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Surface modification of Ti6Al4V based on ultrasonic surface rolling processing and plasma nitriding for enhanced bone regeneration
Accepted Manuscript Surface modification of Ti6Al4V based on ultrasonic surface rolling processing and plasma nitriding for enhanced bone regeneration
Ke Ren, Wen Yue, Hongyu Zhang PII: DOI: Reference:
S0257-8972(18)30614-5 doi:10.1016/j.surfcoat.2018.06.039 SCT 23490
To appear in:
Surface & Coatings Technology
Received date: Revised date: Accepted date:
23 March 2018 5 June 2018 20 June 2018
Please cite this article as: Ke Ren, Wen Yue, Hongyu Zhang , Surface modification of Ti6Al4V based on ultrasonic surface rolling processing and plasma nitriding for enhanced bone regeneration. Sct (2018), doi:10.1016/j.surfcoat.2018.06.039
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ACCEPTED MANUSCRIPT Surface modification of Ti6Al4V based on ultrasonic surface rolling processing and plasma nitriding for enhanced bone regeneration Ke Ren a, Wen Yue a, *, Hongyu Zhang b, *
School of Engineering and Technology, China University of Geosciences (Beijing), Beijing
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100083, China b
State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua
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University, Beijing 100084, China
* Corresponding authors: [email protected] (W. Yue)
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[email protected] (H. Zhang)
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Abstract Titanium alloy has been widely used in dentistry, orthopedic surgery and other medical fields. Surface morphology and structure of titanium alloy play an important role in cell adhesion and proliferation. The main purpose of this study is to investigate the biocompatibility and
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osteogenesis performance of Ti6Al4V modified using ultrasonic surface rolling processing (USRP) and plasma nitriding (PN). The raw materials were nominated as untreated, and the Ti6Al4V samples treated by USRP (with a processing duration of 1 h and 3 h) and PN were
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named as USRP1, USRP2, USRP1+PN, and USRP2+PN, respectively. The Ti6Al4V samples were characterized by SEM, AFM, and surface profilometer for surface morphology and 3D
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surface topography, and USRP1+PN showed the highest surface roughness (268 nm). The water contact angle measurements demonstrated that USRP2 had more favorite wettability
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(75.7°). The XRD analysis indicated the enhancement of nanocrystallization for the samples
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treated by USRP and the formation of a nitride layer for the samples treated by USRP and PN.
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The interactions between the Ti6Al4V samples and MC3T3-E1 cells were investigated in vitro by SEM, CCK-8 test, alkaline phosphatase (ALP) activity assay, and Alizarin Red S
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staining. The SEM images and CCK-8 tests revealed that all the Ti6Al4V samples were biocompatible to MC3T3-E1 cells, and the results of ALP activity assay and Alizarin Red S staining showed a better osteogenesis performance for the samples treated by USRP and PN. The present study indicates that USRP and PN may act as an effective method for Ti6Al4V to induce bone regeneration.
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Keywords: titanium alloy; ultrasonic surface rolling processing; nitriding; biocompatibility;
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osteogenesis.
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1. Introduction Titanium and its alloys (Ti-alloys), e.g. Ti6Al4V, have been widely applied in orthopedic and dental implants due mainly to their excellent properties, such as robust mechanical properties, corrosion resistance, high strength to weight ratio, and biocompatibility [1-5]. However,
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Ti-alloys usually cannot adequately integrate with bone at the early stage after implantation because of their bioinert surface [6-8]. In order to enhance the surface activity of Ti-alloys based prostheses and to improve their survivorship, various surface modification techniques
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have been utilized [9], such as plasma spraying [10, 11], acid-etching [12] and micro-arc oxidation [13]. However, it is considered that the bonding strength between Ti-alloys and the
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coatings is relatively weak, which limits their life span [14, 15]. Occasionally, toxic metal ions from Ti-alloys based prostheses can be released as a result of the corrosive environment
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[16].
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Surface topographic modification is an important method to enhance the biocompatibility and
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osseointegration of the implants [8]. It has been shown that nano-structured surfaces can enhance cell adhesion, proliferation, differentiation and effective osseointegration [17-21].
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Ultrasonic surface rolling process (USRP) is a novel surface nanocrystallization technique based on severe plastic deformation [22], which is a metal forming process that can result in grain refinement by imposing high strain rates without considerably altering the overall dimension of the material [23-25]. Generally, USRP applies ultrasonic vibration energy to the material surface to generate plastic deformation, and a nanocrystalline layer is produced with enhanced surface hardness and tribological properties [26, 27]. Fan et al. reported that surface
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grain size and hardness of carbon steel were affected by the duration of ultrasonic impact treatment [28]. An increased processing time is one of the reasons that resulted in intensified grain refinement. Additionally, plasma nitriding (PN) has been proposed to be one of the most effective methods that can change surface morphology and structure of Ti-alloys and
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improve their mechanical properties [29]. PN can produce a compound layer of TiN on the top of the matrix and Ti2N beneath, with a hardness of 3000 and 1500 HV, respectively, and a nitriding layer thickness of 10 μm~100 μm [30]. She et al. investigated the effects of nitriding
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temperature on microstructures and vacuum tribological properties of PN-treated titanium, and they concluded that the samples nitrided at the temperature of 850 °C exhibited the most
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excellent wear resistance and the lowest friction coefficient [31]. In another study performed by She et al., USRP was introduced as a pre-treatment means of PN to effectively enhance
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the properties of AISI D2 die steel, e.g. hardness [32]. Therefore, a combination of USRP and
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PN can significantly enhance the mechanical properties of Ti-alloys, which is an important
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factor affecting the lifespan of biomaterials served in human environment. To the best of our knowledge, there have been no reports investigating the biocompatibility and osteogenesis
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performance of Ti-alloys with different nanocrystal surface and nitriding treatment. Therefore, it is considered that an insight into this area is valuable and may predict paramount prospect in a large number of biomedical applications. In the present study, the Ti6Al4V samples were treated by USRP (processing duration: 1 h and 3 h) and PN, and characterized using SEM, AFM, surface profilometer, water contact angle, and XRD for surface morphology, surface topography, wettability, and phase structure.
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Additionally, pre-osteoblastic murine cell lines MC3T3-E1 were employed to evaluate the biocompatibility and osteogenesis properties of the Ti6Al4V samples in vitro by cell adhesion, proliferation, and differentiation. It is hypothesized that the USRP and PN treatments can greatly facilitate osteogenesis and act as potential techniques to enhance bone regeneration of
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Ti-alloys. 2. Materials and methods 2.1. Materials
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Commercial Ti6Al4V sheets (thickness: 3 mm; diameter: 55 mm) were purchased from Baoji Jingcheng Titanium co., Ltd (Baoji, China). MC3T3-E1 cells lines were acquired from
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Shanghai cell bank of Chinese academy of sciences (Shanghai, China). All the chemical and biological regents were purchased from Chemical Store of Tsinghua University (Beijing,
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2.2. Surface modification
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China) and Solarbio Biotech Co., Ltd (Beijing, China).
