Mechanical Properties and in Vitro and in Vivo Biocompatibility of

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Mechanical Properties and in Vitro and in Vivo Biocompatibility of a‑C/a-C:Ti Nanomultilayer Films on Ti6Al4V Alloy as Medical Implants Lingling Li,†,‡ Wenqi Bai,†,‡ Xiuli Wang,*,‡ Changdong Gu,‡ Gong Jin,§ and Jiangping Tu*,‡,§ ‡

State Key Laboratory of Silicon Materials and School of Materials Science and Engineering, Zhejiang University, Hangzhou 310027, China § ZhongAo HuiCheng Technology Co. Ltd., Beijing 100176, China S Supporting Information *

ABSTRACT: Hydrogen-free a-C/a-C:Ti nanomultilayer films are deposited on medical Ti6Al4V alloy using a closed field unbalanced magnetron sputtering under graded bias voltage. The mechanical and tribological properties of the nanomultilayer films are performed on the nanoindentor, Rockwell and scratch tests, and ball-on-disk tribometer. The biological properties are evaluated by cell cytotoxicity, genotoxicity, subchronic systemic toxicity and implant. The hard a-C/a-C:Ti nanomultilayer films on medical alloy exhibit high adhesion strength and excellent tribological properties in both ambient air and Hank’s solution. Biocompatibility results reveal the film no cytotoxity, no genotoxicity, no subchronic systemic toxicity and no contraindications in implant systems. Because of excellent mechanical properties and biosafety, the carbon-based films on medical alloy unveils a prospective application in medical implants. KEYWORDS: amorphous carbon film, multilayer structure, mechanical property, biocompatibility, medical implant films with graded and nanomultilayered structures simultaneously may display high hardness, high toughness and good adhesion strength. To obtain a better understanding of the hard carbon-based nanomultilayer film applied in medical implant, in this present work, a-C/a-C:Ti nanomultilayer (a-C NM) films were deposited on medical Ti6Al4V alloy by magnetron sputtering under graded bias voltage. Microstructure and mechanical properties of the films were investigated, and biocompatibility were carried out to evaluate the biosafety according to ISO 10993 and Organization of Economic Cooperation and Development (OECD) standards.

1. INTRODUCTION It is well-known that amorphous carbon (a-C) film has attracted enormous interests because of their low friction coefficient and wear rate, high hardness, and good chemical inertness.1−5 Meanwhile, in vitro and in vivo research has affirmed that a-C films have favorable biocompatibility,6−8 which makes them become promising materials for biomedical applications, especially for medical implants such as hip joint, knee joint, etc. However, there are still some drawbacks because the residual stress in most hard a-C films is very high which induces the coating spontaneously delaminate if the adhesion is not sufficient.9,10 To resolve these problems, researchers have applied many methods, such as bias voltage grading,11−13 element doping14 and nanomultilayer structuring.9,15,16 Zhang et al. prepared bias grading hydrogen-free diamond-like carbon (DLC) film, which had a moderately high toughness and enhanced adhesion strength compared to constant bias DLC films. Moreover, the hard surface layer with the highest sp3 content in the bias graded film could provide excellent tribological performance.12 Additionally, our previous work indicated that the bias graded Ti-contained a-C gradient composite film on Ti6Al4V alloy exhibited better toughness, adhesion strength and tribological performance in Hank’s solution than that deposited with constant bias voltage.13 To date, there are some works on bias graded deposition but the majority of them are about the a-C films or a-C/carbide nanocomposite films. It can be deduced from previous works that nanomultilayer structure can limit crack propagation, leading to high toughness without reducing the hardness.4,10,17,18 Thereafter it can be inferred that the hard carbon © 2017 American Chemical Society

2. MATERIALS AND METHODS 2.1. Film Preparation. The a-C NM films were deposited by a closed field unbalance magnetron sputtering system (CC800/9 ML, CemeCon, Germany). Three types of samples were prepared: Si (100) wafers were used to characterize film microstructure and chemical composition. Medical Ti6Al4V alloy discs with 20 mm in diameter and 2 mm in thickness were used for mechanical tests and biological evaluation of cell cytotoxicity, genotoxicity, acute systemic toxicity and subchronic systemic toxicity. Medical Ti6Al4V alloy rods with 2 mm in diameter and 6 mm in length were used for implant experiment. All samples used for biocompatibility were sterilized by autoclaving at 120 °C for 20 min. All the substrates were ultrasonically cleaned in acetone for 20 min, in ethanol for 10 min, and then blow-dried by nitrogen to clear impurities on the surface. Prior to deposition, the base pressure of the Received: February 21, 2017 Accepted: May 3, 2017 Published: May 3, 2017 15933

