j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2

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Original Article

3D-printed Ti6Al4V femoral component of knee:

Improvements in wear and biological properties by
AIP TiN and TiCrN coating

JunJie Ni a, Fan Liu b, Gaolin Yang c, Gun-Hwan Lee d, Sung-Min Chung e,

In-Seop Lee a,f, Cen Chen a,*
a
College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, PR China
b
Department of Orthodontics, School of Stomatology, China Medical University, Shenyang, 110002, PR China
c
Institute of Laser Advanced Manufacturing, Zhejiang University of Technology, Hangzhou, 310023, China
d
Department of Surface Technology, Korea Institute of Materials Science, Changwon, 641-831, Republic of Korea
e
Biomaterials R&D Center, GENOSS Co., Ltd., Suwon-si, 443-270, Republic of Korea
f
Institute of Human Materials, Suwon, 16514, Republic of Korea

article info abstract

Article history: As patients are dissatisfied with the excessive weight of Co-based knee femoral compo-
Received 21 May 2021 nents, it is an urgent need to reduce their weight without compromising the excellent
Accepted 28 July 2021 mechanical properties. In the present study, femoral components of knee with Ti6Al4V
Available online 4 August 2021 alloy (86 g) or CoeCr alloy (192 g) were printed by selective laser melting (SLM). In order to
improve the mechanical properties and biocompatibility of 3D-printed Ti6Al4V, TiN and
Keywords: TiCrN coating layers were deposited on the surface of SLM Ti6Al4V using arc ion plating
TiN (AIP) system. The surface characterizations were analyzed by scanning electron micro-
TiCrN scopy (SEM) equipped with energy dispersive spectrometer (EDS), X-ray diffraction (XRD),
Arc ion plating contact angle tensiometer, and contact profilometer. The wear property, hardness value
SLM Ti6Al4V and corrosion resistance in phosphate buffered saline (PBS) were also investigated through
Mechanical properties vickers hardness, sliding wear test, electrochemical impedance spectroscopy and poten-
Biological evaluation tiodynamic polarization. The antibacterial capability and biocompatibility were assessed
with Staphylococcus aureus and mesenchymal stem cells (MSCs) in vitro. Results illustrated
that, after coating SLM Ti6Al4V with thin TiN and TiCrN layers, the hardness, anti-wear
performance and corrosion resistance of SLM Ti6Al4V were significantly improved and
became comparable to SLM CoCr. The TiN and TiCrN coated SLM Ti6Al4V also showed
moderate antibacterial ability, and excellent biocompatibility in comparation with SLM
Ti6Al4V and SLM CoCr.
© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author.
E-mail addresses: chencen@zstu.edu.cn, chencen313@gmail.com (C. Chen).
https://doi.org/10.1016/j.jmrt.2021.07.143
2238-7854/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2
1. Introduction
The total knee arthroplasty (TKA) is a highly efficacious and
cost-effective procedure for patients with knee osteoarthritis.
Currently, there are more than 200,000 TKA surgery in China
every year, which will increase year by year [1]. The common
artificial knee joint is composed of femoral component, tibial
component and tibial tray. The wear between femoral
component and tibial tray during patients’ activity is the
major factor in the reliability and durability of artificial knee
joint [2].CoCr is the preferred material for femoral component
of knee prosthesis due to its good mechanical properties
especially in wear resistance [3,4]. However, the in vitro ex-
periments show that cobalt ion can affect the growth of os-
teoblasts cells [5]. Moreover, patients have shown
dissatisfaction with the excessive weight because of the high
density of Co based alloys [6]. Therefore, there is an urgent
need to reduce the weight of knee femoral component
without sacrificing its mechanical properties.