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2.2.1. Ultrasonic surface rolling processing The chemical composition of the annealed Ti6Al4V sheet is shown in Table 1. The Ti6Al4V
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samples were polished to remove the surface oxide layer using 400# and 1200# sandpaper consecutively, and then ultrasonically cleaned in acetone for 15 min. The USRP treatment was performed using a custom-made device to form a surface nanocrystalline layer. As shown in the schematic Figure 1, ultrasonic vibration was transformed into tens of thousands of strikes per second by a transducer booster, and a Gcr15 cemented carbide ball was used to strike the surface of the Ti6Al4V sample at a frequency of 20 kHz [33]. The experimental
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parameters of the USRP treatment are listed in Table 2, and the Ti6Al4V samples treated with a duration of 1 h and 3 h were grouped as USRP1 and USRP2. The raw Ti6Al4V samples were classified as untreated. 2.2.2. Plasma nitriding
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The Ti6Al4V samples were placed into a LDM 2-25 PN furnace (China Academy of Railway Sciences, Beijing, China) after ultrasonic cleaning with acetone. The samples were connected to the cathode, and the furnace wall acted as the anode. The vacuum chamber was evacuated
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to ultimate vacuum (below 10 Pa), and the leakage rate of the equipment was less than 2 Pa per 15 min prior to the nitriding treatment. The nitriding was carried out at 500 Pa in an NH3
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atmosphere and at a temperature of 850 °C (the temperature of the furnace body) for 10 h. During the PN process, the glow discharge was operated with a potential voltage 700~750 V
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to obtain the prescribed nitriding temperature. Finally, the Ti6Al4V samples were cooled
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down to room temperature in an NH3 atmosphere at 300 Pa, and were grouped as USRP1+PN
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and USRP2+PN.
2.3. Materials characterization
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2.3.1. Surface morphology
Scanning Electron Microscope (SEM) and Atomic Force Microscope (AFM) were used to examine the two-dimensional (2D) and three-dimensional (3D) surface morphology of the Ti6Al4V samples. For SEM observation, the Ti6Al4V samples were sputter-coated with a gold-platinum layer and then evaluated using a Quanta 200 FEG SEM (FEI, Eindhoven, Netherlands) associated with an energy dispersive X-ray analysis (EDX). Regarding with
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AFM examination, the Ti6Al4V samples were deposited on the glass slide and characterized using a MF-3D AFM (Oxford instruments, Oxford, England). The scanning rate was 1 Hz, and the scanning size was 20 µm 20 µm. 2.3.2. Surface topography
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The 3D surface topography of the Ti6Al4V samples was examined by a surface profilometer (Nano-Map-D, Aep technology, Silicon Valley, USA) with a scanning size is 500 µm 500 µm. The surface roughness values (Sa) were calculated based on at least three measurements,
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and the average values were obtained. 2.3.3. Wettability
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The Ti6Al4V samples were tested for surface wettability using an OCA-20 contact angle system (Dataphysics Instruments, Filderstadt, Germany) based on the sessile drop method as
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we conducted previously [34, 35]. The water contact angle values of the right and left sides of
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the deionized water droplet were measured, and the experiments were repeated at least three
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times for the calculation of the average value. 2.3.4. XRD analysis
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A D/max 2550 V X-ray diffractometer (XRD, Rigaku Corp, Tokyo, Japan) with a Cu-Ka radiation source (wavelength: 1.54 Å) was adopted to evaluate the phase structure of the Ti6Al4V samples. The XRD pattern was collected over a 2θ range of 30-100 ° and at an increment of 0.04 °/step (1.5 s /step). 2.4. Cell proliferation and adhesion on Ti6Al4V samples 2.4.1. Cell culture
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2.4.2. Cell proliferation
The Ti6Al4V samples were cut into cylindrical disks with a diameter of 1.5 cm employing a computer numerical control (CNC) wire-cut electric discharge machine, and then placed into
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the wells of 24-well culture plates. The samples were sterilized using 75% ethanol for 1 h followed by phosphate buffer saline (PBS) supplemented with 1% penicillin-streptomycin for
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4 h. The samples were further sterilized under UV irradiation for another 6 h before the cell experiment.
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A density of 2×104 cells/well was applied for the vaccination of MC3T3-E1 cells, and the
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culture plates were gently transferred to a CO2 incubator for cell cultivation. After culturing
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for 3 and 5 d, the culture medium was replaced with 400 µL CCK-8 working solution and further incubated for another 3 h. Afterwards, the medium was transferred to 96-well plates
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with 200 µL per well, and the absorbance at 450 nm was evaluated using a microplate reader (Varioskan Flash, Thermo, Waltham, USA). 2.4.3. Cell adhesion After culturing for 5 d, the Ti6Al4V samples were rinsed with PBS to remove the unattached cells, and fixed with 2.5% glutaraldehyde solution. Subsequently, the cells were dehydrated progressively via a series of alcohol with different concentrations (30%, 50%, 70%, 80%,
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90%, 95%, and 100%), each for 5 min. After drying, the samples were sputter-coated with a gold-platinum layer and evaluated using the SEM for cell adhesion on the material. 2.5. Osteogenesis capability The in vitro osteogenesis capability of the Ti6Al4V samples was evaluated based on alkaline
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phosphatase (ALP) activity and Alizarin Red S staining characterized in normal medium and osteogenesis induction medium. 2.5.1. ALP activity
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MC3T3-E1 cells were seeded on the Ti6Al4V samples at a density of 1×105 cells/well, and cultured in a 24-well culture plate for 7 and 14 d. At the predetermined time point, the culture
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medium was decanted, and the cells were rinsed gently three times with PBS and then lysed in 500 µL of 0.2% Triton X-100. Lysates were sonicated after being centrifuged at 10,000
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rpm for 8 min at 4 °C. 50 µL of supernatant was collected and mixed with 150 µL working
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solution (BioAssay Systems, Hayward, USA). The conversion of p-nitrophenylphosphate
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into p-nitrophenol in the presence of ALP was determined by measuring the absorbance at 405 nm using the microplate reader. The ALP activity was calculated via the equation from a
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previous publication [36].
2.5.2. Alizarin Red S staining After culturing on the Ti6Al4V samples in both normal medium and osteogenesis induction medium for 7 and 14 d, the MC3T3-E1 cells were fixed with 2% glutaraldehyde for 15 min and then 0.5% Alizarin Red S was added. After 15 min of incubation, the Ti6Al4V samples were washed with distilled water to remove any weakly adhered Alizarin Red S. Imaging was
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performed via single lens reflex camera (Canon inc, Tokyo, Japan). Subsequently, 10% cetylpyridinium chloride (CPC, J&K Scientific Ltd., Beijing, China) was added to dissolve the adhered Alizarin Red S on the surface of the samples, and the solution was measured to obtain absorbance at 540 nm by the microplate reader.
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2.6. Statistical Analysis
The results of experiment data were shown as mean value ± standard deviation, and analyzed by Statistical Product and Service Solutions 19.0 software (SPSS, Chicago, IL, USA). Each
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experiment was repeated at least three times. Statistical analysis was performed by using one-way analysis of variance (post hoc multiple comparison: least-significant difference test).
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The statistical significance was set at p < 0.05.