DOI: 10.1021/acsami.7b02552 ACS Appl. Mater. Interfaces 2017, 9, 15933−15942

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ACS Applied Materials & Interfaces sputtering system was evacuated to 4 × 10−3 Pa, and then argon gas was introduced to keep the working pressure of 0.2 Pa. Substrates were etched by Ar+ bombardment at a bias voltage of −500 V for 30 min, in order to remove the oxides and adsorptions. During deposition, rotation speed of substrate holder was kept at 5 rpm. First, a thin Ti buffer layer was deposited onto the substrates for 10 min, with a bias voltage of −200 V, and then a C−Ti transition layer with gradient content ratio of Ti/C was deposited by gradually increasing the graphite target current from 0.2 to 2.5 A, and decreasing the titanium target current from 2.5 to 1 A for 60 min with a bias voltage of −100 V. Subsequently, the a-C layer was deposited by facing the graphite target and the a-C:Ti layer was prepared by codeposition of graphite and titanium target. The a-C:Ti layer and a-C layer were deposited alternatively. Through the nanomultilayer deposition process, the current of graphite target was kept at 2.5 A and the current of titanium target was kept at 1 A, a-C NM film was deposited with the bias voltage range from −50 to −150 V at a rate of −5 V for every 6 min. Detailed parameters for the deposition of the graded bias nanomultilayer films are summarized in Table S1. In this work, the total thicknesses of all nanomultilayer films are around 2 μm. The depositing parameters, such as target current density, depositing time, and duty cycle, have been optimized in previous works.3,13 The surface and cross-section morphologies of nanomultilayer films were characterized by scanning electron microscope (SEM, Hitachi S4800, Japan). Microstructure observation was carried out by transmission electron microscopy (TEM, Tecnai G2F30 S-Twin, USA). The cross-section samples were prepared by mechanical polishing and Ar ion-milling (Gatan 691, USA). The atomic bonding ordering of films was analyzed by Raman spectroscopy (LABRAM HR-800) with wavenumber shift among 4000 to 100 cm−1 in excitation line of 514.5 nm. The bonded structures of films were characterized by X-ray photoelectron spectroscopy (XPS) using ESCALab 220i-XL electron spectrometer, operating with a monochromated Al−Kα X-ray radiation source in a base pressure of 10−7 Pa. The binding energy was referenced to C 1s line at 284.6 eV from adventitious carbon. The hardness and Young’s modulus of the films were measured using a nanoindentor (Agilent technologies, G-200, USA) with Berkovich diamond indenter. The maximum indentation depth was kept less than 10% of the film thickness to minimize substrate effects. Six indentations in each sample configured on different areas were performed to have reliable statistics. Adhesion tests were performed on the films through scratch and Rockwell tests. Standard scratch tests were carried out with a conventional scratch tester (WS-2002, China). For these scratch tests, a diamond pin (0.2 mm in radius) was drawn across the surface of the film at a constant linear velocity of 4 mm min−1, while increasing the load linearly from 0 to 80 N. Standard Rockwell tests were performed using hardness tester (HR-150A, Xinnuo Testing Instrument Co., China) at a load of 100 kg using a Rockwell indenter of 0.2 mm in diameter to assess the vertical adhesion of the films. The scratch and Rockwell craters were observed by optical microscope (Nikon Eclipse ME600D, Japan). Tribological properties of the multilayer films were performed on a ball-on-disk tribometer (WTM-1E, China) at room temperature. Si3N4 ceramic ball (4 mm in diameter, hardness HV= 1550) was used as the counter body. The tests were carried out at a normal load of 5 N at a sliding velocity of 0.2 m s−1 in ambient air (50% RH) and in Hank’s solution. The coefficient of friction was monitored continuously during the tests by a linear variable displacement transducer and recorded on a data acquisition computer attached to the tribometer. The wear traces of the films were observed by optical microscope after test duration for 60 min. Composition of Hank’s solution is documented elsewhere.9 2.2. Extract Preparation. All extract samples of a-C NM films were prepared according to ISO 10993−12. The detailed procedure was described in the Supporting Information. 2.3. In Vitro Biocompatibility Tests. 2.3.1. Cell Cytotoxicity. Cytotoxicity test was performed according to ISO 10993−5 and evaluated by MTT (SM).19 L-929 mouse fibroblasts cells (ATCC, USA) were used for cell cytotoxicity tests. Roswell Park Memorial