Ti6Al4V alloy is an extensively used material for light-
weight structure and load-bearing implants [7], due to its good
corrosion resistant, high strength to density ratio, and excel-
lent biocompatibility [8]. Over the last decades, additive
manufacturing (AM), such as electron beam melting (EBM) and
selective laser melting (SLM), has been increasingly interested
to produce patient oriented Ti6Al4V implants with 3D com-
plex architectures and well-designed pores [9]. However, poor
wear resistance has limited its applications [10,11]. Wear on
articulating surface often induce the metal ion release and
increase the risk of inflammation. Protective coatings can be
used to solve such problems [12,13].
Surface coating can reduce the wear of substrate materials
to improve the stability of prosthetic implants [14]. In the
previous studies, metal nitride coatings were widely studied
because of their excellent mechanical properties [15,16]. It is
reported that the nitride coatings can apparently reduce the
release of toxic ion from Ti6Al4V [17]. Especially, TiN [13] and
TiCrN [18] coatings get a lot of attention owing to high hard-
ness and good resistance to abrasion. Also, the TiN and TiCrN
coatings have been proved to improve the antibacterial ability
and biocompatibility [19].
Currently, many techniques such as magnetron sputtering
[20], plasma spraying [21] and vacuum-arc deposition [22]
have been applied to deposit metal nitride coatings. The arc
ion plating (AIP) which have strong merits of high ionization
degree is widely used for depositing TiN and TiCrN coatings
[23]. Although many researches have investigated the me-
chanical properties of TiN and TiCrN coatings on wrought
Ti6Al4V [24,25], few reports have been published about the
application of TiN and TiCrN coatings on 3D-printed Ti6Al4V
to improve tis mechanical properties and biological behaviors.
In this study, CoCr and Ti6Al4V femoral components and
discs were fabricated by SLM. TiN and TiCrN coatings were
synthesized on SLM Ti6Al4V through AIP techniques. The
mechanical properties, corrosion resistance, antimicrobial
capability and cyto-compatibility of TiN and TiCrN coated SLM
Ti6Al4V are systematically investigated to compare with SLM
CoCr.
2323
2. Materials and methods
2.1. Chemicals and materials
Phosphate buffered saline (PBS), live/dead viability/cytotox-
icity kit and phalloidin-TRITC were acquired from Thermo
Fisher Scientific (Waltham, MA, USA). Mesenchymal stem
cells (MSCs) from SpragueeDawley rat and MSC growth me-
dium were purchased from Cyagen Biosciences (Soochow,
China). Penicillin, streptomycin, 40 ,6-diamidino-2-
phenylindole (DAPI) and Cell Counting Kit-8 (CCK-8) were
obtained from Beyotime Biotechnology (Shanghai, China).
Ti6Al4V powders were purchased from Supra Alloys Inc.
(Camarillo, CA, USA), while CoCr powders were acquired from
LPW Technology (LPW Technology Ltd, Runcorn, UK).
2.2. Preparation of substrate
Femoral components of knee and disc-samples (20 mm
diameter, 2 mm thickness) were fabricated with TiAl4V or
CoCr powders by SLM system (EOSINT-M280, EOS GmbH,
Munich, Germany) based on previously reported procedure
[26]. Briefly, Ti6Al4V powder (30 mm diameter) and CoCr pow-
der (15e45 mm diameter) was selected as basic materials. The
models were firstly designed by CAD software, sequentially
outputted into STL data, and built layer by layer through laser
beam scanning. The SLM process parameters were as follows:
(1) for SLM Ti6Al4V, laser power: 400 W; scanning velocity:
700 mm/s; (2) for SLM CoCr, laser power: 130 W; scanning
velocity: 900 mm/s.
2.3. Deposition of TiN and TiCrN coating
Prior to coating, SLM Ti6Al4V components or discs were pol-
ished using SiC sandpaper from 600 meshes to 4000 meshes
sequentially, then cleaned with acetone, ethanol, and distilled
water. TiN and TiCrN coatings were deposited by AIP on pol-
ished SLM Ti6Al4V according to previous study [27]. The vac-