3.1. Surface morphology
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3. Results and discussion
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Figure 2 shows the SEM images of the Ti6Al4V samples treated by USRP and PN. It could
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be observed that the samples had quite different micro-structure surface morphologies, which may affect cell-material interactions as a result of the various surface contact area and surface
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energy [37]. The surface morphology of the untreated sample was relatively smooth, and the other samples appeared much rougher with the presence of many peaks and valleys on the surface. Severe surface deformation was observed for the Ti6Al4V samples of the USRP2 group due to the increased processing duration. In addition, the EDX analysis (measured by area distribution, Table 3) for the samples of the USRP+PN group showed nitrogen element following PN treatment (USRP1+PN: 43.87%; USRP2+PN: 48.40%), which indicated that a
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nitride layer had successfully formed on the surface. The 3D surface morphology of the Ti6Al4V samples characterized by AFM is demonstrated in Figure 3. Obviously, there were more peaks and valleys present on the surface of the samples treated by USRP and PN than the untreated samples. In addition, the samples of the
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USRP1+PN group and USRP2+PN group exhibited much rougher surfaces in comparison with the other samples, which was in accordance to the results of 2D surface morphology. 3.2. Surface topography
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The surface topography of the Ti6Al4V samples is evaluated by 3D surface profilometer, and the average surface roughness values were presented in Figure 4. As expected, significant
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difference of surface roughness was observed for the samples. The surface roughness values for the samples of the USRP1+PN group and USRP2+PN group were around 268 nm and
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202 nm, respectively, which was significantly higher than those of other groups (p<0.01). It
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was considered that there were more grain boundaries and defect densities for the samples of
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the USRP2+PN group than those of the USRP1+PN group, and the grain boundaries could promote the formation of nitride precipitates and decrease the size of nitride particles [32].
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This might be one potential reason why the surface roughness value for the samples of the USRP1+PN group was the largest. The surface roughness of the untreated samples was ~60 nm, and an increase was observed following USRP processing (USRP1 group: 70.5 nm, p<0.05; USRP2 group: 77.6 nm, p<0.01). 3.3. Wettability Water contact angle measurement is performed to characterize the surface wettability of the
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Ti6Al4V samples, which plays an important role in cell-material interactions via absorption of proteins to the surface [25], and the results are shown in Figure 5. The untreated samples and those of the USRP1+PN group were slightly hydrophobic with water contact angles of 98.4° and 96.7°, respectively, whilst all the other samples were hydrophilic. Following USRP
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processing, water contact angles reduced to 82.7° (USRP1 group) and 75.7° (USRP2 group), indicating that USRP processing had a positive effect on surface wettability of the samples due to grain refinement [25]. However, water contact angles increased to 96.7° (USRP1+PN
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group) and 81.6° (USRP2+PN group) after PN treatment. This phenomenon was caused by the formation of TiN and Ti2N during PN treatment, which could decrease the surface energy
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of the Ti6Al4V samples and consequently increase water contact angle. Tang et al. reported in their study that a lot of compounds from air and body fluid formed on the surface of
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stainless steel following plasma treatment, resulting in the decrease of surface wettability and
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3.4. XRD analysis
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surface free energy [37].
The Ti6Al4V samples are measured with XRD to confirm the phase structure, and the results
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are presented in Figure 6. The α-Ti and β-Ti diffraction peaks for the samples of the USRP1 group and USRP2 group were broader and weaker than those of the untreated samples, and this was caused by lattice dislocation and grain refinement effect at the atomic level based on the Scherrer equation [33]. This indicated that the enhancement of nanocrystallization of the Ti6Al4V samples was successfully achieved after USRP processing. A similar trend could be observed at the TiN peaks (43° and 62°) and the Ti2N (37°) peaks for the samples of the
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USRP1+PN group and USRP2+PN group, suggesting that a nitride layer formed on the surface of the Ti6Al4V samples [38]. 3.5. Cell proliferation The in vitro cytotoxicity of the Ti6Al4V samples evaluated via CCK-8 is shown in Figure 7.
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The relative viability of the MC3T3-E1 cells on all the samples increased apparently from 3 d to 5 d, and the smallest value was obtained for the untreated samples (~25%), which was significantly lower than all the other groups (day 3, p<0.05; day 5, p<0.01). On day 5, the
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relative viability for the samples of the USRP1 group and USRP2 group was similar (~125%), although there were obviously different nanocrystal surface morphologies from the SEM
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images. The Ti6Al4V samples of the USRP1+PN group and USRP2+PN group presented a better biocompatibility on day 5 than the other groups, with the relative cell viability values
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approximately 220% and 160%, respectively. In particular, a significant statistical difference
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(p<0.01) was obtained between the Ti6Al4V samples of the USRP1+PN group and those of
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the USRP1 group. It is considered that presumably the rougher surface formed by USRP and PN could not only increase the specific surface area of the samples but also absorb more
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hydroxyl groups into the surface, which may facilitate the increase of relative viability of MC3T3-E1 cells [39-42]. Eisenbarth et al. investigated fibroblast cells adhesion process on Ti6Al4V and Ti30Ta materials with different surface roughness, and they found that cell contact guidance was enhanced with the increase of surface roughness [39]. Additionally, the study performed by Anselme et al. indicated that human osteoblast grew faster on Ti-alloys with a rougher surface [41]. In our study, the Ti6Al4V samples treated by USRP and PN
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demonstrated excellent biocompatibility in comparison with the untreated samples, and these results were in good accordance to previous studies. 3.6. Cell adhesion MC3T3-E1 cells adhesion on the Ti6Al4V samples after culturing for 5 d is evaluated by
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SEM, and the results are presented in Figure 8. It was observed that the cells exhibited a uniform distribution on the surface, and the stretched parts as well as thin branches of the cells were perpendicular to the valleys for the samples treated by USRP processing (red
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arrows). In addition, it was clear that the cells showed mature focal adhesion and increased spreading on the surface of the samples treated by USRP and PN, indicating an excellent
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interaction between the cells and the Ti6Al4V samples [43]. When the MC3T3-E1 cells were seeded on the surface of Ti6Al4V samples, the cells interacted with the substrate via integrins,
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which associated with cytoskeletal elements forming focal adhesion. Consequently, numerous
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focal adhesion present on the Ti6Al4V samples of the USRP1+PN group and USRP2+PN
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group enabled formation of filopodia-like structures through cytoskeleton arrangement. The MC3T3-E1 cells seeded on the Ti6Al4V samples of the USRP1 group and USRP2 group
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displayed a flat morphology, and this could be predicted for a smoother surface [44]. The overall results showed that the Ti6Al4V samples treated by USRP and PN exhibited favorable biocompatibility and interactions to MC3T3-E1 cells. 3.7. ALP activity Osteogenic differentiation of MC3T3-E1 cells seeded on the Ti6Al4V samples is assessed by examining the levels of ALP activity, which is an early osteoblast marker. The results were
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collected after culturing in induced osteogenesis medium for 7 d and 14 d, as shown in Figure 9. Obviously, the ALP activities of the tissue culture plate (TCP) and Ti6Al4V samples on day 14 were all larger than those on day 7. The Ti6Al4V samples showed an increased ALP activity than TCP on either day 7 or day 14, especially for the USRP1+PN (p<0.01) and the
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USRP2+PN groups (p<0.05). On day 7, the ALP activity of untreated samples was around 10 IU/L, which was similar to the Ti6Al4V samples of the USRP1 group and slightly larger than the Ti6Al4V samples of the USRP2 group. On day 14, the ALP activity of untreated samples