Institute (RPMI) 1640 with 10% fetal bovine serum as blank control, RPMI 1640 with 0.5% phenol as positive control and polystyrene as negative control. The dimension of the polystyrene was the same as the medical Ti6Al4V alloy discs. Cell proliferation rates were calculated by mean optical density (OD) value of each group in comparison with blank control group. The experiments were carried out three times in order to confirm reproducibility. 2.3.2. Genotoxicity. 2.3.2.1. Bacterial Reverse Mutation (Ames) Test. The in vitro gene reverse mutation test in bacteria utilized four strains of Salmonella typhimurium (S. typhimurium) TA97a, TA98, TA100, and TA102 (Moltox, USA) in accordance with the guidelines recommended by OECD 471 and ISO 10993−3 (as seen in the Supporting Information). Groups range from low, middle and high doses (32, 800, 20000 μg/plate) of the extract were selected to assess the potential reverse mutation. Plate incorporation was performed with the extract and bacteria exposed to the substance with or without metabolic activation (S9). Normal saline was served as negative control, 2,4,7-trinitro-9-fluorenon, methylmethanesulfonate and 2nitrofluorene (Accu Standard, USA) were used as positive controls are listed in Table S2. Tryptophan-independent revertant colonies were scored after experiments. Experiments were repeated at least twice and for each concentration was tested in triplicate. 2.3.2.2. Mammalian Cell Gene Mutation. The L5178Y TK+/− mouse lymphoma cell line (ATCC, USA) was undertaken to assess the potential of a-C NM films to induce in vitro gene mutation in mammalian cells. The test concentrations of 100, 50, and 25% were evaluated. Serum-free RPMI 1640 as a blank control. Five μg mL−1 cyclophosphamide (sigma, USA) with S9 and 10 μg mL−1 methyl mesylate (sigma, USA) without S9 were used as positive controls. Procedure was in accordance with the guidelines recommended by OECD 476 and ISO 10993−3 (as seen in the Supporting Information). The mutant frequency (MF) was derived from the number of mutant colonies in selective medium, the number of colonies in nonselective medium and calculated following published definitions.20,21 2.3.2.3. Chromosome Aberration. The V79 Chinese hamster cell line obtained from typical culture preservation commission, Chinese academy of sciences (Shanghai, China). The chromosome aberration study was assessed in accordance with the methods described in OECD 473 and ISO 10993−3 (see in Information). We selected 100% extract as the highest concentration, 50 and 25% as middle dose and lowdose, respectively. Any structural aberrations were recorded, including chromosome-type breaks and exchange calculated as incidence per 100 metaphase cells. 2.4. In Vivo Biocompatibility. 2.4.1. Animal. Kunming mice, weighing between 20.3−25.7 g were used for acute systemic toxicity test. Japanese white rabbits aged 6 months and weighing 2.6 ± 0.4 kg were used for subchronic toxicity test and implant experiment. All animals in the studies were obtained from the zoological animal center of Sichuan University (Sichuan, China). They were kept in individual cages and adaptation under temperature controlled conditions with a 12 h light/dark cycles and ad libitum access to water and food. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) and approved by Animal Care and Use Committee of the Sichuan province (Permit Number: SYXK (Chuan) 2013−17). 2.4.2. Acute Systemic Toxicity. Acute systemic toxicity evaluation was carried out according to ISO 10993−11 (as seen in the Supporting Information). To ensure the welfare of mice, we checked bodyweight loss, food/water consumption, changes in activity, and behavior of the animals daily as a clinical indication. 2.4.3. Subchronic Toxicity. The subchronic systemic toxicity test was evaluated according to ISO 10993−11, using extract solution (as seen in the Supporting Information). Japanese white rabbits were randomized to two groups: experimental group and control group with 8 animals each group (half male and half female). At the end of the test, body weight, hematology, blood biochemistry, organ weights, and histopathology were examined. 15934


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ACS Applied Materials & Interfaces

at ∼1560 cm−1. The D band is resulted from the breathing modes of sp2 atoms in rings and the G band is caused by the bond stretching of all pairs of sp2 atoms in both rings and chains.22,23 The value of Id/Ig of the film is 1.66. Further analysis of the effect of Ti element on bonding structure of the nanomultilayer films is obtained from XPS. For detecting variation of bonding structure with depth of biasgraded nanomultilayer, Ar ion etching was taken and the etching rate was based on the TiO2 thin film (standard) of about 2 nm min−1. XPS tests were taken at depth of about 40 nm (first etching 20 min), 600 nm (second etching 300 min) and 900 nm (third etching 450 min). At each etched depth, five XPS tests at 1 min etching interval were taken to obtain bonding information on both a-C layer (high carbon content) and a-C:Ti layer (high titanium content). Figure 2a−d show deconvoluted C 1s spectra of a-C and a-C:Ti layer after first etching, a-C layer after second etching and third etching, respectively. All of them exhibit four peaks which are located at binding energy of 288.9 ± 0.2, 285.2 ± 0.1, 284.4 ± 0.1, and 282.8 ± 0.1 eV, representing C−O bond, sp3 hybridization of carbon bond, sp2 hybridization of carbon bond and C−Ti bond, respectively.9,24,25 As shown in Figure 2a, b, compared to that of a-C layer, the sp3 fraction of a-C:Ti is obviously lower due to the effect of Ti atoms,13 as peak intensity ratio of sp3/sp2 decreases from 0.64 to 0.42. Moreover, the peak intensity ratio of sp3/sp2 decreases with depth, as shown in Figure 2a, c, and d, from 0.64 after first etching to 0.50 after second etching, and then further decreases to 0.41 after third etching, which indicates that the bias-graded film has an gradient structure, that is, sp3 fraction gradually increases from bottom of the nanomultilayer part to the surface. Figure 2eshows Ti 2p peak of film after the first etching, which is deconvolved by Gaussian fitting into six subpeaks: 464.5 ± 0.1 eV (fwhm = 3 eV), 462.1 ± 0.1 eV (fwhm = 3 eV), 461 ± 0.1 eV (fwhm = 1.9 eV), 458.5 ± 0.1 eV (fwhm = 1.8 eV), 456.5 ± 0.1 eV (fwhm = 2.2 eV), and 455 ± 0.1 eV (fwhm = 1.4 eV), among which 464.5 ± 0.1 and 458.5 ± 0.1 eV correspond to Ti−O bond.10 Ti 2p3/2 at 455 ± 0.1 eV and Ti 2p1/2 at 461 ± 0.1 eV can confirm the formation of TiC in the a-C NF film.26 Additionally, two unassigned peaks at 456.5 ± 0.1 eV and 462.1 ± 0.1 eV are supposed to be nonstoichiometric Ti−C bond, which may result from ion bombardment damaged carbide phase,16,27 or the mutual diffusion of Ti and C atoms at the interface which do not strictly follow stoichiometric ratio of bonding.3 Furthermore, proportion of peak area of Ti−O bond in Ti 2p peak is obviously higher than that of C−O bond in C 1s peak, indicating that oxygen atoms mainly bond to titanium atoms. The presence of C−O bond and Ti−O bond is attributed to residual oxygen in the deposition chamber. 3.2. Mechanical Properties. The a-C NM film deposited under graded bias voltage has a moderate hardness (H) of ∼20 GPa and a relatively low elastic modulus (E) of ∼179 GPa. The H3/E2 and H/E values of the film is 0.250 and 0.112. According to previous work, H3/E2 and H/E are suggested as an indicator of toughness, namely the resistance to plastic deformation and wear resistance, respectively.9,28,29 The film with high H3/E2 and H/E values indicates that nanomultilayer deposited under graded bias voltage may display high toughness. Adhesion strength of the a-C NM films was measured using scratch and Rockwell tests. Figure 3a shows the scratch traces on the bias-graded nanomultilayer films. The critical load of the film is higher than 80N and the whole scratch trace is without any obvious fragment or delamination. The load used in

