhang, X. Zhou, Z. Ren, N. Sahai, Y. Dong and C. Ye) are grateful for the financial support of this research by the start-up funds provided by the College of Engineering and the College of Polymer Science and Engineering at The University of Akron. Scratch hardness experiments were performed with equipment purchased using funding from the US Army Research Office under contract/grant number W911NF1610549.
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