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was around 13 IU/L, which was slightly larger than the Ti6Al4V samples of the USRP1 group and similar to the Ti6Al4V samples of the USRP2 group. These results indicated that
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the USRP processing did not contribute much to the osteogenic differentiation of MC3T3-E1 cells. Interestingly, it was demonstrated that the ALP activity for the Ti6Al4V samples of the
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USRP1+PN group was around 12 IU/L on day 7 and 18 IU/L on day 14, which was greatly
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larger than that of all other samples, especially in comparison with the untreated samples and
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those of the USRP1 group (day 7, p<0.05; day 14, p<0.01). Although the difference between the Ti6Al4V samples of the USRP2+PN group and other groups (untreated, USRP1 group,
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and USRP2 group) was not obvious, an increased tendency in the ALP activity values could be identified, which indicated that PN had a positive effect on the osteogenic differentiation of MC3T3-E1 cells. The enhanced osteogenic differentiation of the Ti6Al4V samples treated by USRP and PN may be attributed to the rough structure of TiN and Ti2N layers formed during PN [45, 46]. Vercaigne et al. evaluated the effect of plasma-sprayed titanium implants on trabecular bone
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healing in goat, and they found that plasma spraying resulted in different surface roughness and structures, which played a major role in bone healing [44]. Mussano et al. also reported that the osteogenic response was promoted when increasing the surface roughness of a silicon nitride-titanium nitride (Si3N4-TiN) composite [45]. On the other hand, it was indicated that
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nitrogen containing functional groups might form on TiN and Ti2N in the environment of induced osteogenesis medium, and they promoted the growth of osteoblasts by the interaction with hyaluronan, which was a negatively charged organic compound of extracellular matrix
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[43]. In the study performed by Mussano et al., it was shown that the titanium surface coated
osteogenic differentiation [43]. 3.8. Alizarin Red S staining
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with NH2-Ti by plasma surface activation and plasma polymers could significantly accelerate
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The mineralization of extracellular matrix of MC3T3-E1 cells (cultured in normal medium or
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induced osteogenesis medium for 7 d and 14 d) to the Ti6Al4V samples is determined via
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Alizarin Red S staining, which is a characteristic for late-stage osteogenic differentiation [47], and the results are presented in Figure 10 and Figure 11. On day 7, almost no mineralization
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was observed for TCP cultured in normal medium, whereas the Ti6Al4V samples all showed mineralization, although a better calcium mineral deposition was obtained for the untreated samples in comparison with those of all the other groups (USRP1 group, USRP2 group, USRP1+PN group, and USRP2+PN group). Additionally, both TCP and Ti6Al4V samples cultured in induced osteogenesis medium demonstrated an enhanced sign of calcium mineral deposition than those cultured in normal medium. On day 14, a similar trend was observed
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for the Ti6Al4V samples cultured in normal medium and induced osteogenesis medium to the results on day 7, and overall mineralization was greatly enhanced for both TCP and Ti6Al4V samples than those on day 7. The semi-quantitative evaluation of Alizarin Red S staining as shown in Figure 12 supported the above observation, and in particular the absorbance values
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for the Ti6Al4V samples of the USRP1+PN group were the largest on day 14 cultured either in normal medium or induced osteogenesis medium. These results indicated that the Ti6Al4V samples treated by USRP and PN were beneficial for late-stage osteogenic differentiation.
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4. Conclusions
In the present study, the Ti6Al4V samples were treated by USRP and PN to generate surfaces
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with different surface roughness, and the biocompatibility and osteogenesis properties of the samples were investigated in vitro via cell adhesion, proliferation, and differentiation using
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MC3T3-E1 cells. The results demonstrated that after USRP and PN processing the Ti6Al4V
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samples formed a nanocrystalline structure as well as a TiN and Ti2N layer covering the
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original surface, which greatly promoted cell adhesion, proliferation, and differentiation due to the increased surface roughness and surface chemistry property. In addition, the 3D surface
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topography of the Ti6Al4V samples could be further modified by adjusting the parameters of USRP and PN processing in order to adapt various biological applications. In conclusion, the USRP and PN treatments may present a promising technique to enhance bone regeneration of Ti-alloys. 5. Acknowledgements This study was financially supported by National Natural Science Foundation of China
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(Grant no. 51675296, 41572362), Tsinghua University Initiative Scientific Research Program (Grant no. 20151080366), Fundamental Research Funds for the Central Universities (53200859657), Research Fund of State Key Laboratory of Tribology, Tsinghua University
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(Grant no. SKLT2018B08) and Beijing Nova Program (Z171100001117059).
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nanofiber structures for effective guided bone regeneration membranes, Mater Sci Eng C. 78
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Table 1 Nominal compositions of the elements of Ti6Al4V (wt.%). Ti
Al
V
Fe
O
Si
C
N
H
Content
Bal.
5.5-6.8
3.5-4.5
0.3
0.2
0.15
0.1
0.05
0.01
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Element
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Table 2 The parameters of USRP to generate nanocrystallization on Ti6Al4V surface. Amplitude
Load
Spindle speed
Feed rate
Tip diameter
frequency (kHz)
(µm)
(N)
(rpm)
(mm/rev)
(mm)
20
30
300
150
0.05
15
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Vibration
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Table 3 EDX analysis of the different Ti6Al4V samples (at. %) USRP1
USRP2
USRP1+PN
USRP2+PN
Ti
74.92
73.32
73.60
48.45
44.78
Al
9.85
9.47
9.32
0.18
0
C
12.54
14.42
14.19
7.50
6.82
N
0
0
0
43.87
48.40
V
2.69
2.79
2.89
0
0
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untreated
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Figure legends Figure 1: Sketch of the ultrasonic surface rolling processing (USRP) device. Figure 2: SEM images and EDX analysis of different Ti6Al4V samples. Scale bar: 10 µm. Figure 3: AFM images showing the surface profiles of different Ti6Al4V samples.
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Figure 4: Surface roughness of different Ti6Al4V samples. Error bar represents mean value ± standard deviation (n=3). *p<0.05, **p<0.01: compared with the untreated samples. Figure 5: Water contact angles and images of different Ti6Al4V samples.
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Figure 6: XRD patterns showing the phase structure of different Ti6Al4V samples. Figure 7: MC3T3-E1 cells proliferation on different Ti6Al4V samples evaluated by CCK-8
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on day 3 and day 5. Error bar represents mean value ± standard deviation (n=3). *p<0.05, **p<0.01: compared with the untreated samples; ^^p<0.01: compared with the samples of the
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USRP1 group.
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Figure 8: MC3T3-E1 cells adhesion on different Ti6Al4V samples on day 5 observed by
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SEM images. The scale bar in the figures: upper row 200 µm; lower row 50 µm. Figure 9: ALP activities of MC3T3-E1 cells on different Ti6Al4V samples cultured in
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induced osteogenesis medium on day 7 and day 14. Error bar represents mean value ± standard deviation (n=3). *p<0.05, **p<0.01: compared with the TCP samples; ^p<0.05, ^^p<0.01: compared with the untreated samples; &p<0.05, &&p<0.01: compared with the samples of the USRP1 group. Figure 10: Alizarin Red S staining images of MC3T3-E1 cells on different Ti6Al4V samples cultured in normal medium (up) and induced osteogenesis medium (down) on day 7.
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Figure 11: Alizarin Red S staining images of MC3T3-E1 cells on different Ti6Al4V samples cultured in normal medium (up) and induced osteogenesis medium (down) on day 14. Figure 12: Semi-quantitative evaluation of Alizarin Red S staining of MC3T3-E1 cells on different Ti6Al4V samples cultured in both (A) normal medium and (B) induced osteogenesis
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medium on day 7 and day 14. *p<0.05, **p<0.01: compared with the TCP samples.
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Highlights
(1) Ti6Al4V was treated by ultrasonic surface rolling processing (USRP) and plasma nitriding (PN), which could modify the surface roughness and surface chemistry.
treatment were firstly investigated in this study.