2.4.4. Implant Experiment. The experiment was carried out in according to ISO 10993−6 (as seen in the Supporting Information). After one-week adaptation, 18 rabbits were used for this experiment with uncoated Ti6Al4V rod as control. Specimens were taken out and bone tissues were examined for histopathology after experiment. 2.5. Statistical Analysis. Data were analyzed by the Statistical Package for Social Sciences 17.0 software (SPSS 17.0, USA) and presented as mean ± standard deviation (S.D). The statistical difference was evaluated by one-way ANOVA. P < 0.05 and P < 0.01 were considered to indicate a statistically significant difference.

3. RESULTS 3.1. Microstructure of Films. The cross-section SEM micrograph of the as-deposited film confirms the multilayered structure due to alternatively deposition of a-C layer and a-C:Ti layer (Figure S1), besides a typical columnar structure is observed at both the multilayer and gradient layer parts which can be attributed to the growth mode of the film. In addition, the surface of the bias-graded film presents lots of fine homogeneous clusters with some small gaps between each other (inset in Figure S1). The detailed layer morphology and crystal phase of the film are analyzed from cross-sectional TEM. As shown in Figure 1, the film is composed of three distinct

Figure 1. TEM image of a-C NM film.

segments: a Ti interlayer of about 200 nm, a C−Ti gradient interlayer of about 400 nm and a-C NM of about 1400 nm. It is demonstrated that the bilayer period of the film is about 33 nm, and thickness ratio of the a-C:Ti layer versus a-C layer is about 1:2. The interfaces between adjacent layers are moderately sharp. In fact, unlike fine crystalline multilayer films, for example some nitride multilayers, distinct interfaces are difficult to form in this a-C NM film. The nanomultilayer films deposited at constant bias voltage have the similar bilayer period and thickness ratio due to the same deposition parameters except for bias voltage. The HRTEM image displays detail information on a-C:Ti layer. Nanocrystalline TiC embedded in a-C matrix can be found in the a-C:Ti layer, forming a nanocomposite structure, whereas the a-C layer exhibits an amorphous structure. Atomic bonding structure of the as-deposited films was studied by Raman spectroscopy due to its sensitivity to various forms of carbon. The spectrum shown in Figure S2 is fitted into two Gaussian bands: the D peak at ∼1360 cm−1 and the G peak 15935


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Figure 2. (a, b) C 1s of a-C layer and a-C:Ti layer after first etching; (c) C 1s of a-C layer after second etching; (d) C 1s of a-C layer after third etching; and (e) Ti 2p of a-C:Ti layer after first etching XPS spectra of a-C NM film and their deconvolutions.

solution is smaller than that in ambient air, suggesting that the film with bias-graded structure exhibits comprehensive tribological properties in Hank’s solution. To explore the chemical and microstructure change in worn surface, we employed EDAX and Raman spectroscope. Contents of each element remain almost constant before and after tribo-tests in both ambient air and Hank’s solution. Furthermore, line scanning analysis for wear trace of the biasgraded film in Hank’s solution was carried out. Along the whole trace the composition only varies in a very small range, implying that no obvious chemical transformation occurs during tribological tests in both ambient air and Hank’s solution. Table 1 lists the Raman analysis of the worn surface under different circumstances. For all circumstances Id/Ig values are very close. However, for wear trace in ambient air, fwhmG is slightly lower, which may indicate the formation of graphitized transfer layer during sliding in ambient air. Meanwhile, fwhmG of the wear trace in Hank’s solution increases faintly, suggesting that graphitized transfer layer is not well generated during the friction process. Thus, lubrication function of the solution may become a factor of low wear rate in Hank’s solution.

Figure 3. Optical images of (a) scratch track and (b) Rockwell crater of a-C NM films.