uum arc sources are composed of a titanium (99.9%) target or a
titaniumechromium target with 25% Cr composition. SLM
Ti6Al4V were mounted on a substrate holder with 10 rpm
speed to uniform the coating layers. After being pumped down
to 1  105 torr, chamber pressure was reduced to 101 torr by
introducing Ar. The deposition conditions were as follows:
working pressure: 3  103 torr; voltage: 50 V; current: 60 A;
time: 4 h; gas feeding rate (Ar: N2): 100: 200 (TiN); gas feeding
rate (Ar: N2): 50: 300 (TiCrN); target distance: >250 mm.
2.4. Surface characterization
The surface microstructure and element distribution were
characterized by scanning electron microscopy (SEM, Quanta
200; FEI Company, Philips, Netherlands) with energy disper-
sive spectrometer (EDS). The phase composition of different
samples was measured by an X-ray diffractometer (XRD,
Philips PW1700). 2q range of XRD analyses was set from 10 to
80 with speed of 2 /min. Water contact angle was performed
by a video contact angle tensiometer (DSA25, Kruss, Hamburg,
2324 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2
Germany). The surface roughness was measured by using a
contact profilometer (Dektak XT, Bruker, Berlin, Germany).
2.5. Mechanical evaluation
The wear properties of the coatings were analyzed by using a
pin-on-disk-type tribometer. Si3N4 ball (6.3 mm) was used as
friction counterpart to investigate dry sliding wear properties.
The conditions were as follows: contact load: 5 N; rotational
speed: 100 rpm/min; radius: 2 mm; time: 1200 s. Worn surfaces
were observed with optical microscope (IX53, Olympus,
Tokyo, Japan), while worn tracks were evaluated by a 2D
profiler (SJ-301, Mitutoyo, Kanagawa, Japan). The microhard-
ness of the coatings was performed with 25 g load on a HV-
1000 tester in an elapsed time of 15 s.
2.6. Electrochemical corrosion studies
The corrosion properties were determined through electro-
chemical impedance spectroscopy (EIS) and potentiodynamic
polarization in PBS solution. A three-electrode cell system was
applied in electrochemical measurements by electrochemical
workstation (CHI600E, Shanghai Chenhua): platinum as
counter electrode, standard calomel electrode (SCE) as the
reference electrode. Prior to measurements, the samples were
soaked in PBS solution for 60 min to establish open circuit
potential (OCP). EIS measurements were accomplished by
applying a sinusoidal potential perturbation of 5 mV at the
OCP and a sweep from 10 mHz to 100 kHz in logarithmic
decrement. The potentiodynamic polarization curves were
measured from 1.0 to 1.5 V with a scan rate of 5 mV/s.
2.7. In vitro antibacterial assessment
The bacteriostatic properties were investigated with Staphy-
lococcus aureus. All samples were sterilized using ultraviolet
light for 30 min in 75% ethanol and distilled water. Different
samples were placed in 12-well plates, and 1 mL of the sus-
pension of bacterial (105 CFU/mL) were added on the sample
surface. After 24-h cultivation, 200 mL suspension of each well
were transferred into solid medium for another 24-h incuba-
tion. Then the number of bacteria colonies was counted
Fig. 1 e Photographs of non-coated SLM CoCr, TiN and TiCrN coated SLM Ti6Al4V knee femoral components; and SLM CoCr,
SLM Ti6Al4V, TiN or TiCrN coated SLM Ti6Al4V discs.
according to GB/T 4789.2. The antibacterial rates were calcu-
lated as follows [28]:
Antibacterial rate (%) ¼ [(CFU of control groups - CFU of
experimental groups) / CFU of control groups]  100%.
2.8. Cell culture
MSCs were thawed and cultured in MSC growth medium with
5% CO2 at 37 C. MSCs were collected and sub-cultured every
three days, at a density of 2  105 cells/mL. Cells of passage
2e6 were used for further cell experiments.
2.9. Indirect cytotoxicity
Cytotoxicity was evaluated through CCK-8 and live/dead kit,
where MSCs were indirectly exposed to extract solution for