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(2) The biocompatibility and osteogenesis properties of Ti6Al4V following USRP and PN
(3) USRP and PN treatment may represent a feasible and promising technique for improving
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the bone regeneration capacity of Ti6Al4V.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Ke Ren, Wen Yue, Hongyu Zhang PII: DOI: Reference:
S0257-8972(18)30614-5 doi:10.1016/j.surfcoat.2018.06.039 SCT 23490
To appear in:
Surface & Coatings Technology
Received date: Revised date: Accepted date:
23 March 2018 5 June 2018 20 June 2018
Please cite this article as: Ke Ren, Wen Yue, Hongyu Zhang , Surface modification of Ti6Al4V based on ultrasonic surface rolling processing and plasma nitriding for enhanced bone regeneration. Sct (2018), doi:10.1016/j.surfcoat.2018.06.039
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ACCEPTED MANUSCRIPT Surface modification of Ti6Al4V based on ultrasonic surface rolling processing and plasma nitriding for enhanced bone regeneration Ke Ren a, Wen Yue a, *, Hongyu Zhang b, *
School of Engineering and Technology, China University of Geosciences (Beijing), Beijing
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a
100083, China b
State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua
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University, Beijing 100084, China
* Corresponding authors: [email protected] (W. Yue)
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[email protected] (H. Zhang)
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Abstract Titanium alloy has been widely used in dentistry, orthopedic surgery and other medical fields. Surface morphology and structure of titanium alloy play an important role in cell adhesion and proliferation. The main purpose of this study is to investigate the biocompatibility and
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osteogenesis performance of Ti6Al4V modified using ultrasonic surface rolling processing (USRP) and plasma nitriding (PN). The raw materials were nominated as untreated, and the Ti6Al4V samples treated by USRP (with a processing duration of 1 h and 3 h) and PN were
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named as USRP1, USRP2, USRP1+PN, and USRP2+PN, respectively. The Ti6Al4V samples were characterized by SEM, AFM, and surface profilometer for surface morphology and 3D
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surface topography, and USRP1+PN showed the highest surface roughness (268 nm). The water contact angle measurements demonstrated that USRP2 had more favorite wettability
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(75.7°). The XRD analysis indicated the enhancement of nanocrystallization for the samples
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treated by USRP and the formation of a nitride layer for the samples treated by USRP and PN.
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The interactions between the Ti6Al4V samples and MC3T3-E1 cells were investigated in vitro by SEM, CCK-8 test, alkaline phosphatase (ALP) activity assay, and Alizarin Red S
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staining. The SEM images and CCK-8 tests revealed that all the Ti6Al4V samples were biocompatible to MC3T3-E1 cells, and the results of ALP activity assay and Alizarin Red S staining showed a better osteogenesis performance for the samples treated by USRP and PN. The present study indicates that USRP and PN may act as an effective method for Ti6Al4V to induce bone regeneration.
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Keywords: titanium alloy; ultrasonic surface rolling processing; nitriding; biocompatibility;
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osteogenesis.
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1. Introduction Titanium and its alloys (Ti-alloys), e.g. Ti6Al4V, have been widely applied in orthopedic and dental implants due mainly to their excellent properties, such as robust mechanical properties, corrosion resistance, high strength to weight ratio, and biocompatibility [1-5]. However,
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Ti-alloys usually cannot adequately integrate with bone at the early stage after implantation because of their bioinert surface [6-8]. In order to enhance the surface activity of Ti-alloys based prostheses and to improve their survivorship, various surface modification techniques
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have been utilized [9], such as plasma spraying [10, 11], acid-etching [12] and micro-arc oxidation [13]. However, it is considered that the bonding strength between Ti-alloys and the
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coatings is relatively weak, which limits their life span [14, 15]. Occasionally, toxic metal ions from Ti-alloys based prostheses can be released as a result of the corrosive environment
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[16].
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Surface topographic modification is an important method to enhance the biocompatibility and
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osseointegration of the implants [8]. It has been shown that nano-structured surfaces can enhance cell adhesion, proliferation, differentiation and effective osseointegration [17-21].
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Ultrasonic surface rolling process (USRP) is a novel surface nanocrystallization technique based on severe plastic deformation [22], which is a metal forming process that can result in grain refinement by imposing high strain rates without considerably altering the overall dimension of the material [23-25]. Generally, USRP applies ultrasonic vibration energy to the material surface to generate plastic deformation, and a nanocrystalline layer is produced with enhanced surface hardness and tribological properties [26, 27]. Fan et al. reported that surface
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grain size and hardness of carbon steel were affected by the duration of ultrasonic impact treatment [28]. An increased processing time is one of the reasons that resulted in intensified grain refinement. Additionally, plasma nitriding (PN) has been proposed to be one of the most effective methods that can change surface morphology and structure of Ti-alloys and
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improve their mechanical properties [29]. PN can produce a compound layer of TiN on the top of the matrix and Ti2N beneath, with a hardness of 3000 and 1500 HV, respectively, and a nitriding layer thickness of 10 μm~100 μm [30]. She et al. investigated the effects of nitriding
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temperature on microstructures and vacuum tribological properties of PN-treated titanium, and they concluded that the samples nitrided at the temperature of 850 °C exhibited the most
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excellent wear resistance and the lowest friction coefficient [31]. In another study performed by She et al., USRP was introduced as a pre-treatment means of PN to effectively enhance
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the properties of AISI D2 die steel, e.g. hardness [32]. Therefore, a combination of USRP and
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PN can significantly enhance the mechanical properties of Ti-alloys, which is an important
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factor affecting the lifespan of biomaterials served in human environment. To the best of our knowledge, there have been no reports investigating the biocompatibility and osteogenesis
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performance of Ti-alloys with different nanocrystal surface and nitriding treatment. Therefore, it is considered that an insight into this area is valuable and may predict paramount prospect in a large number of biomedical applications. In the present study, the Ti6Al4V samples were treated by USRP (processing duration: 1 h and 3 h) and PN, and characterized using SEM, AFM, surface profilometer, water contact angle, and XRD for surface morphology, surface topography, wettability, and phase structure.
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Additionally, pre-osteoblastic murine cell lines MC3T3-E1 were employed to evaluate the biocompatibility and osteogenesis properties of the Ti6Al4V samples in vitro by cell adhesion, proliferation, and differentiation. It is hypothesized that the USRP and PN treatments can greatly facilitate osteogenesis and act as potential techniques to enhance bone regeneration of
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Ti-alloys. 2. Materials and methods 2.1. Materials
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Commercial Ti6Al4V sheets (thickness: 3 mm; diameter: 55 mm) were purchased from Baoji Jingcheng Titanium co., Ltd (Baoji, China). MC3T3-E1 cells lines were acquired from
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Shanghai cell bank of Chinese academy of sciences (Shanghai, China). All the chemical and biological regents were purchased from Chemical Store of Tsinghua University (Beijing,
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2.2. Surface modification
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China) and Solarbio Biotech Co., Ltd (Beijing, China).