Rockwell tests is 100 kg, and no evident crack and film delamination can be observed (Figure 3b). Tribological behaviors of the hard carbon-based nanomultilayer films in ambient air and Hank’s solution were evaluated by ball-on-disk test at an applied load of 5 N. The film with graded bias has low average coefficient of friction of 0.12 in ambient air and 0.08 in Hank’s solution. The running-in stage is not so obvious in Hank’s solution. The film with biasgraded structure has relatively low coefficient of friction (0.1). From optical morphologies of wear traces of the nanomultilayer films, it can be confirmed that the width of wear trace in Hank’s 15936


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each extract group in the number of strains of the colonies was no more than 2 times of negative control group which reveal no dose−response relationship. Positive control group in the number of strains of the colonies was 3 times more than negative control group. 3.3.2.2. Mammalian Cell Gene Mutation. Cell MF exposed to different concentrations of 100, 50, and 25% groups (with or without metabolic activation) are never higher than 2 times of negative control (58.55 × 10−6 and 42.28 × 10−6), which show no dose−effect relationship (Figure 5). Appropriate positive

Table 1. Raman Analysis of the Worn Traces of a-C NM Films D peak position (cm−1)

G peak position (cm−1)

Id/Ig

fwhmG (cm−1)

1372.7 1378.3

1552.2 1560.5

1.66 1.65

139.1 124.6

1373.8

1557.2

1.65

145.3

area without friction wear trace in ambient air wear trace in Hank’s solution

3.3. In Vitro Biocompatibility. 3.3.1. Cell Cytotoxicity. Figure 4 shows cell proliferation rates after 24 h exposure to

Figure 5. Cell-mutation test of extracts of test specimens (a-C NM film coated Ti6Al4V discs) in L5178Y TK+/− cells with and without metabolic activation (S9). Experiments were performed three times and in duplicate per concentration. The significance level observed is *p < 0.05 in comparison with positive controls. Figure 4. Cytotoxicity test results obtained with the extract solution method. Cell proliferation rates (%) after 24 h exposure to extracts of the test specimens (a-C NM film coated Ti6Al4V discs). Experiments were performed three times and in duplicate per concentration. The significance level observed is *p < 0.05 in comparison with the blank control group.

controls show a distinct increase in induced mutant colonies, indicating the sensitivity and validity of the assay, and MF value of positive controls is more than 2 times of negative control. 3.3.2.3. Chromosome Aberration. In a 6 h treatment with or without S9, there were no aberrant metaphases in negative control and all test groups. There was no statistically significant increase in frequencies of aberrant metaphases in any of the extract groups when compared with negative control groups (Table S3). On the other hand, there was a statistically significant increase (P < 0.01) in the frequency of aberrant metaphases in positive control when compared with negative control group. In 24 h treatment without S9, there were no aberrant metaphases in extract groups. Extracts did not induce chromosomal aberrations in V79 cell strain after treatment in absence or presence of metabolic activation under experimental conditions of 100, 50, and 25%. 3.4. In Vivo Biocompatibility. 3.4.1. Acute Systemic Toxicity. Acute systemic toxicity after injection of extract was determined for testing the suitability.30,31 There was no death incidence and no remarkable difference in general conditions such as behavioral (movement) activity, respiratory illness, abdominal irritation, eyelid prolapse, and normal behavior between control and experimental animals (Table S4). As

extracts of test specimen. The cell proliferation rates of negative and positive control groups were 96.5% and 11.4% respectively, revealing effectiveness of the control. Cell proliferation rates of 100, 50, 25, and 12.5% groups were 97.1, 98.7, 95.6, and 100.3%, respectively. There is no statistical difference between the extract solution group, negative group and blank control group, whereas the positive group has a significant difference compared with other groups. MTT results show that there is no cytotoxic effects of the a-C NM film on L-929 mouse fibroblasts cells. 3.3.2. Genotoxicity. 3.3.2.1. Bacterial Reverse Mutation (Ames) Test. As presented in Table 2, there is no significant increase in revertant colonies in the strains TA97, TA98, TA100 and TA102 at any dose level, either in the presence or absence of S9, when compared to negative controls. In contrast, there was a significant increase in the number of colonies in all positive control groups. Under the experimental condition, Table 2. Ames Test Dataa TA97a dose (μg/plate) 32 800 20000 negative control positive control a

-S9 108.0 116.7 131.3 126.3 759.0

± ± ± ± ±

TA98 +S9

5.3 20.8 11.4 13.5 43.6

120.7 132.0 131.7 143.3 873.5

± ± ± ± ±

-S9 5.9 11.0 14.3 28.4 11.7

20.0 26.0 21.7 18.0 570.5

± ± ± ± ±

TA100 +S9

5.2 1.4 3.1 5.2 382.8

13.7 17.0 20.7 18.7 1133.5

± ± ± ± ±

-S9 3.1 7.5 6.4 8.3 5.9

124.0 127.7 141.3 126.0 613.5

± ± ± ± ±

TA102 +S9

15.4 18.9 6.7 14.1 306.5

138.0 137.0 131.3 132.3 588.5

± ± ± ± ±

-S9 29.7 19.5 6.7 14.6 2.5

215.3 210.3 211.3 244.0 1168.5

± ± ± ± ±

+S9 39.5 17.0 16.3 16.7 37.1

215.0 221.7 231.3 196.3 1098.0

± ± ± ± ±

7.1 8.5 13.3 7.5 29.7

All values from triplicate plates are expressed as mean ± SD and analyzed by one-way ANOVA. 15937


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ACS Applied Materials & Interfaces shown in Figure 6, experimental mice have no significant changes in body weight compared to control mice.

Figure 8. Effect of a-C NM film-coated Ti6Al4V and uncoated Ti6Al4V implantation on histological findings in implant experiment: (A) 4, (B) 8, and (C) 12 weeks of film samples; (D) 4, (E) 8, and (F) 12 weeks of control samples. Scale bar represents 200 μm.