24 h and 48 h. To prepare extract solution for cytotoxicity test,
SLM CoCr, SLM Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V
discs were immersed in MSC growth medium at 37 C for 72 h.
MSCs were cultured on tissue-culture treated polystyrene
(TCP) overnight, then the cell culture medium was replaced
with extracted solution, respectively. The untreated MSCs
cultured on TCP served as TCP control. CCK-8 can be reduced
into yellow formazan by dehydrogenase in mitochondrial.
The Cell viability can be indirectly demonstrated by
measuring the light absorption value at the wavelength of
450 nm [29]. For quantitative test, CCK-8 reagent was added to
each sample and incubated for 1 h at 37 C. Then, MSCs
viability was determined at 450 nm using an enzyme-labeled
instrument (DNA Export, TECAN, Austria). The live/dead
viability/cytotoxicity kit were used for the qualitative assay.
MSCs were stained by Calcein AM (Green) and EthD-1 (Red) at
4 C for 10 min in the dark, then observed under confocal mi-
croscope (IX81-FV1000, Olympus, Tokyo, Japan).
2.10. Cell proliferation
Discs were sterilized in 75% ethanol and distilled water for
30 min each under ultraviolet light before test. MSCs were
cultured on TCP control, SLM CoCr, SLM Ti6Al4V, TiN and
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2
Fig. 2 e SEM images of as-printed SLM Ti6Al4V (a, b), TiN (c, d) and TiCrN (e, f) coatings on polished SLM Ti6Al4V.
TiCrN coated SLM Ti6Al4V with a density of 2  104 cells/mL to
explore the cell proliferation. The untreated MSCs cultured on
TCP were used as TCP control. After 1, 3, 5 and 7 days of cul-
ture, cell viability was evaluated by CCK-8 at 450 nm. MSCs
were also stained by phalloidin-TRITC and DAPI to observe the
cellular morphology with confocal microscope.
2.11. Statistics
All quantitative tests were obtained with triplicate or quad-
rant repeats, and expressed as mean ± standard deviation
(SD). A one-way ANOVA followed by Tukey's test was used to
the statistical analysis among different groups. P value < 0.05
were considered statistically significant.
3. Results and discussion
3.1. Composition and structure
The macroscopic morphologies of the SLM CoCr, TiN and
TiCrN coated SLM Ti6Al4V femoral components of knee were
showed in Fig. 1. The weight of SLM CoCr femoral component
was 192 g, while the weight of TiN and TiCrN coated SLM
Ti6Al4V femoral components with same size were ~44% of
2325
SLM CoCr. The TiN and TiCrN coated SLM Ti6Al4V maintained
a mirror surface, but displayed noticeable changes in their
color after the vacuum AIP. The as-deposited TiN coating
presented golden color, while the TiCrN coating exhibited
silver.
SLM disc samples in Fig. 1 were prepared for subsequent
characterizations. Typical microstructures of SLM Ti6Al4V
and the TiN, TiCrN coated substrate were examined under
SEM (Fig. 2). Unmelted Ti6Al4V particles were found on as-
printed SLM Ti6Al4V disc, but mostly disappeared by the
following polish. It is due to unmelted particles to be sintered
on surfaces through staircase effect [30]. The top view of
coated surface indicated that the TiN and TiCrN were homo-
geneously coated on polished SLM Ti6Al4V surface. Many
particles and droplets were observed after deposition on SLM
Ti6Al4V. The surface characteristics were consistent with
those of AIP coating technology [31], which was owing to the
deposition of melted droplets with different size on SLM
Ti6Al4V surface [32,33].
The cross-sectional images of SLM Ti6Al4V and obtained
coatings were shown in Fig. 3. The thickness of TiN and TiCrN
coatings were 1.33 ± 0.06 mm and 1.42 ± 0.08 mm, which is
similar to other published report of AIP coatings [19]. Elements
distribution map of SLM Ti6V4V, TiN and TiCrN coated SLM
Ti6Al4V were analyzed by EDS. Obviously, compared with SLM
2326 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2