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2.2.1. Ultrasonic surface rolling processing The chemical composition of the annealed Ti6Al4V sheet is shown in Table 1. The Ti6Al4V
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samples were polished to remove the surface oxide layer using 400# and 1200# sandpaper consecutively, and then ultrasonically cleaned in acetone for 15 min. The USRP treatment was performed using a custom-made device to form a surface nanocrystalline layer. As shown in the schematic Figure 1, ultrasonic vibration was transformed into tens of thousands of strikes per second by a transducer booster, and a Gcr15 cemented carbide ball was used to strike the surface of the Ti6Al4V sample at a frequency of 20 kHz [33]. The experimental
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parameters of the USRP treatment are listed in Table 2, and the Ti6Al4V samples treated with a duration of 1 h and 3 h were grouped as USRP1 and USRP2. The raw Ti6Al4V samples were classified as untreated. 2.2.2. Plasma nitriding
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The Ti6Al4V samples were placed into a LDM 2-25 PN furnace (China Academy of Railway Sciences, Beijing, China) after ultrasonic cleaning with acetone. The samples were connected to the cathode, and the furnace wall acted as the anode. The vacuum chamber was evacuated
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to ultimate vacuum (below 10 Pa), and the leakage rate of the equipment was less than 2 Pa per 15 min prior to the nitriding treatment. The nitriding was carried out at 500 Pa in an NH3
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atmosphere and at a temperature of 850 °C (the temperature of the furnace body) for 10 h. During the PN process, the glow discharge was operated with a potential voltage 700~750 V
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to obtain the prescribed nitriding temperature. Finally, the Ti6Al4V samples were cooled
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down to room temperature in an NH3 atmosphere at 300 Pa, and were grouped as USRP1+PN
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and USRP2+PN.
2.3. Materials characterization
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2.3.1. Surface morphology
Scanning Electron Microscope (SEM) and Atomic Force Microscope (AFM) were used to examine the two-dimensional (2D) and three-dimensional (3D) surface morphology of the Ti6Al4V samples. For SEM observation, the Ti6Al4V samples were sputter-coated with a gold-platinum layer and then evaluated using a Quanta 200 FEG SEM (FEI, Eindhoven, Netherlands) associated with an energy dispersive X-ray analysis (EDX). Regarding with
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AFM examination, the Ti6Al4V samples were deposited on the glass slide and characterized using a MF-3D AFM (Oxford instruments, Oxford, England). The scanning rate was 1 Hz, and the scanning size was 20 µm 20 µm. 2.3.2. Surface topography
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The 3D surface topography of the Ti6Al4V samples was examined by a surface profilometer (Nano-Map-D, Aep technology, Silicon Valley, USA) with a scanning size is 500 µm 500 µm. The surface roughness values (Sa) were calculated based on at least three measurements,
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and the average values were obtained. 2.3.3. Wettability
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The Ti6Al4V samples were tested for surface wettability using an OCA-20 contact angle system (Dataphysics Instruments, Filderstadt, Germany) based on the sessile drop method as
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we conducted previously [34, 35]. The water contact angle values of the right and left sides of
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the deionized water droplet were measured, and the experiments were repeated at least three
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times for the calculation of the average value. 2.3.4. XRD analysis
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A D/max 2550 V X-ray diffractometer (XRD, Rigaku Corp, Tokyo, Japan) with a Cu-Ka radiation source (wavelength: 1.54 Å) was adopted to evaluate the phase structure of the Ti6Al4V samples. The XRD pattern was collected over a 2θ range of 30-100 ° and at an increment of 0.04 °/step (1.5 s /step). 2.4. Cell proliferation and adhesion on Ti6Al4V samples 2.4.1. Cell culture
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2.4.2. Cell proliferation
The Ti6Al4V samples were cut into cylindrical disks with a diameter of 1.5 cm employing a computer numerical control (CNC) wire-cut electric discharge machine, and then placed into
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the wells of 24-well culture plates. The samples were sterilized using 75% ethanol for 1 h followed by phosphate buffer saline (PBS) supplemented with 1% penicillin-streptomycin for
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4 h. The samples were further sterilized under UV irradiation for another 6 h before the cell experiment.
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A density of 2×104 cells/well was applied for the vaccination of MC3T3-E1 cells, and the
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culture plates were gently transferred to a CO2 incubator for cell cultivation. After culturing
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for 3 and 5 d, the culture medium was replaced with 400 µL CCK-8 working solution and further incubated for another 3 h. Afterwards, the medium was transferred to 96-well plates
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with 200 µL per well, and the absorbance at 450 nm was evaluated using a microplate reader (Varioskan Flash, Thermo, Waltham, USA). 2.4.3. Cell adhesion After culturing for 5 d, the Ti6Al4V samples were rinsed with PBS to remove the unattached cells, and fixed with 2.5% glutaraldehyde solution. Subsequently, the cells were dehydrated progressively via a series of alcohol with different concentrations (30%, 50%, 70%, 80%,
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90%, 95%, and 100%), each for 5 min. After drying, the samples were sputter-coated with a gold-platinum layer and evaluated using the SEM for cell adhesion on the material. 2.5. Osteogenesis capability The in vitro osteogenesis capability of the Ti6Al4V samples was evaluated based on alkaline
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phosphatase (ALP) activity and Alizarin Red S staining characterized in normal medium and osteogenesis induction medium. 2.5.1. ALP activity
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MC3T3-E1 cells were seeded on the Ti6Al4V samples at a density of 1×105 cells/well, and cultured in a 24-well culture plate for 7 and 14 d. At the predetermined time point, the culture
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medium was decanted, and the cells were rinsed gently three times with PBS and then lysed in 500 µL of 0.2% Triton X-100. Lysates were sonicated after being centrifuged at 10,000
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rpm for 8 min at 4 °C. 50 µL of supernatant was collected and mixed with 150 µL working
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solution (BioAssay Systems, Hayward, USA). The conversion of p-nitrophenylphosphate
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into p-nitrophenol in the presence of ALP was determined by measuring the absorbance at 405 nm using the microplate reader. The ALP activity was calculated via the equation from a
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previous publication [36].
2.5.2. Alizarin Red S staining After culturing on the Ti6Al4V samples in both normal medium and osteogenesis induction medium for 7 and 14 d, the MC3T3-E1 cells were fixed with 2% glutaraldehyde for 15 min and then 0.5% Alizarin Red S was added. After 15 min of incubation, the Ti6Al4V samples were washed with distilled water to remove any weakly adhered Alizarin Red S. Imaging was
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performed via single lens reflex camera (Canon inc, Tokyo, Japan). Subsequently, 10% cetylpyridinium chloride (CPC, J&K Scientific Ltd., Beijing, China) was added to dissolve the adhered Alizarin Red S on the surface of the samples, and the solution was measured to obtain absorbance at 540 nm by the microplate reader.
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2.6. Statistical Analysis
The results of experiment data were shown as mean value ± standard deviation, and analyzed by Statistical Product and Service Solutions 19.0 software (SPSS, Chicago, IL, USA). Each
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experiment was repeated at least three times. Statistical analysis was performed by using one-way analysis of variance (post hoc multiple comparison: least-significant difference test).
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The statistical significance was set at p < 0.05.