Figure 6. Body weight of acute systemic toxicity assessment. Weighting data at 0, 24, 48, and 72 h in each group (male and female groups).

active osteoblasts, fibroblasts cells and mesenchymal cells on the surface of new bone can be seen at bone implant junction (Figure 8a, d). For experimental site, although the trabecular bone is less than control site at 4 weeks, there are a large number of fibro collagenous tissue are developed and osteoblasts are active. The amounts of new bone increase postoperatively and the woven bone are transferred into lamellar bone with the increase of osteons at 8 weeks (Figure 8b, e). At 12 weeks after implant surgery, most of the woven bone are substituted by lamella which has engulfed the implant at the junction area (Figure 8c, f). The density of new bone increases gradually suggesting that new bone has formed with a good bond between host bone and implant.34,35 No visible signs of inflammation or bone graft rejection being found.

3.4.2. Subchronic Systemic Toxicity. There were no significant changes in food and water consumptions of repeated administration in 28 days, and no deaths and no clinical signs were observed throughout the study. All animals did not produce any statistically significant difference in body weight (Table S5). Before injection, a small number of hematological parameters (WBC) and blood biochemistry (ALP, GLU, TG) of experimental group reached statistical significance when compared with controls, but there was no clear trend with treatment and consequently, which were attributed to normal biological diversity.32,33 There were no significant differences of body weight, hematology, blood chemical and viscera coefficient between experimental and control animals (both female and male rabbits) (Tables S5−S8). From the histological images shown in Figure 7, all animals show no abnormal alterations of heart, liver, kidney, paranephros, spleen, gonad and ovary caused by dose, indicating that there is no significant difference between control and experimental groups. 3.4.3. Implant Experiment. Implant experiment was to evaluate whether there would be any adverse reactions in the implant sites caused by the samples after a period of observation time. During the observation time, all animals had gained weight normally and did not show any adverse effects like edema, regional lymphadenopathy or loss of hair. New bone is formed around the implant in all cases and there is neither evidence of immune responses, tissue necrosis nor osteolysis are observed after experiment. Histological images of cross section of diaphysis in the femoral site of the control and experimental specimens can be seen in Figure 8. At 4 weeks, cortical bone wall containing numerous osteons is adjacent to the bone marrow interspersed among trabeculae, plenty of

4. DISCUSSION 4.1. Microstructure and Mechanical Properties. Amorphous carbon films as surface modification of materials can offer a hard, wear resistant, low friction, and inert surface, which make them have great potential for biomedical application. However, its drawbacks of high intrinsic stress and low adhesion are a key problem which can cause implant failure. A bias-graded multilayer structure carbon film is designed in this work to solve these problems due to the multilayer structure can deflect or reduce the crack propagation and decrease the stress concentration. Additionally, bias-graded method is another effective method to achieve high hardness, good toughness as well as high adhesion strength.13 The a-C NM film on Ti6Al4V alloy has appropriate hardness of 20 GPa, high toughness and high adhesion strength of about 80 N. The enhanced hardness is mainly ascribed to the presence of nanocrystalline TiC and plenty of interfaces, which can hinder

Figure 7. Effect of extracts on histological findings in mice during 28 days repeated dose toxicity study. Experimental groups: (A) heart, (B) liver, (C) kidney, (D) paranephros, (E) spleen, (F) gonad, (G) ovary. Control groups: (H) heart, (I) liver, (J) kidney, (K) paranephros, (L) spleen, (M) gonad, (N) ovary. Scale bar represents 20 μm. 15938


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ACS Applied Materials & Interfaces dislocation movements. Moreover, improved adhesion strength of the film is resulted from the bias-graded structure which ensures no abrupt change in composition and high toughness as above-mentioned. Compared to a previous work,13 the adhesion strength of bias-graded nanomultilayer is obviously higher than that of bias-graded nanocomposite films, which may result from nanomultilayer structure in which the interfaces become the sites of energy dissipation and crack deflection.10 Additionally, bias-graded a-C NM film exhibits low friction and high wear resistance in both ambient air and simulated body fluid (Hank’s solution) because of the graphitelike tribolayer and lubrication function of Hank’s solution. From the EDAX analysis of wear track in Hank’s solution it can be deduced that the bias-graded a-C NM film possesses good chemical inert (Figure S3). Multilayer structure can significantly enhance the mechanical and tribological properties of aC based film. 4.2. In Vitro Biocompatibility. As one of the most promising medical implant materials, the a-C NM film on Ti6Al4V alloy has shown significant advantage of mechanical properties over other candidates. To access to medical application, a series of testing for assuring the safety of product is necessary. In this work, biosafety and efficacy of a-C NM films were evaluated systematically by ISO 10993 and OECD guideline. In vitro cell cytotoxicity test is a rapid, simple, convenient and sensitive quantitative assay, which is often as a necessary item for initial biocompatibility evaluation of the medical devices.36−39 The toxicity of the material to the cell can be sensitive indicated by MTT. In normal culture medium, cells proliferate by mitosis, although if there are any external factors influence the growth, the cell adhesion, activity and proliferation rate will be decreased and the cells even be killed.40,41 Cell type is an important consideration for cytotoxicity test. In our work, L-929 mouse fibroblasts cells were chose because they were wildly used for cytotoxicity test of biomaterials42−45 and recommend by ISO 10993−5. The cell proliferation rate is also related with the concentration of the extracts. Four different extracts were chosen in the cell cytotoxicity tests. The procedure gives us hints about the cytotoxicity effect of the film to cell. MTT results can be accepted as an early indicator of the safety of the a-C NM films. Ames is a method with high sensitivity, specificity to detect whether a substance has genotoxicity.46,47 S. typhimurium mutant (histidine defect type) strain cannot grow in the medium without histidine, but if there is mutation agent in the medium, the salmonella mutant can back mutation to wild type (phenotype) and grow up in the medium without histidine.48 A substance is regarded as positive if induced at least an increase of 2-fold compared with positive control in mean revertant numbers, a dose−response relationship is observed.49,50 In this test, no mutagenic effect was observed, the extracts were considered as no reverse mutation inducement in S. typhimurium under experimental conditions. Ames can evaluate mutagenicity of a biomaterial in a short-term, but there are some uncertainties of false negative results caused by strain variation.51 Another uncertainty factor is that the gene system of microbe is simpler than mammal cause. Mammalian cell gene mutation and chromosome aberration test are necessary to comprehensively evaluate the genetic toxicity. L5178Y TK+/− is one of the cell lines recommend by OECD guideline No. 476 for TK gene mutation test. Cells deficient in thymidine kinase (TK) due to the mutation TK+/− to TK−/−