Fig. 3 e Cross-sectional SEM images and elements distribution map of SLM Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V.

Ti6Al4AV, the TiN coating had higher content of N atoms;

while TiCrN coated SLM Ti6Al4V had higher content of Cr
atoms on out lateral. The EDS confirmed successful deposition
of TiN and TiCrN coatings on SLM Ti6Al4V surface.
The XRD patterns of coated and as-printed SLM Ti6Al4V
were shown in Fig. 4. Typical peaks belonging to SLM Ti6Al4V
were displayed with many phases mainly of (101) and (002).The
TiN coated SLM Ti6Al4V had TiN phase with strong (111) grains
which is normally appeared from coatings deposited by AIP
[23]. While, a preferred orientation (200) and relative weak peak
(111) appeared as for TiCrN coated SLM Ti6Al4V. Many studies
have shown that adding Cr element will change the crystal
phase [18]. In addition, peaks belong to SLM Ti6Al4V were also
observed for coated samples because X-ray can penetrate the
thin coating layers and reach the substrates.
The contact angle and surface roughness of SLM Ti6Al4V,
TiN and TiCrN coated SLM Ti6Al4V were shown in Table 1. The
contact angle of SLM Ti6Al4V substrate (65.57 ± 2.21 )
Fig. 4 e XRD profile of the SLM Ti6Al4V, TiN and TiCrN
increased when coated with TiN (70.70 ± 1.55 ) and TiCrN
coated SLM Ti6Al4V.
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2 2327

rate of SLM CoCr, SLM Ti6Al4V, TiN and TiCrN coated SLM
Table 1 e The roughness and contact angle of SLM
Ti6Al4V. The SLM Ti6Al4V discs (wear rate ¼ 1980  106 mm3/
Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V. Values
represented the mean ± SD (n ¼ 3). Nm) had the lowest wear resistance. The wear resistance
substantially improved after deposited with TiN (wear
Sample Ra (mm) Contact angle ( )
rate ¼ 535  106 mm3/Nm) and TiCrN (wear
SLM Ti6Al4V 4.35 ± 0.02 65.57 ± 2.21 rate ¼ 34  106 mm3/Nm) coatings (P < 0.05). It is worth noting
TiN Coating 1.39 ± 0.07 70.70 ± 1.55
that the TiCrN coated SLM Ti6Al4V showed better resistant to
TiCrN Coating 1.11 ± 0.06 76.20 ± 2.89
wear than SLM CoCr (wear rate ¼ 505  106 mm3/Nm)
(76.20 ± 2.89 ). As-printed SLM Ti6Al4V had the highest (P < 0.05), which may contribute to the addition of Cr element.
roughness of 4.35 ± 0.02 mm, but decreased to the average Fig. 5D displayed the surface hardness of SLM CoCr, SLM
surface roughness of 1.39 ± 0.07 mm, 1.11 ± 0.06 mm after pol- Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V. After AIP
ished and coated with TiN, TiCrN, respectively. deposition, measurable improvement of surface hardness
had achieved for TiN (2100 HV) and TiCrN (2200 HV) coated
3.2. Mechanical and wear properties SLM Ti6Al4V, which was much higher than uncoated SLM
Ti6Al4V (400 HV) and SLM CoCr (417 HV) (P < 0.05). Although
The wear properties were examined by optical microscope the weight of TiN and TiCrN coated SLM Ti6Al4V femoral
and 2D profiles in Fig. 5A and B. Wear resistance were components was ~50% lower than SLM CoCr, they presented
explored through wear track. The track width and depth of the comparable mechanical properties to CoCr-based
as-polished SLM Ti6Al4V was widest and deepest. After components.
deposited with TiN and TiCrN coatings, the grooves dimin- Even though, the mechanical properties of 3D-printed
ished. The groove of TiCrN coated SLM Ti6Al4V was even Ti6Al4V were significantly improved by applying thin TiN and
small than non-coated SLM CoCr. Fig. 5C displayed the wear TiCrN coating layers, the wear resistance and coating