3.1. Surface morphology
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3. Results and discussion
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Figure 2 shows the SEM images of the Ti6Al4V samples treated by USRP and PN. It could
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be observed that the samples had quite different micro-structure surface morphologies, which may affect cell-material interactions as a result of the various surface contact area and surface
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energy [37]. The surface morphology of the untreated sample was relatively smooth, and the other samples appeared much rougher with the presence of many peaks and valleys on the surface. Severe surface deformation was observed for the Ti6Al4V samples of the USRP2 group due to the increased processing duration. In addition, the EDX analysis (measured by area distribution, Table 3) for the samples of the USRP+PN group showed nitrogen element following PN treatment (USRP1+PN: 43.87%; USRP2+PN: 48.40%), which indicated that a
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nitride layer had successfully formed on the surface. The 3D surface morphology of the Ti6Al4V samples characterized by AFM is demonstrated in Figure 3. Obviously, there were more peaks and valleys present on the surface of the samples treated by USRP and PN than the untreated samples. In addition, the samples of the
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USRP1+PN group and USRP2+PN group exhibited much rougher surfaces in comparison with the other samples, which was in accordance to the results of 2D surface morphology. 3.2. Surface topography
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The surface topography of the Ti6Al4V samples is evaluated by 3D surface profilometer, and the average surface roughness values were presented in Figure 4. As expected, significant
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difference of surface roughness was observed for the samples. The surface roughness values for the samples of the USRP1+PN group and USRP2+PN group were around 268 nm and
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202 nm, respectively, which was significantly higher than those of other groups (p<0.01). It
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was considered that there were more grain boundaries and defect densities for the samples of
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the USRP2+PN group than those of the USRP1+PN group, and the grain boundaries could promote the formation of nitride precipitates and decrease the size of nitride particles [32].
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This might be one potential reason why the surface roughness value for the samples of the USRP1+PN group was the largest. The surface roughness of the untreated samples was ~60 nm, and an increase was observed following USRP processing (USRP1 group: 70.5 nm, p<0.05; USRP2 group: 77.6 nm, p<0.01). 3.3. Wettability Water contact angle measurement is performed to characterize the surface wettability of the
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Ti6Al4V samples, which plays an important role in cell-material interactions via absorption of proteins to the surface [25], and the results are shown in Figure 5. The untreated samples and those of the USRP1+PN group were slightly hydrophobic with water contact angles of 98.4° and 96.7°, respectively, whilst all the other samples were hydrophilic. Following USRP
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processing, water contact angles reduced to 82.7° (USRP1 group) and 75.7° (USRP2 group), indicating that USRP processing had a positive effect on surface wettability of the samples due to grain refinement [25]. However, water contact angles increased to 96.7° (USRP1+PN
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group) and 81.6° (USRP2+PN group) after PN treatment. This phenomenon was caused by the formation of TiN and Ti2N during PN treatment, which could decrease the surface energy
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of the Ti6Al4V samples and consequently increase water contact angle. Tang et al. reported in their study that a lot of compounds from air and body fluid formed on the surface of
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stainless steel following plasma treatment, resulting in the decrease of surface wettability and
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3.4. XRD analysis
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surface free energy [37].
The Ti6Al4V samples are measured with XRD to confirm the phase structure, and the results
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are presented in Figure 6. The α-Ti and β-Ti diffraction peaks for the samples of the USRP1 group and USRP2 group were broader and weaker than those of the untreated samples, and this was caused by lattice dislocation and grain refinement effect at the atomic level based on the Scherrer equation [33]. This indicated that the enhancement of nanocrystallization of the Ti6Al4V samples was successfully achieved after USRP processing. A similar trend could be observed at the TiN peaks (43° and 62°) and the Ti2N (37°) peaks for the samples of the
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USRP1+PN group and USRP2+PN group, suggesting that a nitride layer formed on the surface of the Ti6Al4V samples [38]. 3.5. Cell proliferation The in vitro cytotoxicity of the Ti6Al4V samples evaluated via CCK-8 is shown in Figure 7.
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The relative viability of the MC3T3-E1 cells on all the samples increased apparently from 3 d to 5 d, and the smallest value was obtained for the untreated samples (~25%), which was significantly lower than all the other groups (day 3, p<0.05; day 5, p<0.01). On day 5, the
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relative viability for the samples of the USRP1 group and USRP2 group was similar (~125%), although there were obviously different nanocrystal surface morphologies from the SEM
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images. The Ti6Al4V samples of the USRP1+PN group and USRP2+PN group presented a better biocompatibility on day 5 than the other groups, with the relative cell viability values
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approximately 220% and 160%, respectively. In particular, a significant statistical difference
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(p<0.01) was obtained between the Ti6Al4V samples of the USRP1+PN group and those of
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the USRP1 group. It is considered that presumably the rougher surface formed by USRP and PN could not only increase the specific surface area of the samples but also absorb more
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hydroxyl groups into the surface, which may facilitate the increase of relative viability of MC3T3-E1 cells [39-42]. Eisenbarth et al. investigated fibroblast cells adhesion process on Ti6Al4V and Ti30Ta materials with different surface roughness, and they found that cell contact guidance was enhanced with the increase of surface roughness [39]. Additionally, the study performed by Anselme et al. indicated that human osteoblast grew faster on Ti-alloys with a rougher surface [41]. In our study, the Ti6Al4V samples treated by USRP and PN
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demonstrated excellent biocompatibility in comparison with the untreated samples, and these results were in good accordance to previous studies. 3.6. Cell adhesion MC3T3-E1 cells adhesion on the Ti6Al4V samples after culturing for 5 d is evaluated by
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SEM, and the results are presented in Figure 8. It was observed that the cells exhibited a uniform distribution on the surface, and the stretched parts as well as thin branches of the cells were perpendicular to the valleys for the samples treated by USRP processing (red
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arrows). In addition, it was clear that the cells showed mature focal adhesion and increased spreading on the surface of the samples treated by USRP and PN, indicating an excellent
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interaction between the cells and the Ti6Al4V samples [43]. When the MC3T3-E1 cells were seeded on the surface of Ti6Al4V samples, the cells interacted with the substrate via integrins,
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which associated with cytoskeletal elements forming focal adhesion. Consequently, numerous
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focal adhesion present on the Ti6Al4V samples of the USRP1+PN group and USRP2+PN
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group enabled formation of filopodia-like structures through cytoskeleton arrangement. The MC3T3-E1 cells seeded on the Ti6Al4V samples of the USRP1 group and USRP2 group
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displayed a flat morphology, and this could be predicted for a smoother surface [44]. The overall results showed that the Ti6Al4V samples treated by USRP and PN exhibited favorable biocompatibility and interactions to MC3T3-E1 cells. 3.7. ALP activity Osteogenic differentiation of MC3T3-E1 cells seeded on the Ti6Al4V samples is assessed by examining the levels of ALP activity, which is an early osteoblast marker. The results were
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collected after culturing in induced osteogenesis medium for 7 d and 14 d, as shown in Figure 9. Obviously, the ALP activities of the tissue culture plate (TCP) and Ti6Al4V samples on day 14 were all larger than those on day 7. The Ti6Al4V samples showed an increased ALP activity than TCP on either day 7 or day 14, especially for the USRP1+PN (p<0.01) and the
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USRP2+PN groups (p<0.05). On day 7, the ALP activity of untreated samples was around 10 IU/L, which was similar to the Ti6Al4V samples of the USRP1 group and slightly larger than the Ti6Al4V samples of the USRP2 group. On day 14, the ALP activity of untreated samples