are resistant to cytotoxic effects of pyrimidine analogue trifluorothymidine (TFT).52 Thymidine kinase-proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not.21 A substance is regarded as positive if induced either a reproducible concentration-related increase in the MF or a reproducible positive response at least one of the tested concentrations. A response is regarded as biologically significant if a mutation frequency induced at least one of the concentrations is 2 times higher than the mean spontaneous mutation frequency in the experiment.53,54 The substance is therefore deemed to be mutagenic if there is a reproducible concentration-related increase in the mutation frequency. There are no statistical evaluation of the mutation rates in negative control, and all extracts do not induce mutations in the cell line L5178Y with and without of metabolic activation. For chromosome aberration test, relevant OECD guideline No. 473 recommends testing 5-fold higher concentrations in absence of cytotoxicity. However, initial maximum dose was selected based on results of a solubility test. Guideline requirement to score at least three concentrations were fulfilled in experiments with 24 h sampling time. In the 24 h sampling time, no doses induce aberrant metaphases, so the relationship of theoretical dose−response could not been observed.55,56 The validity of the results is not limited by dose selection or any other chosen experimental conditions.57,58 In comparison with positive controls, extracts did not cause any biologically relevant or statistically significant enhancement, which indicates that there is no potential mutagenicity of a-C NM films. The highest dose level used conforms to the maximum dose level recommended by relevant guideline and all other aspects of the chosen experimental conditions are also in line with guideline requirements, so that the test results are valid. 4.3. In Vivo Biocompatibility. Acute systemic toxicity test result preliminary indicates that the extract is not induce acute systemic toxicity. In subchronic systemic toxicity test, repeated exposure to extracts in 28 days had been utilized for determination of adverse effects of samples in laboratory animals. Hematology, blood chemical, and viscera coefficient analysis reflects the clinical risk evaluation of the a-C NM film on medical Ti alloy, the results indicate that there are not gender and no adverse effects of the samples under experimental condition. It is established a good toxicological evaluation for efficacy and safety of the products based on the a-C NM film in rabbits. Histological images of the implant experiment show that there are a few inflammatory cells, but with extending the time, inflammatory cells are gradually reduced at 8 weeks and 12 weeks. Inflammatory cell is a generally phenomenon usually occurred at the beginning of a recovery period after surgery. Histological image results are consistent with the case which presents in surgery studies.59−61 Histological findings indicate the reorganization of bone tissue around implantation site, which is similar to the previous work.62 Results of the implant experiment presented in this study support the presumption that the a-C NM films can be successfully applied in medical implant.

5. CONCLUSIONS The a-C NM film deposited on a medical Ti alloy has appropriate hardness and toughness, high adhesion strength, 15939