Fig. 5 e Mechanical properties of SLM CoCr, SLM Ti6Al4V, TiN and TiCrN coated Ti6Al4V: (A) optical microscope images of
worn surfaces, (B) the profiles of wear tracks, (C) wear rate, and (D) surface hardness. Values represented the mean ± SD
(n ¼ 3). *, P < 0.05 compared with SLM CoCr. #, P < 0.05 compared with SLM Ti6Al4V.
2328 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2
Fig. 6 e Electrochemical behaviors in PBS solution: (A) Nyquist plots and (B) polarization curves. In Fig A, (a) SLM CoCr, (b)
SLM Ti6Al4V, (c) TiN coating, (d) TiCrN coating, (e) equivalent circuit for SLM CoCr and SLM Ti6Al4V, and (f) equivalent circuit
for TiN and TiCrN coatings. In Fig B, (a) SLM CoCr, (b) SLM Ti6Al4V, (c) TiN coating, and (d) TiCrN coating.
hardness are still lower than those on wrought Ti6Al4V
[34e36]. Such differences mainly relate to the better wear
resistance and hardness of the wrought substrate than that of
SLM substrate according to published study reported by Lyu
[37]. Future research should focus on the improving the me-
chanical properties by modulating AIP parameters.
3.3. Corrosion analysis
Electrochemical impedance spectroscopy (EIS) and potentio-
dynamic polarization were used to test the bio-corrosion
resistance of coated and uncoated samples. PBS solution
were used to imitate the physiological environment. SLM CoCr,
SLM Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V were soaked
in PBS solution for 60 min to achieve a consistent OCP value.
Fig. 6A demonstrated Nyquist plots of different samples.
The diameter of semi-circles of TiN and TiCrN coated SLM
Ti6Al4V were larger than that of uncoated SLM Ti6Al4V, which
indicated that they had higher corrosion protection. SLM CoCr
illustrated largest semi-circles compared to other samples.
The electrical equivalent circuit for EIS plots were presented in
Fig. 6A (e) (for coated samples) and (f) (for uncoated samples),
which demonstrated the electrochemical characteristics of
surface. The charge transfer resistance (Rct) results calculated
from Fig. 6A (e) and (f) were compared in Table 2. An increase
in Rct value indicated a higher corrosion resistance. The Rct
value of TiCrN (3.60  106 U/cm2) and TiN (6.61  105 U/cm2)
coated SLM Ti6Al4V were much higher than that of SLM
Ti6Al4V (3.95  104 U/cm2), but slightly lower than SLM CoCr
(6.51  106 U/cm2). The Rct value of TiN and TiCrN coating are
Table 2 e Electrochemical parameters of SLM CoCr, SLM
Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V immersed in
PBS solution.
Sample Rct/(U/cm2) Ecorr (V) Icorr (mA/cm2)
SLM CoCr 6.51  106
0.215 0.90
SLM Ti6Al4V 3.95  104 0.507 1.42
TiN Coating 6.61  105 0.342 1.20
TiCrN Coating 3.60  106 0.272 1.04
higher than the published study reported by Thampi [18],
which may induced by relative small surface roughness.
Potentiodynamic polarization test was further performed
to evaluate the corrosion behavior of TiN and TiCrN coatings.
Fig. 6B showed the polarization curves of different samples
and the related corrosion potential (Ecorr) and current density
(Icorr) were presented in Table 2. Ecorr indicates the driving
force of the corrosion process. SLM CoCr had the highest Ecorr
among all samples. Compared with uncoated SLM Ti6Al4V,
the higher Ecorr of TiN or TiCrN indicated that the corrosion
process become more noble by the nitride coatings. The Icorr
can directly reflect the corrosion rate under Ecorr [38]. The Icorr
values were 0.90 mA/cm2 for SLM CoCr,1.42 mA/cm2 for un-
coated SLM Ti6Al4V, 1.20 mA/cm2 for the TiN coating, and
1.04 mA/cm2 for the TiCrN coating, respectively. Compared
with SLM Ti6Al4V, the Icorr of TiN and TiCrN coated SLM
Ti6Al4V were lower, which means better corrosion resis-
tance. Although the AIP technical brought vast improvement
of the corrosion resistance, the corrosion resistance of SLM
CoCr was still the best. The lower corrosion resistance of TiN
and TiCrN coated SLM Ti6Al4V than SLM CoCr are probably
due to some small defects on the thin coating, which will
lead to the exposure of substrate. Increasing the uniformity
of the coating will be a considerable choice to improve the
corrosion resistance of TiN and TiCrN coated SLM Ti6Al4V
[39].
3.4. Antibacterial assay
S. aureus takes part in more than half of implant infections
[40]. The antibacterial capability was investigated through
plate counting method in Fig. 7. The number of S. aureus on
TiCrN coating was the smallest among all experiment
groups (P < 0.05). The bacteriostatic properties with
different samples were ranked as follows:
TiCrN > TiN > SLM Ti6Al4V > SLM CoCr. It is reported that
nitride coatings could limit the metabolism and prolifera-
tion of bacteria [19]. Thus, TiN and TiCrN coated SLM
Ti6Al4V have participated in a physical obstruction to avoid
bacteria adhering on SLM Ti6Al4V surface. What is more,
the relative smooth surface of TiN and TiCrN could also
limit the bacterial adhesion.
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2 2329