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was around 13 IU/L, which was slightly larger than the Ti6Al4V samples of the USRP1 group and similar to the Ti6Al4V samples of the USRP2 group. These results indicated that
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the USRP processing did not contribute much to the osteogenic differentiation of MC3T3-E1 cells. Interestingly, it was demonstrated that the ALP activity for the Ti6Al4V samples of the
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USRP1+PN group was around 12 IU/L on day 7 and 18 IU/L on day 14, which was greatly
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larger than that of all other samples, especially in comparison with the untreated samples and
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those of the USRP1 group (day 7, p<0.05; day 14, p<0.01). Although the difference between the Ti6Al4V samples of the USRP2+PN group and other groups (untreated, USRP1 group,
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and USRP2 group) was not obvious, an increased tendency in the ALP activity values could be identified, which indicated that PN had a positive effect on the osteogenic differentiation of MC3T3-E1 cells. The enhanced osteogenic differentiation of the Ti6Al4V samples treated by USRP and PN may be attributed to the rough structure of TiN and Ti2N layers formed during PN [45, 46]. Vercaigne et al. evaluated the effect of plasma-sprayed titanium implants on trabecular bone
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healing in goat, and they found that plasma spraying resulted in different surface roughness and structures, which played a major role in bone healing [44]. Mussano et al. also reported that the osteogenic response was promoted when increasing the surface roughness of a silicon nitride-titanium nitride (Si3N4-TiN) composite [45]. On the other hand, it was indicated that
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nitrogen containing functional groups might form on TiN and Ti2N in the environment of induced osteogenesis medium, and they promoted the growth of osteoblasts by the interaction with hyaluronan, which was a negatively charged organic compound of extracellular matrix
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[43]. In the study performed by Mussano et al., it was shown that the titanium surface coated
osteogenic differentiation [43]. 3.8. Alizarin Red S staining
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with NH2-Ti by plasma surface activation and plasma polymers could significantly accelerate
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The mineralization of extracellular matrix of MC3T3-E1 cells (cultured in normal medium or
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induced osteogenesis medium for 7 d and 14 d) to the Ti6Al4V samples is determined via
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Alizarin Red S staining, which is a characteristic for late-stage osteogenic differentiation [47], and the results are presented in Figure 10 and Figure 11. On day 7, almost no mineralization
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was observed for TCP cultured in normal medium, whereas the Ti6Al4V samples all showed mineralization, although a better calcium mineral deposition was obtained for the untreated samples in comparison with those of all the other groups (USRP1 group, USRP2 group, USRP1+PN group, and USRP2+PN group). Additionally, both TCP and Ti6Al4V samples cultured in induced osteogenesis medium demonstrated an enhanced sign of calcium mineral deposition than those cultured in normal medium. On day 14, a similar trend was observed
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for the Ti6Al4V samples cultured in normal medium and induced osteogenesis medium to the results on day 7, and overall mineralization was greatly enhanced for both TCP and Ti6Al4V samples than those on day 7. The semi-quantitative evaluation of Alizarin Red S staining as shown in Figure 12 supported the above observation, and in particular the absorbance values
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for the Ti6Al4V samples of the USRP1+PN group were the largest on day 14 cultured either in normal medium or induced osteogenesis medium. These results indicated that the Ti6Al4V samples treated by USRP and PN were beneficial for late-stage osteogenic differentiation.
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4. Conclusions
In the present study, the Ti6Al4V samples were treated by USRP and PN to generate surfaces
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with different surface roughness, and the biocompatibility and osteogenesis properties of the samples were investigated in vitro via cell adhesion, proliferation, and differentiation using
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MC3T3-E1 cells. The results demonstrated that after USRP and PN processing the Ti6Al4V
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samples formed a nanocrystalline structure as well as a TiN and Ti2N layer covering the
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original surface, which greatly promoted cell adhesion, proliferation, and differentiation due to the increased surface roughness and surface chemistry property. In addition, the 3D surface
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topography of the Ti6Al4V samples could be further modified by adjusting the parameters of USRP and PN processing in order to adapt various biological applications. In conclusion, the USRP and PN treatments may present a promising technique to enhance bone regeneration of Ti-alloys. 5. Acknowledgements This study was financially supported by National Natural Science Foundation of China
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(Grant no. 51675296, 41572362), Tsinghua University Initiative Scientific Research Program (Grant no. 20151080366), Fundamental Research Funds for the Central Universities (53200859657), Research Fund of State Key Laboratory of Tribology, Tsinghua University
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(Grant no. SKLT2018B08) and Beijing Nova Program (Z171100001117059).
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Table 1 Nominal compositions of the elements of Ti6Al4V (wt.%). Ti
Al
V
Fe
O
Si
C
N
H
Content
Bal.
5.5-6.8
3.5-4.5
0.3
0.2
0.15
0.1
0.05
0.01
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Table 2 The parameters of USRP to generate nanocrystallization on Ti6Al4V surface. Amplitude
Load
Spindle speed
Feed rate
Tip diameter
frequency (kHz)
(µm)
(N)
(rpm)
(mm/rev)
(mm)
20
30
300
150
0.05
15
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Vibration
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Table 3 EDX analysis of the different Ti6Al4V samples (at. %) USRP1
USRP2
USRP1+PN
USRP2+PN
Ti
74.92
73.32
73.60
48.45
44.78
Al
9.85
9.47
9.32
0.18
0
C
12.54
14.42
14.19
7.50
6.82
N
0
0
0
43.87
48.40
V
2.69
2.79
2.89
0
0
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untreated
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Figure legends Figure 1: Sketch of the ultrasonic surface rolling processing (USRP) device. Figure 2: SEM images and EDX analysis of different Ti6Al4V samples. Scale bar: 10 µm. Figure 3: AFM images showing the surface profiles of different Ti6Al4V samples.
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Figure 4: Surface roughness of different Ti6Al4V samples. Error bar represents mean value ± standard deviation (n=3). *p<0.05, **p<0.01: compared with the untreated samples. Figure 5: Water contact angles and images of different Ti6Al4V samples.
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Figure 6: XRD patterns showing the phase structure of different Ti6Al4V samples. Figure 7: MC3T3-E1 cells proliferation on different Ti6Al4V samples evaluated by CCK-8
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on day 3 and day 5. Error bar represents mean value ± standard deviation (n=3). *p<0.05, **p<0.01: compared with the untreated samples; ^^p<0.01: compared with the samples of the
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USRP1 group.
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Figure 8: MC3T3-E1 cells adhesion on different Ti6Al4V samples on day 5 observed by
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SEM images. The scale bar in the figures: upper row 200 µm; lower row 50 µm. Figure 9: ALP activities of MC3T3-E1 cells on different Ti6Al4V samples cultured in
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induced osteogenesis medium on day 7 and day 14. Error bar represents mean value ± standard deviation (n=3). *p<0.05, **p<0.01: compared with the TCP samples; ^p<0.05, ^^p<0.01: compared with the untreated samples; &p<0.05, &&p<0.01: compared with the samples of the USRP1 group. Figure 10: Alizarin Red S staining images of MC3T3-E1 cells on different Ti6Al4V samples cultured in normal medium (up) and induced osteogenesis medium (down) on day 7.
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Figure 11: Alizarin Red S staining images of MC3T3-E1 cells on different Ti6Al4V samples cultured in normal medium (up) and induced osteogenesis medium (down) on day 14. Figure 12: Semi-quantitative evaluation of Alizarin Red S staining of MC3T3-E1 cells on different Ti6Al4V samples cultured in both (A) normal medium and (B) induced osteogenesis
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medium on day 7 and day 14. *p<0.05, **p<0.01: compared with the TCP samples.
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Highlights
(1) Ti6Al4V was treated by ultrasonic surface rolling processing (USRP) and plasma nitriding (PN), which could modify the surface roughness and surface chemistry.
treatment were firstly investigated in this study.
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(2) The biocompatibility and osteogenesis properties of Ti6Al4V following USRP and PN
(3) USRP and PN treatment may represent a feasible and promising technique for improving
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the bone regeneration capacity of Ti6Al4V.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12