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(8) Yang, P.; Huang, N.; Leng, Y. X.; Chen, J. Y.; Fu, R. K.; Kwok, S. C.; Leng, Y.; Chu, P. K. Activation of Platelets Adhered on Amorphous Hydrogenated Carbon (a-C:H) Films Synthesized by Plasma Immersion Ion Implantation-Deposition (PIII-D). Biomaterials 2003, 24, 2821−2829. (9) Falub, C. V.; Müller, U.; Thorwarth, G.; Parlinska-Wojtan, M.; Voisard, C.; Hauert, R. In Vitro Studies of the Adhesion of DiamondLike Carbon Thin Films on CoCrMo Biomedical Implant Alloy. Acta Mater. 2011, 59, 4678−4689. (10) Cai, J. B.; Wang, X. L.; Bai, W. Q.; Wang, D. H.; Gu, C. D.; Tu, J. P. Microstructure, Mechanical and Tribological Properties of a-C/aC:Ti Nanomultilayer Film. Surf. Coat. Technol. 2013, 232, 403−411. (11) Voevodin, A. A.; Capano, M. A.; Laube, S. J. P.; Donley, M. S.; Zabinski, J. S. Design of a Ti/TiC/DLC Functionally Gradient Coating Based on Studies of Structural Transitions in Ti-C Thin Films. Thin Solid Films 1997, 298, 107−115. (12) Zhang, S.; Bui, X. L.; Fu, Y.; Butler, D. L.; Du, H. Bias-Graded Deposition of Diamond-Like Carbon for Tribological Applications. Diamond Relat. Mater. 2004, 13, 867−871. (13) Cai, J. B.; Wang, X. L.; Bai, W. Q.; Zhao, X. Y.; Wang, T. Q.; Tu, J. P. Bias-Graded Deposition and Tribological Properties of TiContained a-C Gradient Composite Film on Ti6Al4V Alloy. Appl. Surf. Sci. 2013, 279, 450−457. (14) Gulbiński, W.; Mathur, S.; Shen, H.; Suszko, T.; Gilewicz, A.; Warcholiński, B. Evaluation of Phase, Composition, Microstructure and Properties in TiC/a-C: H Thin Films Deposited by Magnetron Sputtering. Appl. Surf. Sci. 2005, 239, 302−310. (15) Strondl, C.; van der Kolk, G. J.; Hurkmans, T.; Fleischer, W.; Trinh, T.; Carvalho, N. M.; de Hosson, J. T. M. Properties and Characterization of Multilayers of Carbides and Diamond-Like Carbon. Surf. Coat. Technol. 2001, 142−144, 707−713. (16) Miyake, S.; Shindo, T.; Suzuki, M. Nanomechanical and Boundary Lubrication Properties of Titanium Carbide and DiamondLike Carbon Nanoperiod Multilayer and Nanocomposite Films. Surf. Coat. Technol. 2013, 221, 124−132. (17) Chen, R.; Tu, J. P.; Liu, D. G.; Yu, Y. L.; Qu, S. X.; Gu, C. D. Structural and Mechanical Properties of TaN/a-CNx Multilayer Films. Surf. Coat. Technol. 2012, 206, 2242−2248. (18) Wieciński, P.; Smolik, J.; Garbacz, H.; Kurzydłowski, K. J. Failure and Deformation Mechanisms During Indentation in Nanostructured Cr/CrN Multilayer Coatings. Surf. Coat. Technol. 2014, 240, 23−31. (19) Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65, 55−63. (20) Clements, J. The Mouse Lymphoma Assay. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2000, 455, 97−110. (21) Moorebrown, M. M.; Clive, D.; Howard, B. E.; Batson, A. G.; Johnson, K. O. The Utilization of Trifluorothymidine (TFT) to Select for Thymidine Kinase-deficient (TK−/−) Mutants from L5178Y/ TK+/− Mouse Lymphoma Cells. Mutat. Res. 1981, 85, 363−378. (22) Casiraghi, C.; Ferrari, A. C.; Robertson, J. Raman Spectroscopy of Hydrogenated Amorphous Carbons. Phys. Rev. B: Condens. Matter Mater. Phys. 2005, 72, 1−14. (23) Wang, Q. Z.; Zhou, F.; Zhou, Z. F.; Yang, Y.; Yan, C.; Wang, C. D.; Zhang, W. J.; Li, L. K.-Y.; Bello, I.; Lee, S.-T. Influence of Ti Content on the Structure and Tribological Properties of Ti-DLC Coatings in Water Lubrication. Diamond Relat. Mater. 2012, 25, 163− 175. (24) Niakan, H.; Yang, Q.; Szpunar, J. A. Structure and Properties of Diamond-Like Carbon Thin Films Synthesized by Biased Target Ion Beam Deposition. Surf. Coat. Technol. 2013, 223, 11−16. (25) Wang, Q. Z.; Zhou, F.; Zhou, Z. F.; Yang, Y.; Yan, C.; Wang, C. D.; Zhang, W. J.; Li, L. K.-Y.; Bello, I.; Lee, S.-T. Influence of Carbon Content on the Microstructure and Tribological Properties of TiN (C) Coatings in Water Lubrication. Surf. Coat. Technol. 2012, 206, 3777−3787. (26) Mani, A.; Aubert, P.; Mercier, F.; Khodja, H.; Berthier, C.; Houdy, P. Effects of Residual Stress on the Mechanical and Structural

and good tribological properties in both ambient air and Hank’s solution. Biological evaluation presented in vitro and in vivo studies supports the presumption that the a-C NM film is no cytotoxic, no genotoxicity, no subchronic systemic toxicity, and no contraindications in rabbit bone implant. This newly designed film on medical alloy with excellent mechanical properties and biocompatibility prospect a good candidate for biomaterials, especially for implant such as knee and hip joints.

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ASSOCIATED CONTENT

* Supporting Information S

EThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b02552. Experimental details, SEM image, Raman spectrum, and EDAX analysis of wear track of a-C/a-C:Ti nanomultilayer films, in vitro and in vivo biocompatibility test data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.L.W.). Tel.: +86 571 87952856. Fax: +86 571 87952573 *E-mail: [email protected] (J.P.T.). ORCID

Changdong Gu: 0000-0001-8286-263X Jiangping Tu: 0000-0002-7928-1583 Author Contributions †

L.L.L. and W.Q.B. contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (2016YFC1101900 and 2016YFF0204305).

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REFERENCES

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