Fig. 7 e (A) Re-cultivated S. aureus colonies on agar plates of diff erent surface, and (B) antibacterial rates of diff erent surface
after being incubated for 24 h. Values represented the mean ± SD (n ¼ 3 or 4). *, P < 0.05 compared with SLM CoCr. #, P < 0.05
compared with SLM Ti6Al4V.

3.5. MSC biocompatibility
The biocompatibility of TiN and TiCrN coated SLM Ti6Al4V
determines whether they can be applied to the human body.
The indirect cytotoxicity of TiN or TiCrN coated SLM Ti6Al4V
was measured with MSCs firstly. Fig. 8A depicted the
viability of MSCs cultured in the extracted solution of SLM
CoCr, SLM Ti6Al4V, TiN and TiCrN coated samples for 24 h
and 48 h by CCK-8. The MSCs grew well in the extract so-
lutions of all samples (>70%, ISO: 10993-5:2009(9)). It is
noteworthy that the percentages of MSCs viability in the
extract solution of TiN or TiCrN coated SLM Ti6Al4V even
reached 91.6% and 106.2% after 48 h, respectively. The cell
viability showed no statistically differences after 24 h and

Fig. 8 e Indirect cytotoxicity of MSC in extracted solutions of SLM CoCr, SLM Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V for
24 and 48 h. (A) Survival percentage relative to TCP control. Values represented the mean ± SD (n ¼ 3 or 4). * P < 0.05
compared with TCP control. # P < 0.05 compared with SLM CoCr. (B) Staining of MSCs with Calcein-AM and EthD-1. Scale bar:
200 mm.
2330 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2

Fig. 9 e MSC proliferation on SLM Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V for 1, 3, 5 and 7 days. (A) CCK-8 results at
OD450. Values represented the mean ± SD (n ¼ 3 or 4). *, P < 0.05 compared with TCP control. #, P < 0.05 compared with SLM
CoCr. (B) Phalloidin-TRITC and DAPI staining. Scale bar: 200 mm.

48 h incubation of all samples (P > 0.05). Calcein-AM (green)
and EthD-1 (red) staining can directly reflect the survival of
MSCs. As shown in Fig. 8B, nearly all MSC cells were alive,
and almost no dead cells were observed after 24 h and 48 h.
The staining results revealed that the TiN and TiCrN coated
SLM Ti6Al4V showed good cytocompatibility. Thus, as the
coating materials for knee femoral components, TiN and
TiCrN coating layers could be applied to human body
without serious toxicity.
Having proved that TiN and TiCrN coating had no cyto-
toxicity to MSCs, MSCs were cultured on TCP control, SLM
CoCr, SLM Ti6Al4V, TiN and TiCrN coated SLM Ti6Al4V for 1,
3, 5 and 7 days to evaluate t their influence on MSC
proliferation.
Fig. 9A showed the cell viability of MSCs on represen-
tative samples. It revealed an increase in the cell viability
with incubation time for all sample groups. After 1-, 3-, 5-
and 7-day culture, the cell viability of TiN, TiCrN coated
SLM Ti6Al4V were slightly lower compared with TCP con-
trol but had no statistically differences (P > 0.05). MSCs on
TiCrN showed slightly higher cell activity than on TiN
(P > 0.05). At Day 5, the cell viability of SLM CoCr was
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 4 : 2 3 2 2 e2 3 3 2
significantly lower than TiCrN coated SLM Ti6Al4V
(P < 0.05). After 7-day culture, MSCs on coated and un-
coated SLM Ti6Al4V exhibited higher cell viability than
those on SLM CoCr (P < 0.05).
To observe cell morphology, cytoskeleton actins were
stained by phalloidin-TRITC in red and the nuclei were stained
by DAPI in blue. As shown in Fig. 9B, MSCs cytoskeleton was
well distributed on all sample groups at day 1. Usually, the
MSCs grow randomly orientation on substrates [41]. The MSCs
appeared align growth from day 3 to day 5 only on TiN and
TiCrN coated SLM Ti6Al4V, but not on TCP control, SLM CoCr
and SLM Ti6Al4V. This cell alignment may be due to the
grooves after polishing [42]. Compared with the SLM Ti6Al4V
samples, the MSCs on TiN and TiCrN coated samples reflected
higher cell viability. After exposed to body fluids, the metallic
parts of implants will be oxidized and corroded, which leads to
the release of toxic elements. The biocompatibility of SLM
Ti6Al4V was increased owing to the cytocompatibility and
stability of TiN and TiCrN coatings which can also decrease
the release of toxic elements from Ti6Al4V.
4. Conclusions
In this study, CoCr and Ti6Al4V femoral components and discs
were fabricated by SLM. TiN (1.33 mm), and TiCrN (1.42 mm)
coating layers were deposited on SLM Ti6Al4V using AIP sys-
tem and the mechanical properties and biocompatibility were
investigated. TiN and TiCrN coated SLM Ti6Al4V had excellent
hardness (TiN ¼ 2100 HV, TiCrN ¼ 2200 HV) and wear resis-
tance (535  106mm3/Nm (TiN), 34  106 mm3/Nm (TiCrN),
respectively). The TiN and TiCrN coated SLM Ti6Al4V had
higher corrosion potential and Rct, lower corrosion current
density than those of the uncoated SLM Ti6Al4V. In vitro
experiment showed that the TiN and TiCrN coatings were
antibacterial (>50%), highly biocompatible, and noncytotoxic.
Although the weight is ~50% lower than SLM CoCr, TiN and
TiCrN coated SLM Ti6Al4V displayed comparable mechanical
properties, better antibacterial ability and biocompatibility
than SLM CoCr. Therefore, the TiN and TiCrN coated SLM
Ti6Al4V might be promising to serving as the material for
femoral components.
Declaration of Competing Interest
We declare that we have no financial and personal relation-
ships with other people or organizations that can inappropri-
ately influence our work, there is no professional or other
personal interest of any nature or kind in any product, service
and/or company that could be construed as influencing the
position presented in, or the review of, the manuscript entitled.
Acknowledgments
This research was supported partly by Zhejiang Provincial
Natural Science Foundation of China (LY20E010006), partly by
National Natural Science Foundation of China (51502265,
81701033).
2331
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